Synthetic Biology Exam 2

Primer Design

Primers can be made for any DNA sequences -Primers are *oligonucleotides/ ssDNA* Appropriate Length -Depends on purpose -Good primer is roughly 20bp *Annealing Temp: 55*C-70*C* -Depends on function of length and GC content -Want *higher GC content* because GC binds better than AT -*Higher the GC content, higher the annealing temp* -Want the temperature to be in a good range ~ex) 20bp, 30% GC, 45*C annealing = not good ~ex) 20bp, 55% GC, 55*C annealing = good *Design Considerations* -Only 1 primer binding site per location in template -No strong Primer Dimers -PCR rxn usually has 2 different primers -Primer Dimer is when 2 different primers bind and make dsDNA, producing no amplification -Does not have string secondary structure -Primer should not fold on itself

Cloning based on Homologous Recombination (Ligation Independent Cloning)

Primers for these 3 methods can be interchangeable/ same primers for all: -CPEC = High Fidelity Pol -SLIC = T4 Polymerase, Exonuclease -Gibson = Exonuclease, Polymerase, Ligase Difference is only in the enzymes used to do the cloning Sometimes, one method works better than other -reason is somewhat unknown -CPEC make have mis-priming events -SLIC has timing issues as to when nucleotides should be added -Gibson is done isothermally, and some Pols may not completely extend in that temperature

Base Editing with CRISPR/dCas9

Rather than cutting DNA and having new insertions, dCas9 can directly change single bp -done by fusing dCas9 to enzyme Cytidine Deaminase -changes C to U -U is copied or repaired to T There are other base editing enzymes that exist Functions, but has low efficiency -means you have to do many screens to find cell with desired mutation Problem with base editors is *significant off-targeting can occur* -Off-targeting with Cas9 can occur, so makes sense off-targeting can occur in base editing with dCas9 -significant problem if one wishes to tale Cas9 into therapeutic realm (don't want to cause potentially bad mutation)

Take Aways (Paper - Paper Based)

Riboswitches/toeholds can be used as biosensors in vitro Cheap and easy to use diagnostic tools (point of the whole paper) -there are some companies working on this technology Not as sensitive as Ab-based method (like ELISA or Western Blot) -Cheaper - Ab cost $4k-$30k -Faster - Ab take 2-6 months to make Easier to use in field or out of the lab -transferring technology from lab to field is difficult, so authors were successful in showing proof of this concept -Caveat is they did not use real samples, only purified samples of RNA Limited to viruses -authors only looked at RNA and DNA -Eukaryotic or prokaryotic cells that could cause disease are not amenable to this method Hypothetical ways to detect pathogens -Speculative, because authors didn't test anything else other than RNA

Preventing Background and False Positives in Ligation Independent Cloning

*Background minimized with gel purification and DpnI digestion* Types of Purification: -Dephosphorylation in Restriction Enzyme Cloning is one ex to reduce background noise (in ligation dependent cloning) -Gel Purification is ligation independent ex, where you purify desired DNA band DpnI is restriction enzyme that only digests methylated DNA -DNA isolated from cells is methylated -methyl group is attached to DNA, which E. coli naturally does -PCR product is not methylated -DNA made in vitro does not have bacterial mechanism of methylation DpnI digests the plasmid used as template, but not PCR product -only plasmid DNA is digested because it was methylated by bacteria -plasmid digested because it was used as a template -PCR product is not digested because it does not have methyl groups 4bp Recognition Site is abundant -Odds of finding the specific 4bp combination in constructs is very high -Generally, many cuts in plasmid are made

PCR Components

*Buffer* =maintains pH and provides salts -Mg salt needed in all PCR (usually in buffer) *Template* =DNA that is copied *Primers* =short ssDNA that binds to template where copying will initiate -Location/orientation of primers is important (determines what will get copied) *Nucleotides* =building blocks of DNA *Polymerase* =enzyme that copies the DNA *PCR Machine/Thermal Cycler* =precisely controls temperature -Machine didn't become commonplace until 1990s -In the past, had to move samples manually for each step *Thin-walled Tubes* =allows efficient heat transfer -8 tubes with thin walls, connected and fit inside of thermal cycler

Other Cas9 Enzymes

*Cas12 and Cas14* -both are sgRNA guided and cleave targeted dsDNA -initial cleavage is very specific for target sequence -then becomes indiscriminate nuclease for ssDNA -there is not a lot of ssDNA inside of bacteria, so this is not an issue *Cas13* -sgRNA guided and cleaves targeted ssRNA -then indiscriminate ssRNA cleavage occurs, where cas13 becomes indiscriminate nuclease for ssRNA after initial specific cut -this is bad because mRNA is ssRNA, so chewing up all the ssRNA makes the cell very sick because mRNA is not transcribed

Additional Cas Proteins that Cleave dsDNA

*Cas9 Nuclease* -composed of 2 separate domains that have nuclease activity (RuvC and HNH cleave either strand for DSB of DNA) -sgRNA and PAM site immediately next to it *Cas12a Nuclease* -like cas9 with sgRNA and RuvC complex (RuvC has different partner - Nuc) -PAM site is different in that it is one opposite side of sgRNA *Cascade and Cas3* -more complicated than cas9 and cas12a in that has multiprotein components -several proteins have to come together for nuclease function *Overall idea is that there are additional Cas proteins other than Cas9, which allows for wide array of applications*

Video: DNA Cloning and Recombinant DNA

*DNA Cloning* = making identical copies of a piece of DNA -DNA is usually genes, which is expressed as a protein *Process* 1) Cutting the gene out of the DNA -*Restriction Enzymes* to cut gene out -Restriction Enzymes recognize specific sequences in DNA - 2 RE needed, one at each end 2) Gene is cut out and pasted into plasmid -*Plasmid* = piece of genetic material that sits outside chromosome, but can replicate alongside the chromosome and express genes/proteins -Cut out gene has *overhangs* from Restriction Site cut off region, which base pairs with nucleotides in plasmid -Overhangs make it easier for gene and plasmid to react -*DNA Ligase* = glues/connects backbones of gene into plasmid 3) Plasmid is inserted into an organism to express proteins -E. coli is organism of choice for many -Obtain vial with a lot of E. coli, plasmids put into solution -Heat shock given, influencing bacteria to take up plasmid -Bacteria grown on plate which contains selective agents/ antibiotics -Only bacteria that incorporated plasmid will grow (plasmid contains resistance gene alongside gene of interest) -Colonies grown can be grown further or utilized for something else -Bacteria will be antibiotic resistant and express the protein of interest

Restriction Enzyme Cloning

*Digestion and Ligation must process separately* -Restriction Site remains after gene is ligated into backbone -Digestion must be done in isolation, then ligation -as RS remains, if you tried to do everything in one reaction, RS on desired vector would be continuously cut -both done separately so vector not cut again *Annealing and Ligation are performed simultaneously* Process: -Backbone = vector the insertion will get cloned into -Restriction Sites = EcoRI, XhoI, and HinDIII, which are specific recognition sites that leave overhangs -gene that we want to clone is flanked by EcoRI and HinDIII -Restriction enzymes are added and cut at recognition sites -cleaves the DNA and leaves the overhang -backbone removes portion between restriction sites, and gene of same size is inserted after being cut -same restriction sites on backbone and gene have sticky ends that make complementary bp to bind/anneal -Ligation uses Ligase to bind gene into backbone *PCR Product can also be cloned* -restriction sites added to primer -Again, restriction site cannot be in the same DNA that you're cloning -primer add the restriction site to PCR product -ES cannot be located anywhere in the genes or plasmid you're cloning into, or it will be cut in other places

Video: Gel Electrophoresis

*Gel Electrophoresis* = used for DNA, RNA< proteins, and any macromolecule -involves gel, electric charge, and migration of macromolecules through gel due to charge -moving away from cathode/negative towards anode/positive *Agarose Gel* = polysaccharide gel from seaweed -samples taken and put into wells/divots in gel *Buffer* = water with salts -Prevents pH from going out of bounds, which may affect DNA or charge of DNA *DNA has a negative charge at typical pH* -due to negative charges on phosphate backbone *When Charge is Applied* -Smaller molecules with greater net charge move fastest in gel -Larger molecules move slowly -Means bands at the top of the gel have larger fragments DNA -Bands at bottom of gel have smaller fragments of DNA -Bands are not a singly strand of DNA -Are collection of DNA strands with similar length *DNA Ladder* = separates in a specific manner in standard solution -Bands formed used as standard comparison for samples -Ladder Bands give *approximate* DNA length that migrated in gel *Ethidium Bromide* = dye that helps visualize DNA on gel -Becomes fluorescent when UV light is applied -Intercalating Agent = wedges between DNA -Carcinogenic

PCR (in vitro) vs DNA Replication (in vivo)

*In Cell* -DNA helicase separates strands -RNA Primer initiates replication -Leading ad lagging strand synthesis *In PCR* -Temperature denatures and separates strands -DNA primer initiates replication -Whole strand synthesized, no leading or lagging *Exponential amplification in both because copies are copied*

PCR Conditions

*Initial Denaturation* = 1-10 min, depending on Pol -94*C-98*C, depending on Pol *Denaturation* = 15-60 sec, depending on Pol -94*C-98*C, depending on Pol *Annealing* = 15-60 sec, depending on Pol -Different Pols have different buffers, and different buffers have different ionic strengths -50*C-72*C, depending on *sequence of primers* and Pol *Extension* = 15-60 sec *per kb*, depends on *Pol speed* -68*C-72*C, depends on Pol *Final Extension* (Optional) = 5 minutes -Used to complete any copies of DNA that haven't been fully extended -68*C-72*C, depends on Pol *Cooling* -infinite amount of time -4*C

Recombineering (Paper - Genome-scale Engineering)

*Most popular of the methods discussed in the paper* Similar to HR -idea is the same in that you have an area of homology on some type of DNA element, and it recombines into your cell -Big differences is the *presence of helper proteins* Facilitated by Helper Proteins repurposed from lambda bacteriophage -Lambda Red = Beta, Gam, and Exo -*increase the efficiency of integration exponentially* Help proteins are so good that you only need as little as 40 bp of homology to target specific location -40bp of homology can be induced in PCR primers/ programmed right into oligos -order 2 primers with 40 bp overhangs that target specific location in chromosome -also has 20bp that are homologous to antibiotic marker -can amplify marker so it has homology to targeted region on both sides, only only about 40 bp -don't have to do cloning sequence of adding upstream and downstream homology (only have to order primers and PCR amplify markers) -*all you have to do is PCR amplify and transform into your cells* *Recombineering is highly efficient* Recombinase can be used to remove markers (such as antibiotic resistance genes) -to remove marker, gene must be flanked by recognition sites known as Flp Recombinase Recognition Site/ FRT -FRT is flanking marker gene from very beginning, where the template contained resistance gene flanked by FRT -Recombinase can be activated at the end where recognition sites combine and Excise gene/marker in middle -*Flp Recombinases are most often used to do things like this/ get rid of marker* Overall: -Recombineering combines HR with help of Helper Proteins taken from phage known as Lambda Red proteins, which increase efficiency greatly by reducing amount of homology needed -also combined with recombinase/recognition sites to eventually remove marker -*Very popular and efficient way of doing gene deletions* One downside is FRT recognition site is left after excision of marker -problem with leaving it is that it can recombine with other recognition sites that may have been left in the genome from another reaction -there is a limited number of times this method can be used, and limited number of scar sites that can accumulate in chromosome

Video: Introduction to NEBuilder HiFi DNA Assembly (Similar to Gibson Assembly Technique, but has some proprietary improvements)

*NEBuilder HiFi DNA Assembly* = Effective method for assembly of multiple DNA fragments Advantages over Gibson Assembly Master Mix: -Higher efficiency -Higher accuracy -Ability to assembly both 5' and 3' restriction enzyme mismatches -Ability to bridge 2 dsDNA fragments with ssDNA -No licensing fees from NEB *Process* = Single Tube isothermal reaction with NEBuilder HiFi Assembly Master Mix -Method utilizes adjacent DNA fragments with complimentary ends -Complimentary ends added through PCR -Overlapping ends needed for NEBuilder HiFi DNA Assembly Master Mix -Incubated 15min-1hr at 50*C -During incubation, Master Mix's 3 Enzyme activities work on fragments ~1) 5' to 3' exonuclease activity creates single stranded 3' overhangs ~2) Complimentary sequences anneal, creating dsDNA of interest ~3) High fidelity DNA Pol extends 3' ends -DNA Ligase seals remaining nicks Results in fully sealed, dsDNA can serve as template for: -PCR, RCA, other molecular biology applications such as direct transformation *NEBuilder* HiFi DNA Assembly Master Mix *can also join 2 ds nonoverlapping DNA fragments together with an overlapping ssDNA oligonucleotide* NEB has been successfully used to: -Reliably join up to 6 DNA fragments in single molecule -Create library (gRNA or linker insertion) Assembly Overall: -makes quick work of large multi-fragmented assemblies -Useful for more routine applications such as cloning

CRISPR/Cas9 Targeting - RNA Guided

*Native CRISPR Targeting* -crRNA = RNA acquired from viral injection, from Spacer SNA/Protospacer -tracrRNA = has homology with repeats on Protospacer, which forms a complex -complex formed is essential dsRNA, which associates with Cas9 -if there is target that associated with crRNA, will get binding and cleavage by Cas9 protein *Engineered CRISPR* -sgRNA = no more formation of complex from 2 RNAs -Doudna lab made sgRNA, which was to mimic the crRNA-tracrRNA Complex, but made from one piece of RNA Improvement because it condensed the system to 2 parts instead of 3 -simpler and easier

Cas13

*TArgets ssRNA rather than dsRNA* Can be used to deplete transcripts -instead of preventing transcription (like dCas9), can directly target mRNA -not perfect because there is indiscriminate ssRNA nuclease activity, after initial cleavage of specific target -can make cell very sick, but can be overcome with new technology Prevent translation with dCas13 -like how you remove nuclease activity from cas9, also do it with Cas13 -dCas13 binds to mRNA and prevents binding or ribosome for translation cas13 can also do Base Editing with RNA -done by tethering enzymes to dCas13 and targeting RNAs -can change sequence site specific location (like dCas9 and cytidine deaminase) Used to do Live RNA Imaging -dCas13 can be tethered to GFP, to visibly show where dCas13 binds Detects RNA - SHERLOCK -allows you to detect RNA -used in clinicals settings such as Zika

PCR Troubleshooting

*Vary the Annealing Temperature* -Temp too Low = nonspecific binding (primers bind to random areas) -Temp too High = inefficient amplification (primers won't bind) *Try a Different Pol* -Some Pols are better at different reactions -High Fidelity vs Low Fidelity/Taq *Additives to Reduce Secondary Structure/Production of Primer Dimers* -Primer Dimers = primers anneal to each other -DMSO or Glycine Betaine can prevent secondary structures -Problem is when you add these, you can also reduce the activity and change annealing temperature -Changes fidelity of enzymes and decreases annealing temperature *Bad PCR* Smear =combination of all bands into indistinguishable blobs Nonspecific Amplification =Indistinct bands (not smeared, just light or not specific band) -If annealing temp increased, nonspecific product is gone

Gel Electrophoresis

-Separated DNA based on size and charge of fragment -Smaller molecules migrate faster -Larger pieces on top of gel, small pieces at bottom of gel -DNA has negative charge and migrates towards Anode/positive electrode Setup: -Gel rig with gel inside -Make gel in lab by melting agarose into plate and solidifying -Comb makes wells where sample is inserted -Electrodes put into either side of gel rig, voltage applied -Buffer is applied to prevent extreme change in pH/charge Nucleic Acid Due binds to DNA (often fluorescent) -DNA Ladder = DNA of known size and concentration -lets you determine how big the experimental samples are DNA resolution depends on agarose % -Higher the agarose %, the smaller the pores are Can be used to purify DNA or separate DNA by size -literally cut out band of interest/major product -dissolve gel using chemicals and heat -use silica column for purification *Useful for separating DNA pieces after PCR or digestion*

Polymerase Chain Reaction/PCR

-Used to amplify/copy DNA in vitro -Essential for many molecular biology and synthetic biology processes -Developed in 1980s, through tech that has been previously described History: -1970s = 1st reports of replication of ssDNA from template using synthetic primers and DNA polymerase -1976 - thermostable DNA Pol taken from Thermus aquaticus -1983 = Kary B Mullis invented PCR technique -1985 = introduction of thermal cycler that automats PCR process

Ebola Virus Detection - application of biosensor tot he real world (Paper - Paper Based)

12 targets were designed -designed 12 toehold sequences/targets from Ebola virus which were specific to 2 strains - idea was in presence of mRNA of entire virus, would get unfolding and translation of LacZ (the repressed gene) Most targets responded Assay was developed in 12 hours -paper wanted to do all of this in 24 hours or less -but they started the clock after the primers arrived (which would have taken days) -for all 12 biosensors, there was some type of response to Sudan strain -response varied from 10-fold to 80-fold -Zaire strain had little less response for some, but peak of 80-fold for some of the biosensors -showing the biosensor system did work in detecting Ebola virus strains Detection was specific to the virus strain -authors want to see if their system could accurately differentiate between Zaire and Sudan strain -authors mixed different toeholds and exposed them to each strain -results showed there was pretty good detection that was specific to strain (able to differentiate between strains with system) Unclear how different the sequences are: -authors mentioned some strains tested were 3bp different in size, but in terms of actual sequence, it was unclear how different they were -something that is very different is more likely to be specific than 2 strains that are very similar -but Reisch believes the 2 strains must be pretty similar, so ability of toeholds to differentiate the 2 strains is cool

Integrase/ Recombinase (Paper - Genome-scale Engineering)

= Enzymes that recombine at specific sites or sequences -usually have *short recognition site* (20bp) -often not naturally occurring, meaning do not naturally exist in chromosome of some type of cell -good things because recognition sequence can be added to chromosome, and used for manipulation purposes Like restriction enzymes, except recombinase is catalyzed - not endonucleases, are recombinases/integrases *Site specific but not site programmable* -*Specificity comes from protein, not nucleic acid* -have recognition site that you cannot change Result depends on recognition site location and orientation Types of results: 1) Excision -when you express Cr or Flp recombinases, you have a recognition site on left or right side of the gene -both recognition sites oriented same way (face same direction) -will get recombination between the 2 sites -whatever is in middle will be removed -will get construct where 1 recognition site still remains 2) Inversion -Recognition sites flank the gene of interest, but in opposite orientation -when Cre or Flp recombinase is activated, gene in middle is flipped/ change orientation 3) Insertion -there is recognition site on chromosome and on a plasmid that contains the gene of interest -when recombinase activated, whole plasmid is integrated into genome 4) Translocation -recombination between 2 separate recognition sites occurs through recombinase -2 different genes from 2 different strands combine into 1 sequence For Genome Editing: -this method is difficult for genome editing because these sites are not natural -10bp Recognition Sites must first be inserted into chromosome -before you do desired editing, must do an initial editing process to add recognition site -very large fragments of DNA can be recombined -want to use Integrase/Recombinase because can put in large fragments of DNA with the method -method is also efficient -*most common it to excise a gene with Flp recombinase* Downside: -there are only a few well-characterized integrases -they also have very strict site specificity (which is good because you don't want off-targeting, but bad for modification)

Boolean Logic Gates (Paper - Genetic programs)

= Mathematical basis of computing Ability to make "biocomputers" depends on ability to apply Boolean Logic to cells Manuscript used AND Gates -both inputs must be "on" for output to also be "on" - 0 + 0 = 0 - 0 + 1 = 0 - 1 + 0 = 0 - 1 + 1 = 1 2 Simplest Logic Gates 1) YES Gate -input 0, have output 0 -input 1, have output 1 2) NOT Gate -input 0, have output 1 -input 1, have output 0

Intron Retro-transposition (Paper - Genome-scale Engineering)

=DNA elements that integrate through RNA intermediates at non-random sites -*site specificity occurs from nucleic acids, not protein* Can be programmed for site specificity, but has some restrictions Efficiencies vary depending on sites -not widely used -commercialized system is somewhat expensive Limited payload size -in contrast to integrases, only 1kb can be inserted into genome -can be used to insert Recognition Sites for recombinase -then integrase/recombinase can be used to insert something larger

Type IIs Restriction Enzymes (different from Type II Restriction Enzymes)

=Enzyme cleaves DNA at defined location *outside the recognition site* -cute side is outside of the recognition site and makes sticky ends Cut site can be any sequence -you can design sticky ends -can have 2 cut sites that will have sticky ends that will anneal to each other 3-4bp overhangs made Popular Enzymes = BbsI, BasI, BsmBI, and SapI -each is a little different where they cut -region where they cut outside of recognition site is also different -but each still makes 4bp overhangs -*cleaving outside of restriction site is useful in producing your own sticky ends* *Golden Gate Cloning* = restriction enzyme cloning -uses Type IIs Enzymes -create your own sticky ends

Restriction Enzymes

=Proteins that cleaved DNA *Type II Restriction Enzymes* = Cleave at defined location at or near recognition site -create sticky end with overhangs or blunt ends -Sticky Ends = has overhands on one side of dsDNA -Blunt Ends = no overhangs or protrusions -HinDIII discovered in 1970 - led to Nobel Prize in 1980s -Recognition Sites are often *palindromes* -same sequence in forward direction as reverse -Over 3k known, over 600 commercially available

USER Cloning

=Uracil Specific Excision Repair Uracil can be incorporated into oligonucleotides/ DNA primer -U is then incorporated into PCR products Enzyme nicks DNA at Uracil to create unique sticky ends/overhangs -USER Enzyme cuts out Uracil from dsDNA and anything downstream of Uracil -produces single stranded overhang around Target DNA -as Uracil is the only thing USER recognizes, flanking sequence can be anything you want it to be -can design whatever sequence you want in primers -sticky ends are made, which can assemble into the vector *Cons* -USER Primers are ore expensive and take longer to come in -Special enzymes needed (not all DNA Pols can accept a U in primers)

Switches were Orthogonal (Paper - Paper Based)

All 8 of the toehold riboswitches ended up being orthogonal Orthogonal = only induced by cognate trigger RNA -authors mixed and matched each of the switch RNAs with other trigger RNAs -only see fluorescence of toehold switch with cognate RNA -linear fluorescence shows orthogonality

Touch Down PCR (variation of PCR)

Annealing temperature is decreased throughout run Starts high for more specific binding Decreases each cycle for increase efficiency -decrease by 1 degree in each cycle -method is built into newer CPR machines

CRISPR/Cas9 Repurposed

Any DNA sequence can be targeted if *Protospacer Adjacent Motif/PAM* site is present -can target specific pieces of DNA by changing sequence of 20bp at end of sgRNA -20bp DNA sequence provides target specificity for CRISPR/Cas9 system -20bp encoded on plasmid in bacteria The only requirement is that *PAM must be present in the target sequence* -when target DNA from invading virus is incorporated into CRISPR array, there is no PAM site next to it -no PAM site is the reason why the host chromosome is not cleaved -therefore, *target DNA that you want cleaved can be any kind of sequence, just needs to be next to a PAM site* *CRISPR/Cas9 is a Site programmable endonuclease* -can essentially be targeted anywhere in the chromosome -but only be functional if target is next to PAM site

Assembly PCR (variation of PCR)

Assembles DNA primers into dsDNA -Overlapping ssDNA sequences that are reverse complements of each other There are fragments of DNA that overlap but have gaps -DNA Pol goes and fills gaps in during PCR extension -Specific primers can then bind to targeted ends of DNA -PCR amplification of targeted/assembled DNA then occurs

GFP Expression from Freeze-Dried Extract (Paper - Paper Based)

Authors tested S30 cell extract, S30 T7 cell extract, PT7 system First testing constitutive promoters expressing GFP in cell-free extracts -S30 and S30 T7 were cells grown in lab and lysed -PT7 already cell-free Cell-free extract generally just lysed cells -taking cells and concentrating down -lysing membranes to release internal contents Results shown in 2 ways for constitutive promoters: -constitutive means it is always on 1) BF = Bright Field -BF - spot of cell mixture put under visible light -F - spot under fluorescent light -can see green fluorescence when under fluorescent light, and BF looks the same under visible light -only fluorescent when inducer/plasmid added 2)Graph -shows full change of GFP expression in control and experimental sample -around 4-fold change in GFP expression when DNA is supplied, compared to no DNA (lowest change in S30) -PT7 system purchased had biggest fold change Authors THEN tested Inducible GFP Expression from cell-free extracts -introduced TetR protein (aTc inducer) into system -GFP in S30 and S30 T7, and mCherry in PT7 Authors noted repression could be improved by adding additional TetR before freeze drying -if you added purified protein to extract, was able to bring down leakiness seen in cell-free/tetO GFP graph Results in 2 ways for Inducible GFP Expression: -Inducible is checking to see if an off system can be turned on 1) BF -F= inducer/aTc, where inducer added had fluorescence, and no inducer added had no fluorescence -tetO GFP looks green under fluorescence -tetO mCherry looks red under fluorescence 2) Graphs -there was better fold change with tetO mCherry after reconstitution -shows system does work in inducible setting, when inducer is added to system to express the protein

System Orthogonality (Paper - Genetic programs)

Authors tested system orthogonality after making improvements Orthogonality of Protein-Protein Interactions -tested each of the different parts with cognate and noncognate chaperones -should ONLY have RFP turn on and have highest fold change when sicA is provided to invF -authors still saw some crosstalk between ipgC (chaperone) and invF-psicA (TF-promoter) Orthogonality of Protein-DNA Interactions -there was high orthogonality between protein-DNA, where the parts worked as they should

Printed Array (Paper - Paper Based)

Authors trying to show you can do multiple reactions on the same piece of paper -can simply print a grid pattern on paper using common ink -reagents are spotted and freeze-dried, you can specifically test each one with a sample or trigger RNA Standard printer can create barrier to allow multiple reactions on single piece of paper -authors did pattern of samples of different colors -showed no bleed over from one well to next One single piece of paper could do up to 25 assays

Take Homes (Paper - Genetic programs)

Boolean Logic Gates can be created in bacterial cells Can take several inputs and program a cellular response -response was RFP in paper One of the big caveats with this type of research: -*Context dependence* matters when trying to make the cell behave in a different way than it wants to -It is different when comparing RFP and something else -essentially, we measure RFP now, but if we put a different output response to measure, may not function the same way Identifying parts to use in logic gates is difficult -Often, parts don't work as expected -Reason why the authors had to reengineer certain parts to circuit would work better -engineering takes a lot of time (years just to optimize parts so circuit can be built) 5,600 NOR Gates were used to create Apollo aa guidance system -possible in machine because all electronic gates are physically separated -not yet possible in bacteria, because you need 5,600 orthogonal NOR Gates to make the circuit (as of 4 years ago, 4 NOR Gates is the most we've done)

Input Logic gate - need orthogonality to be good sot another part can be added (Paper - Genetic programs)

By turning on pBAD (chaperone) and pTac (TF), you get chaperone and TF to turn on pipaH promoter and express sicA chaperone -inducible promoter was added as 3rd part to drive expression of invF (TF) -created a 3-input logic gate, where it needs sicA made by original 2 inputs and invF made from 3rd input to get an output of RFP Authors saw 4.5-fold induction over the highest off state (0-1-1) -System functions but there is a degree of leakiness -with no induction there was a low readout of RFP -with induction of all 3 (1-1-1), there was a high readout of GFP -shows the system does work pretty well, but depending on the application, 4.5-fold change may not be good enough

Video: Biologist Explains One Concept in 5 Levels of Difficulty - CRISPR

CRISPR = new area of biomedical science enabling gene editing and allowing us to understand genetic basis of many diseases (ex. autism & cancer) Level 1 - Child -CRISPR = tools scientists use to edit or change genomes -Genome = instruction manual that makes people -sometimes there are mistakes in the genome that make people sick -CRISPR can be used to erase/fix them Level 2 - Teen -CRISPR = way to edit the genome -molecular pair of scissors that can go through the genome, to specific places, and edit/make cuts -Most common mutations in cancer is P53, where CRISPR can be used to target tumor cells and fix P53 mutation for cures Level 3 - College Student -CRISPR = revolutionary gene editing tool -not like the ones before because it is more precise and affordable -works with Cas9 protein complex and sgRNA, where sgRNA tells Cas9 where to go and cut -CRISPR is precise because many different sgRNAs can be made for specific portions of the genome where we wish to edit -CRISPR can be used in human cells, but there are ethical problems such as editing in Germ Line where traits can be passed on Level 4 - Grad Student -CRISPR for gene editing in humans is controversial -depends on use in Somatic Cells or germ Line Cells -editing Germ line is unethical because these traits can be passed down and have unintended/unforeseen consequences -there is also no consent from the embryo/eventual person that would have change from CRISPR -If CRISPR does make it to marker, who will have access is an ethical issue -Will CRISPR be cost prohibitive? Will only select few be able to use it? Will people use it for non-therapeutic reasons? -people are afraid CRISPR will be used in frivolous ways to change a person's traits, but science doesn't know these details yet Level 5 - Expert -Expert is looking at the effect of human variation on CRISPR/Cas reagents -we use sgRNAs for precision of CRISPR, but what happens if there is a mutation at a specific site that differs between 2 people? -maybe the site won't be as efficient, or won't be able to bring Cas to the correct site and cut -Reference genome that was published in 2001 was just one person's genome, and there are many differences between individuals -studying to see if CRISPR will still cut at these sites with variation preset (may not cut), or worse CRISPR may cut somewhere else because it matches elsewhere -CRISPR has the ability to do crazy things out of the box, science just hasn't gotten there yet -CRISPR has created a whole open field of opportunities that may lead to greater things -it is a misconception that people think CRISPR is just one enzyme of Cas9 -CRISPR started with just cas9, but now many Cas have been found across various bacteria with different targetable regions

Video: Jennifer Doudna - Genome Engineering with CRISPR/Cas9

CRISPR = hallmarks of acquired immunity in bacteria -there are repeating DNA segments that are interspaced with Spacer DNA that are the history of previous infections of bacteriophages -CRISPR System allows sequences from viruses to be integrated into bacterial genome, which helps protect against future infection 3 Steps to acquire immunity in bacteria: 1) Adaptation -integration of short viral DNA molecules into CRISPR Locus 2) crRNA Biogenesis -crRNA is transcribed portion of CRISPR Locus that forms complex with Cas9 protein to bp with matching viral sequences 3) Interference -crRNA matches viral DNA, and inhibits viral DNA, stopping infection Streptococcus pyogenes -Has CRISPR system that has single gene encoding protein known as Cas9, was shown to be required for CRISPR system -found out Cas9 is dual-RNA-guided dsDNA endonuclease -Cas9 has ability to interact with DNA and generate DSB in sequences that match crRNA -crRNA interacts with tracrRNA, where the 2 come together and form structure that recruits Cas9 protein -scientists came up with idea wo change system so there is single protein (Cas9) and single guide RNA that could control CRISPR system to have precise editing ability -2 RNAs were linked together to make sgRNA Programmed Cas9 Cleaves DNA at Specified Sites -they generate short sgRNA that recognized different sites in DNA molecule -this was incubated with 2 different restriction enzymes: Sal1 which cuts DNA upstream of desired site, and Cas9 -in each of the reaction lanes, there were different sized fragments made form doubly digested plasmid -Size of DNA corresponds to cleavage at different sites directed by sgRNA for Cas9 system -different sizes indicated Cas9 system worked Genome editing begins with dsDNA Cleavage: -after DSB is generated, breaks in cell are detected and repaired by 2 types of pathways: 1) Nonhomologous End Joining (NHEJ) -ends of DNA are chemically ligated back together with small insertion or deletion at site of break 2) Homologous Repair -donor repair molecule has sequences that match those flanking sides of DSB, which brings in new DNA -we can no generate DSB at sites we choose with CRISPR/Cas9 system and insert desired DNA for genome editing Genome Targeting Technologies: -Zinc Finger Nuclease/ ZFN -TAL Effector Nuclease/TALEN -2 programmable ways to generate DSB in DNA that rely on *protein-based recognition of DNA sequences* -proteins that are modular and generated in different combinations of modules to recognize different DNA sequences -Requires a lot of protein engineering to do this (difficult) -CRISPR-Cas9 is easier because it is *RNA programmed protein*, where it is controlled by sgRNA -system is simple enough to use where anyone with basic molecular biology training can take advantage of system to do genome engineering -Cas9 system can also be "multiplexed"

Video: What is CRISPR?

CRISPR was first identified in E. coli -CRISPR = Clustered Regularly Interspaced Short Palindromic Repeats -Short Palindromic Repeats = short segments of DNA 20-40bp in length -Palindromes - read same left to right -Clustered Regularly Interspaced = identical repeats, one after another -repeats are interspaced with Spacer DNA in between -Spacer DNA is not identical, each is unique from each other -Spacer DNA is important because it matches viral/bacteriophage DNA -Region also contains number of genes that associated with CRISPR = CRISPR Associated Genes/Cas Genes Cas Genes make Cas Proteins -Helicases = unwind DNA -Nucleases = cut DNA -scientists found this is the immune system for bacteria, to fight bacteriophage Normally: -bacteriophage would inject DNA and would hijack the cell -make more viral components, burst, and kill the cell With CRISPR: -Cas Complex is made, Spacer DNA is transcribed to make crRNA that sits inside Cas Complex -Complex then breaks viral DNA apart, preventing infection If there is viral DNA injected that does not match Spacer DNA -CRISPR/Cas system makes a different protein that takes viral DNA, breaks it apart, and copies into new Spacer DNA with CRISPR system -Spacers are essentially history of old infections that won't infect the bacteria again Scientists thought if we could hijack the system, could possible deactivate genes or insert new genes -CRISPR/Cas9 - from Streptococcus pyogenes -they only had one Cas protein = Cas9 -has nuclease to cut DNA -has 2 long strips of RNA: crRNA and tracrRNA -crRNA = fits into Cas, spacer segment that matches viral DNA -tracrDNA = holds crRNA in place -hold thing together forms complex that can break down DNA Lab modified cas9 system by puttinf their own RNA/DNA in place of crRNA -developed a way tp connect crRNA and tracrRNAby creating "trcrRNA-crRNA Chimera" Chimera is known as single guide RNA/sgRNA -new system had sgRNA and Cas9 complex -sgRNA is created that has corresponding bit of RNA that matches DNA in desired location -sgRNA allows programmable specificity, telling CRISPR/Cas9 where to cut -essential goal is to inactivate the gene If you want to insert a gene, need 3 parts: -Cas9 -sgRNA -Host RNA that you want to insert CRISPR/Cas9 system works in living cells, and can cut DNA in multiple different places -can be used to fix mutations or genes within a person -*overall, CRISPR system was identified in bacteria and modified for humans*

CRISPR Bacterial Immunity

CRISPR was identified in genome sequences over last 20-30 years -function was unclear for long time -eventually found to be a system of bacterial immunity Steps of Immunity: 1) Foreign DNA Acquisition -prokaryotic cell is attacked by bacteriophage that injects viral DNA -viral DNA comes into cell -some components of CRISPR Locus (Cas genes and proteins) chop viral DNA and integrate it into the bacterium's chromosome -for every piece of DNA that has been incorporated (Spacer DNA), there are repeat sequences next to the Spacer -Area known as the CRISPR Locus 2) CRISPR RNA Processing -whole CRISPR Locus is transcribed and creates pre-crRNA -large single unit is then processed into individual crRNA units 3) RNA-guided Targeting of Viral Element -individual RNA units can then interact with Cas9 protein -any time the specific piece of DNA that matches the crRNA comes into bacteria, Cas gene can cleave DNA and inactivate it, preventing bacteriophage from infecting cell

Nested PCR (variation of PCR)

Composed of 2 PCR Reactions 1) First reaction can be dirty -many nonspecific products 2) Second reaction is nested -primers bind internal to first product -amplifying small portions of DNA within first "dirty" DNA *Nested reaction results in cleaner amplifications*

CRISPR/dCas9 Uses

Can do many things by tethering accessory proteins to dCas9 Dynamic control can be achieved through several techniques Gene Regulators -Transcriptional Repression or Activation -Epigenome Editing Dynamic Control -Chemical induction ~split dCas9 into 2 unites, tether to different domains, and have inducer that makes 2 units come together to form whole dCas9 system ~has inactive version followed by active version, that can either block or initiate transcription -Optogenetics ~using light to activate proteins, where dCas9 can be tethered to light responsive proteins ~with addition of light, would make active dCas9 Chromatin Interaction -dcas9 can be used to remodel chromatin structure and help to study the function of chromatin in certain situations within eukaryote Some of these methods can be used in bacteria, but proteins in figure generally specific to eukaryotic cells

GFP Expression from Freeze Dried PT7 System (Paper - Paper Based)

Commercially available PT7 "cell-free expression" system was freeze dried and used to transcribe and translate GFP -authors took PT7 system, froze it, water crystals removed with vacuum, and were left with all the enzymes and mixture in dry/powder form -authors then resuspended freeze-dried mixture and compared activity to fresh mixture -provided some GFP DNA and looked for transcription and translation of GFP to measure fluorescence *Found that the freeze-dried system worked very well* -there wasn't a big difference between freeze-dried and fresh sample -both had the same fluorescence/expression of GFP -time course was done where they allowed freeze-dried sample to site for a few days to over a year -there was little decrease in activity over time, but was generally small decrease -*means PT7 system is very stable and capable of being freeze-dried* -surprising because there are MANY components, which still work after being dried and resuspended

CRISPR for Diagnostics

Cas12 and cas13 have indiscriminate nuclease activity -only occurs after the enzyme finds first cognate target and makes first specific cut -Indiscriminate Nuclease Activity can be harnessed to detect specific pieces of nucleic acid, or to give signal when Cas enzyme detects specific nucleic acid Steps: -first design sgRNA to target something specific (on Cas12 or Cas13) -once specific molecule is detected, enzyme has indiscriminate enzyme activity -if you supply labeled pieces of nucleic acid that are essentially random, and Cas12/Cas13 chews up target DNA, random labeled DNA then glows -in absence of indiscriminate nuclease activity, molecules do not glow -after Cas target has been cleaved and other random molecules have been chewed up, they start to glow Used for glowing and chewing up of other molecules as proxy/ readout if initial target has been cleaved -initial target not cleaved, no glowing -initial target is cleaved, glowing because random labeled nucleic acids are cleaved and glow ssDNA Detection -DETECTR ssRNA Detection -SHERLOCK Methods are not incredibly well developed, but has been shown they can detect presence of DNA or RNA from samples you would take in the field -provides quick and cheap diagnostics for field

Part Engineering (Paper - Genetic programs)

Chaperons, TFs, Promoters: -sicA-invF-psicA -ipgC-mxiE-pipaH -exsC-exsA-pexsC The parts required improvements in the paper -parts were not ideal and needed to be reconstructed -invF = misannotated start site -pipaH = -35 box mutated to identify lower background -sicA = random mutations to lower "crosstalk" Summary of Figure: Table B) -looking at output activity, no difference between inducer or no inducer added -saw misannotation in invF TF -translation could initiate at any start codon/ Methionine -original annotate start codon was not read, but an upstream Met was read instead -when invF was corrected, there was much higher output when inducer was added when compared to without inducer Table C) -saw promoter pipaH was leaky -in absence of inducer, there was still high output -Used PCR Mutagenesis to mutate -35 hexamer, and found promoter that produced lower background -Corrected pipaH had less output without inducer Table D) -there was high cross talk between 2 different Chaperones and TFs -sicA and mxiE were from 2 different organisms, but were not completely orthogonal -interaction induced activity from sicA, where you do not want interaction (want completely orthogonal) -Authors mutated protein and screened them to find a sicA that did not have robust response in presence of mxiE Table E) -with orthogonal and modified sicA, there is robust response when paired with complementary TF invF -Authors able to make system more orthogonal compared to others

Synthetic Genomes (Paper - Genome-scale Engineering)

Cost of DNA Synthesis dramatically decreased in last 2-3 decades Can no buy 1-3kb of DNA quickly and cheaply -DNA sequence is not always 100% accurate -when you buy DNA sequence, whether it is Oligo, plasmid, etc., you are not getting just 1 piece of DNA -you get millions of pieces of DNA in one test tube -not all sequences will be identical - will have mutations Next step is to assemble small pieces of DNA into something bigger -combining 1-3kb pieces into much larger fragments >100 kb -any mutations you originally had will combine into bigger piece -chances of mutation combining into larger synthesized genome is high Entire genome of Mycoplasma mycoides has been synthesized this way -there was one single bp mutation in genome that didn't allow bacteria to survive/grow Individual Chromosome of S. cerevisiae been synthesized this way and put into cell to replace native ones -trying to rpelace native chromosome with something completely synthetic

CRISPR/Cas for Genome Editing

DSB enables genome editing to occur 2 Primary Mechanisms for Genome Editing in Eukaryotes -Nonhomologous End Joining/ NHEJ -Homology Directed Repair/ HDR

The Fundamental Requirement for most Genome Editing Techniques = *Double Strand Break/DSB*

DSB is enzymatically catalyzed by Endonucleases Restriction Enzymes are not an example of endonuclease -they are Site Specific, but NOT programmable for any site other than their programmed one Programmable Site Specific Nucleases would allow targeting of any sequence in any part of the Genome -Zinc Finger Nuclease/ZFN - late 1990s -TALENs - early 2010s -CRISPR/Cas9 - mid 2010s

Multiplexed Genome Engineering = Allelic Replacement (Paper - Genome-scale Engineering)

Derivative or recombineering -instead of using 3 Lambda Red Genes/Helper proteins, only need *one* -known as *Single Stranded Binding Protein/ SSB* = *Beta* from Lambda Red -instead of transforming dsDNA like PCR product, you transform single strand or oligonucleotide into cell/chromosome -*Oligo can have mismatch that you want to insert into chromosome, or even deletion where you can delete several kb of DNA from chromosome* Oligo incorporated into chromosome at Replication Fork on lagging strand during DNA replication -single stranded oligonucleotide takes place of/replaces an Okazaki Fragment with help of SSB -oligo is then incorporated into chromosome -highly efficient way of modifying chromosome, but problem is you inherently don't have selection -no resistance marker incorporated in oligo -can be hard to identify which cells have the oligo combined -potential method for doing high throughput genome editing -"Multiplex" because multiple oligos can be incorporated into same cell -many mutations in one step

Single Crossover vs Double Crossover (Paper - Genome-scale Engineering)

Differ in construction and mechanism of HR *Single Crossover* -Uses *Suicide Plasmid* = plasmid cannot replicate in host -many plasmids may replicate in E. coli, but not in other bacteria -Can do cloning and make plasmid construct in E. coli, then transform it into other bacteria where the plasmid will not replicate -difficult to do Suicide Plasmid in E. coli, because almost all plasmids replicate in E. coli -plasmid contains Ori, antibiotic resistance marker, and some type of counter selectable marker (like SacB), and possible oriT if you want to use conjugation to put plasmid into host strain More Detail: Single Crossover because homologous gene will combine and crossover into host genome (only single site of homology) -incorporates the entire plasmid -have both copies of mutant gene and wild type in host, known as Merodiploid -know this happened because antibiotic resistance marker crossed over -only when entire plasmid crossed over will resistance occur in cells -Plasmid cannot replicate in host, so it can't transfer resistance without combining -Counter selectable marker is used -used to select against presence of the gene -sacB gene is expressed, cells become sensitive to sucrose and will die -only cells without sacB gene/plasmid portion will grow -only left with mutant OR wild type gene -have to screen in some way -process is Scar Free -means there is no leftover sequence, only homologous region *Double Crossover* "Double" because there are 2 regions of homology -there are 1 areas of crossover happening at the same time -if successful, would replace homologous gene in genome with resistance marker -sometimes when you do Double Crossover, single crossover may only occur -second crossover has to occur in 2nd step Components: -can use suicide plasmid -would need upstream and downstream homology flanking resistance marker -OR, use Linear DNA (PCR Product) -needs additional proteins and efficiency is low -recombineering is how you would do Linear DNA, not through Double Crossover Resistance Marker remains after Double Crossover is finished in the genome -cane later be removed Want to remove resistance marker because: -if you remove it, you can reuse the marker -can only use marker once per cell, unless you remove it -when removed, cam do another mutation or maintain plasmid within cell

Polymerase Properties

Different companies make different variants of Taq and other polymerases -Change in properties to produce Pol with desired property *Processivity* = rate of DNA extension -Processivity of each Pol is different depending on properties -Need to adapt PCR conditions to specific reactions, because you make not give Pol enough time to fully extend (may get shorter amplicons than intended) *Thermal Stability* = ability of Pol to withstand denaturing temperatures -Required for denaturing step -Taq actually has a pretty low half-life, other Pols are better *Fidelity* = error rate of DNA incorporation -High Fidelity = used for cloning when each sequence must be correct -very few mutations incorporated, makes very correct sequences -Q5 Hotstart & Platinum SuperFi much better than Taq Pol -Low Fidelity = used for screening or sizing -only need to know the size, not sequence -having few mutations won't be an issue -Taq Pol, usually much cheaper *Screening Examples* Blue-White Screening -Uses Beta-galactosidase enzyme -Can figure out how often blue-white fails, which gives indication of how many mutations are present -Low Fidelity enzymes give many mutations, High Fidelity has very few mutations Sanger Sequencing -screening to see mutations -Low Fidelity has high error rate, High Fidelity has low error rate

PCR Steps

Double stranded DNA sample is added to Thermal Cycler 1) *Denaturation* = temperature used to separate strands -Enzymes from most common bacteria not stable at high temps 94*C-98*C -E. coli Pol initially used and replaced every cycle -Thermophilic bacteria have heat stable enzymes -Thermus aquaticus = source of first commonly used Pol -No longer had to add Pol at every annealing step -Could ad Taq once at beginning and remained in PCR 2) *Annealing/Priming* = temperature brought down to 50*C-70*C -Oligonucleotide primers attach/anneal to ends of strands to promote replication of amplicons -Primers should only bind to one location/specific to one thing -Primers bind to original template and copies, allowing exponential amplification of DNA -Primer concentration usually higher than template concentration 3) *Extension* = move up to 72*C for Pol to have optimal activity -DNA Pol extends/makes copies of DNA template

Orthogonality (Paper - Genetic programs)

Electronic circuits are physically separated Inside of a cell, circuits are not separated -everything is mixed "like a burrito" Parts *must be orthogonal* = able to operate without interference from each other

Extension to LacZ Assays (Paper - Paper Based)

Everything the authors did to this point was use fluorescent proteins -problem is that fluorescent proteins require fluorescent light to observe color -goal was to create a system that was easy to use in the field (wanted to switch to colorimetric assay to be used in the field) Beta-Galactosidase (LacZ Genes) Assays can be colorimetric and easier for field use -Beta-Gal would change substrate from yellow to purple -instead of measuring protein, you are measuring an enzymatic reaction that changes the color of the compound from yellow to purple Using the same 8 toehold riboswitches, authors performed assays -quantified protein and saw 10-fold change in worst performing one -200-fold change in best performing one when coupled to translation of LacZ gene

2-Input Gates (Paper - Genetic programs)

Experimental results are various inducer levels -when you don't provide inducers or have very low concentration of inducers to turn inputs on, RFP readout is low -as you increase concentration of inducer, there is increased readout of RFP -important thing is that even when you fully induce Ara and not with aTc, you're still blue (low readout of RFP, red is high) -higher change in RFP readout occurs when 2 input gate is functioning with both Ara and aTc Transfer Functions (modeled) -modeled the promoters to extrapolate the transfer function of each system -saw each of the 2 input NOT Gates seem to work pretty well in different bacteria

Genome Engineering Methods (Paper - Genome-scale Engineering)

Focus of Manuscript -different ways to modify genome in bacteria Table 1 gives comparison of various targeted genome engineering methods -Last manuscript discussed ZFNs, TALENs, and CRISPR/Cas -big difference between ZFN/TALEN and CRISPR is that *ZFN/TALEN uses proteins as site specificity tool*, while *CRISPR uses sgRNA* Tables shows all of the different ways the genome can be targeted in the paper -also shows how hard or easy it is to program the methods -ZFN = moderate programmability, variable efficiency, maybe multiplexable (difficult to target multiple genes at same time) -TALEN = moderate/high programmability, variable efficiency, maybe multiplexable -CRISPR/Cas = high programmability, variable efficiency, maybe multiplexable -Homologous Recombination - high programmability, low efficiency, not multiplexable

Programmable Nucleases

FokI Endonucleases can be guided by proteins -ZFN and TALENs are *guided by proteins* -they themselves do not have nuclease activity, but can be translationally fixed to FokI enzyme which is a nuclease, and ZFN/TALEN would guide it -both ZFNs and TALENs are made by proteins, where protein-DNA interaction is not well defined -there are protein domains that interact with specific bae pairs, but not absolutely specific -specific proteins (protein domains or ZFNs and TALENs) must be assembled to perform -Laborious and difficult to assemble and clone the protein complexes -there are a lot of repeating sequences, and similarity in between the cassettes even if they target different bp -editing/cutting efficiency of FokI paired with ZFNs or TALENs is not great -method was cutting-edge when first made *CRISPR/Cas9 is guided by RNA* -*different from protein guided complexes* -very specific base pairing -single bp binds to single base, as compared to protein guided systems that have 30 amino acids per bp which are encoded by 90 bp (more complex)

Ligation Independent Cloning

General concept is annealing of complementary ends of linear DNA Sometimes called "Seamless Cloning" = No Scar Sites -Scar = any sequence "left-over" after cloning -ex) Restriction sites, distance between RBS and start codon Scar sites aren't necessarily detrimental, but can be -the farther away you put RBS and start codon, the rose the translation initiation rate will be -example of detriment in cloning, because cloning site is too far from RBS, which makes start codon far away from RBS *Linear DNA made with overlapping homology on both ends* -PCR product or linearized plasmid -each dsDNA has homology to each other at the end Denaturing can be used to separate strands -dsDNA denatures and allows homologous ends to anneal *Polymerase can only extend in 5' - 3' direction* -production of 1 dsDNA that combines DNA from 2 different strands -extension can only happen in one direction, so one if the dsDNA may not extend Technique can be applied to whole plasmids -can be used to create an entire plasmid

Dead Cas9/ dCas9

In addition to being a dsDNA cleavage enzyme, couple of point mutations in active site of protein can *alleviate the ability of the enzyme to cleave DNA* -even though it can no longer cut, *site specific nature remains* Enzyme dcas9 can target specific sites and bind, just not cut -people have translationally fused dCas9 with accessory proteins 2 mutations remove nuclease activity -RuvC and NHN active sites Targeting Specificity Remains = programmable DNA Binding Protein -Accessory proteins can be translationally fused to Cas protein -has many interesting and creative applications -ex) GFP fusion to dCas9 to identify location of target DNA in cell -wherever dCas9 binds, there will be GFP expression

Video: SHERLOCK - Detecting Disease with CRISPR

In this system, CRISPR = C2Cs (Cas13a) -kind o like cas9, but different in that it targets RNA instead of DNA -cuts RNA based on sequences of crRNA, so it can be reprogrammed -when it recognizes target, also chews up other RNAs around the target -called the "Collateral Effect of the Enzyme" lab thought they could use this non-specific activity as a way to report whether or not the RNA target is present -is different application of CRISPR system -not using CRISPR to edit genome, but detect and diagnoses biological material SHERLOCK = Specific High-sensitivity Enzymatic Reporter Unlocking -SHERLOCK takes input, a single molecule or DNA or RNA. where the amplification reaction turns it into a large amount of DNA -amplified DNA is then used to create even more RNA -Cas13a recognizes this RNA and can amplify the signal again, generating detectable readout Applied SHERLOCK to real clinical example for Zika -Took raw urine or blood and put into SHERLOCK system -in couple of hours, were able to detect low levels of Zika in actual human samples -Zika is difficult to diagnose because it exists in very low copies, and not much circulates in the body -*shows how sensitive SHERLOCK test is* SHERLOCK is the DNA-RNA equivalent to a pregnancy test -platform allows POC Diagnostic that gives specific readout quickly -time is of the essence, so SHERLOCK allows rapid and accurate diagnosis

AND Logic Gate (Paper - Genetic programs)

Inputs and Outputs are promoters -2 input and 1 output logic gate 2-input AND Gate -Input 1 = drives expression of Chaperone protein -Input 2 = drives expression of Transcription Factor -2 proteins come together and allow transcription of a promoter Output = RFP (in paper) -Product of transcription by promoters Input signal = small molecule inducers -activates the input promoters to produce proteins Control = turning on both inputs with a small molecule *Both proteins need to be expressed for RNA Pol to be active and transcribe*

CRISPR/Cas9 (Paper - Genome-scale Engineering)

Like other nucleases, CRISPR alone does NOT enable genome editing in most bacteria -CRISPR/Cas9 can be used to cut the chromosomes, but doesn't mean you'll get indels or HDR What you can do is combine recombineering or allelic replacement and CRISPR to enrich for mutant population without marker -CRISPR/Cas9 used as counter selectable marker (targets wild type genome) -Oligos are incorporated into cells through electroporation -will have 2 populations of cells : Oligo Incorporated and Wild Type -In most experiments, wild type outnumbers oligo incorporated -can use Cas9 to select against Wild Type cells -Cas9 cleaves wild type DNA and makes DSB, which leads to cell death because DSB are lethal and Cas9 can't form indels or do NHEJ -if oligo incorporated into chromosome, genome is no longer wild type -cas9 cannot cut and cells will survive -theoretically, all of the colonies on plate should be mutant with combined oligos in chromosome

Circular Polymerase Extension Cloning (CPEC)

Linear DNA with overlapping ends mixed together *PCR Reaction setup with high fidelity Pol* -no primers needed because linear PCR pieces primer each other with homology -PCR product is flanked by same homology as vector -cross priming should occur, where homology at different strands primes each other -extension then occurs with DNA Pol, completing insertion of new DNA into vector to make new plasmid Cycle 10-15 times -increases efficient of method *Several pieces can be combined simultaneously* *Pros* -no extra enzymes -little hands-on time (already have enzymes from PCR) *Cons* -weird products can result from "mis-priming" -efficiency may be affected during melting and annealing, where the correct binding is not primer

CRISPR Enables Systems-Level Experiments

Look for Loss or even Gain of Function -by targeting many locations in the cell Chip synthesized oligonucleotides = 1,000s of pieces of ssDNA for a few cents each -thousands of oligos can now be synthesized for very cheap, and they can be used to target every single gene in your genome -one you have the pool of oligos, can clone them into some vector -important thing to nose is that after oligos have been cloned, all stay together in one pool (don't need different strains, all in one culture) -Then screen each one of the targets against you cells in some type of assay -assays can use Genome Editing, Epigenome Editing, or Gene Regulation -After you perform the assay, you would have had some form of control that didn't have the experimental variable, and use Next Gen Sequencing to identify the number of targets that have accumulated or disappeared compared to control -high throughput sequencing enables interrogation of guide-RNA *CRISPR allow systems-level experiments that you couldn't do by other methods before* -both ability to synthesize DNA at low costs and read DNA at low costs enables these really large systems-level experiments that were previously impossible

Input Logic Gate - expansion by addition of another part (Paper - Genetic programs)

Made of 2 different NOD Gates driven by 2 different sets of orthogonal inducers -3rd NOD Gate uses sicA (chaperone) output from 1st NOD Gate, and invF (TF) output from 2nd NOD Gate as new inducers for output of RFP -4-input because there are originally 4 inputs to make sicA and invF (2 inputs for each) 5.1-fold change higher than highest off state (1-0-1-1) -highest background is 5.1-fold lower than the all on case 11 protein "parts" Biggest network ever made -paper was written a few years ago, and it is still the case System functions, but there is a degree of leakiness

Paper Based Synthetic Gene Networks (Paper)

Manuscript is based on the ability to do in vitro transcription and and translation -trying to bring affordable, easy, and safe methods of testing outside the lab *Biosensor* = device that uses a living organism or biological molecules, especially enzymes or antibodies, to detect presence of chemicals - have some type or input that triggers some type of change -this triggers transcription and translation of given output -output is usually fluorescent protein or colorimetric change -Generally 1 input and 1 output -enables biosensors to be used outside of the lab, without cells

Golden Gate Assembly

Many pieces can be combined simultaneously -because restriction sites are outside of recognition site Unique overhangs enable ordered assembly -unique overhangs allow only specific parts to combine -multiple assemblies can occur together if each sticky end is unique Create a library of plasmids with different parts Combinatorial Assembly means you use the same restriction sites to make different assemblies Golden Gate Cloning is still a new technique in Synthetic Biology

Cell-free Transcription and Translation (Paper - Paper Based)

Method has been around for a long time, and there are commercial kits to do it Components required to do Cell-free Transcription and Translation: -Many enzymes -DNA/RNA to be transcribed or translated Why would you want to do cell-free transcription or translation? -Doing transcription and translation in vivo takes a lot of steps and a lot of time -takes longer, but is cheaper to make more protein -In cell-free system, only need to get DNA and mix it with system -can be a lot quicker depending on what you are doing -Downside = more expensive per unit or mg of protein -may want to use Cell-free protein system (CFPS) only if you need to make small quantity of protein -or if you have toxic protein that cannot be made inside of cells

CRISPR/Cas9 Genome Editing

Method of Genome Editing with Cas9 NHEJ, or Indel formation with small insertion or deletion that causes frameshift mutations Cutting with a Repair Template where you try to integrate some DNA -integration does not have to be a new gene, can be a few bp of targeted mutation Larger deletions or rearrangements by cutting multiple places with Cas9 -Dual Cas9 can delete larger fragments of DNA (cutting 2 sites)

Site Directed Mutagenesis PCR (variation of PCR)

Mismatch placed on PCR -mismatch is put into primer -upon amplification, mismatch is incorporated into PCR product Propagated on complementary strand Can be combined with other methods -SOE or CPEC to put mutation in middle of gene

Multiplex PCR (variation of PCR)

Multiple PCR relations happen within the same tube -designed to obtain several PCR products simultaneously *Different size products* can be separated in gel -each is a different sized product, so you can run a gel and know if each amplified -if they are the same size, can't differentiate bands or tell of they were amplified Useful for Genotyping If the primers bind, you'll get product -if the primers don't bind, you won't get product -if you have point mutation in primer and primer only binds to wild type, won't get amplification because last bp of primer did not bind -if you designed primer complementarity to bp, will get amplification ex) Genotyping PCR -primers specific to different mutations are put in -will tell if person has specific gene mutations if amplified

Drawbacks of Restriction Cloning

Must fin compatible restriction sites -*Restriction Sites cannot be inside your gene, or vector* Ligations are generally very inefficient -<1% of vectors and inserts are ligated -sequences may anneal, but overlap are short and may not get ligated -can be difficult to find plasmids that gene was inserted into Some enzymes are better than others -incomplete digestion can lead to increased background -*Strategies can be used to decrease background* -Dephosphorylation of vector is one ex to decrease background 5' end of DNA is normally phosphorylated -for ligation, one or both 5' ends must be phosphorylated -one side *must* have phosphate group attached to 5' for ligation to occur *Dephosphorylation Prevents Self-ligation/background* Normal: -using gel purification to try to get rid of cut region in backbone, but not 100% efficient -when ligation is done, there is mix between desired insert and self -3 plasmids can be made: ~Original insert goes back and makes same plasmid ~Desired insert combines ~Plasmid cut ends ligate with no insertion *Addition of Dephosphorylase Enzyme* -enzyme dephosphorylates/ removes phosphate -removes phosphate on backbone pieces -no phosphate on cut regions of backbone -no phosphate on cut-out region from backbone -phosphate only on desired insert -insert goes into cut region -ligation occurs because only need *ONE* phosphate on one side to ligate -Reduced background noise -only 1 type of plasmid with desired insert is made -greatly increases efficiency of restriction cloning

Nonhomologous End Joining/ NHEJ

NHEJ occurs when 2 pieces of dsDNA have been broken and come back together in single piece Robust system of DSB repair in eukaryotes -system required because unrepaired DSB are lethal in cells NHEJ is error prone -NHEJ leads to gene deletions because mechanisms of repair are error prone -leads to creation of Indels Indels = insertions and deletions -can create Frameshift Mutations, which makes premature stop codons *Most bacteria do NOR posses robust mechanism of NHEJ* -some proteins don't have Ku or LihG proteins, so unable to do NHEJ Process: -Ku proteins bind to end of broken DNA -ligase ligates dsDNA back together -loss of bp occurs, which leads to bp mutations Alternative End Joining Mechanism -another form of NHEJ -catalyzed by different enzymes and results is different -TTA is deleted in intervening sequence -bp deletions do not lead to Frameshift Mutation NHEJ Enables (Examples from Review paper): 1) Indels = error prone repair of DSB -repair can add insertions or deletions that lead to frameshift mutations and premature stop codons 2) Large Deletions = cutting 2 sites some distance apart -targeting 2 positions on chromosome with Cas9, which cases DSB at 2 different locations -NHEJ can join 2 ends distal from each other, leading to deletion of all DNA in between 2 -allows more DNA deletion than indels 3) Insertion = both chromosome and insert can be targeted by Cas9 -you can cut chromosome with Cas9 and supply donor that has recognition sites on Cas9 -since both are cut with Cas9, chromosome and donor DNA can come together, where donor DNA fills what was cut out by Cas9 -method unclear, but is homology independent targeted integration

DNA Cleaving Cas proteins are limited by PAM

PAM is a require sequence -even though it is a required site, it can be limiting Streptococcus pyogenes PAM site is NGG (different for evert bacterium) -frequency of PAM depends on: 1) Length and GC content of PAM 2) GC content of the genome to be edited -lower GC content, less PAM sites inherently in the bacterium Solutions to help solve the problem of being limited by PAM Site 1)Screen Orthologs of Cas9 -these are different proteins from different organisms, but same functions (still RNA-guided DNA cleavage) 2) Evolved and rationally designed Cas9 variants with altered PAM site specificities

Video: Polymerase Chain Reaction/ PCR

PCR used to: -Clone DNA to put gene of interest and other fragments into plasmid -Used in forensics -Medical diagnostics *Process* (Repeats 25-35 Times): -Reaction needs addition of Nucleotides for extension 1) Denaturation (96*C) -dsDNA separated into 2 strands -No enzyme used, only heat 2) Primer Annealing (55*C) -ssDNA cooled down and primers anneal to specific regions -*Primer* = ordered from company, specific to ends of DNA you wish to copy -A lot of primer put into solution to increase chances of primer annealing to DNA 3) Primer Extension (72*C) -DNA heated back up to allow extension -*DNA Polymerase* extends DNA, binding at primer and extending from 5' to 3' -Special polymerase is needed that is heat resistant (ex Taq Pol) -Polymerase will stop eventually or fall off at the next step (return to Primer Annealing or infinite hold at 4*C) *Result After 1 Cycle* -number of DNA molecules is doubled -repeating cycle 35 times is 2^35, which is over 1 billion molecules -2-3 hours, can go from 1 molecule to billions -Billions only if it is highly efficient, which it isn't always

SOE, Extension Overlap, CPEC (variations of PCR)

SOE = Splicing of Overlap Extension CPEC = Circular Pol Extension Cloning Similar concept to PCR -difference is that there is dsDNA with overlapping homology on another dsDNA -homology is added by binding of primers Combines multiple linear DNA fragments with overlapping sequences -primers produce homology on different dsDNA, they denature in PCR step -produces ssDNA of different genes that have some homology to each other -homology then allows ssDNA to prime each other and combine -results in production of one dsDNA that combined 2 different genes -idea behind Ligation Independent Cloning Can also be used to clone

Cas3 Deletions

Similar to Cas9 in that it suts dsDNA and is sgRNA guided -some Cas3 have ability to do long range deletions Some Cas3 complexes catalyze long deletions of varying length -by causing single cut in chromosomes, the complex Cas3 chews back DNA -in paper published in 2018, shows different lengths of DNA deletion can occur -can delete varying lengths of DNA, where some are very long (up to 50k) with single cut site -different from before where Cas9 had to make multiple cuts -Cas3 is able to get rid of a lot of DNA with single cut Mechanism of Repair is Different -Cas complex might actually help to repair DNA

Homologous Recombination/ HR (Paper - Genome-scale Engineering)

Similar to HDR -uses similar mechanism with Donor Template with homology/identical sequence that will combine/insert into chromosome Regions of homology will recombine into chromosome -at least 500bp homology needed for efficient recombination in bacteria -longer homology even more efficient -because there are certain spots in chromosome of bacteria known as "Hot Spots" for recombination -longer sequence you are targeting, higher chance of coming across "Hot Spot" for recombination

Sequence and Ligation Independent Cloning (SLIC)

Single stranded ends created by *T4 DNA Polymerase* -has exonuclease activity -Exonuclease = chews back DNA from outside inward -in absence of additional nucleotides, T4 chews back all DNA -results in production of ssDNA -presence of nucleotides, T4 lays down DNA and makes ssDNA into dsDNA -mechanism can be controlled by amount of nucleotides/type of nucleotides added *Anneal with complementary ssDNA ends* *Cons* -more hands-on -timing of nucleotide addition and T4 chewing back can be unclear

Video: Synthetic Biology - James Collins

Synthetic Biology = new field combining engineers, physicists, molecular biology, and chemists -with the goal of using engineering principles to put together biomolecular components (genes/proteins) into novel biological circuits and pathways -and to use these to reprogram organisms to work in a range of applications Field grew out of an interest in human genome effort -Mid-1990s saw one of the biggest challenges would be to see how parts work in networks and pathways that underlie biology and disease Instead of thinking about reverse engineering or inferring natural networks, thought about what engineers typically do -could they tinker with parts and make something else Collins and his student launched approach to synthetic biology through *circuit schematic* -come up with circuit schematic as an electrical engineer would -model is mathematically to get a sense of how it might function -go find parts that would be biological equivalent components that could make up the circuit -use molecular biology techniques to piece together the parts into plasmids or DNA constructs, and get into cells to see if it functions Genetic Toggle Switch = motivated by electrical engineering -Toggle Switch in electrical engineering is simple form of memory/circuit -can be flipped between 0-1 or on/off state by transient delivery of pulse -Genetic Toggle Switch is 2 interacting genes set up so that each wants to be on -driven by constitutive promoters -set up as a circuit, so protein from Gene A binds to on switch for gene B, shutting gene B down -protein produced by gene B wants to bind to gene A and switch it off -Essentially, each gene wants to be on, but each gene shuts each other off -*mutually inhibitory network* -Can set up the circuit so that it wants to exist in one of 2 stable states: 1)State A - gene A on, gene B off 2) State B - gene A off, gene B on -in principle, can flip between states by transiently delivering chemical and environmental stimulus that will shut off the currently active gene -allows inactive gene to be turn on, which temporarily switches the circuit *The circuit endowed a cell with memory* -made an ability to program cells for a variety of functions in various industries Now have related switches based in RNA that enable us to turn multiple genes on/off dynamically inside cell to redirect metabolic flux Can use synthetic biology to engineer organisms to address a range of therapeutic applications -Ex) Bacteriophages to go after bacterial biofilms

Cas9 Variants with Altered PAM and Targeting Specificity

Table shows variants evolved and mutated from S. pyogenes NGG PAM Site There are also NGG PAM sites from different organisms that can be used in different applications -longer PAM sites can be useful depending on the application (more specific) One issue with CRISPR is *"Off-targeting"* =where if there is a sequence similar to 20bp you are trying to sequence, the other sequence may also be targeted -some PAM sites have been made to help go against this with "Enhanced Specificity"

Quantitative Detection (Paper - Paper Based)

To properly detect the color change in paper and measure the absorbance changes between colors, authors *made their own electrical optic reader* -DIY electronic sensor built with $100 to quantify reactions on paper Did this to prove the point that you can take something relatively cheap and construct it so that you can take it out to the field -Light Box was cheaper, smaller, and easier to use system than machines in lab When more trigger RNA was added to the system, authors detected faster rate with DIY sensor

Riboswitch Based Sensor = Toehold Sensors (Paper - Paper Based)

Toehold Sensor =Riboswitch that uses a second RNA molecule as an inducer/ "trigger" -different from previous riboswitch in lecture in that it uses "trigger" RNA and no small molecules RBS located on loop region of riboswitch sequence -start codon in stem Toehold initiates binding of trigger RNA -upon binding of trigger RNA, toehold will unwind and free the RBS -allows ribosome to bind and initiate translation of repressed gene on the switch 2 Parts: 1) Toehold Region -single stranded and free -allows a' region of trigger RNA to bind to toehold -by binding trigger, rest of sequence will essentially bind/unwind -allows RBS to be translated 2) Gap where start codon is -when bound to trigger RNA/unwound, is able to be translated Authors tested 8 different toehold switches -with trigger RNA, all 8 turned green with GFP -without trigger RNA, toehold sensors did not fluoresce -when toehold switches bound cognate trigger RNA, initiated unfolding of the secondary structure and allows translation Different reports also functioned on both paper and quartz filters -problem with paper is that there is background autofluorescence -quartz does not do this, so dynamic range might be higher if quartz is used to analyze (but is more expensive) -worst case in quartz had over 10-fold higher than background -best case was over 100-fold better than background -shows the system is very efficient

Detection of Full-length mRNA (Paper - Paper Based)

Toehold riboswitches were designed to detect (base pair) with full-length mRNA -initially full length GFP or RFP transcript -going away from small trigger RNA Full length GFP or RFP is the only thing that should bind to toehold region and cause conformational change to free RBS for translation *Authors were successful in using full-length mRNA/transcript* -able to make fluorescent proteins with full length mCherry and GFP -mCherry mRNA gave expression of GFP -GFP mRNA gave expression of mCherry -showing how binding of toehold to trigger had specific output *Antibiotic Resistance Gene transcripts were then tested* -authors sought to detect the presence of antibiotic resistance genes with toehold switch sensors -designed toeholds that would conformationally change by mRNA encoding 4 different antibiotic resistance genes -in each of the cases, there is a fold-change in detection when mRNA is presented, when compared to toehold that did not have mRNA provided -took further look at ampicillin gene and saw the system did give quantitative results -the more trigger mRNA you had, the more output you get *HeLa Cell was extracted and tested* -authors wanted t prove the PT7 system could be used for eukaryotic cells as well -HeLa cells were provided GFP (or not) and fluorescence of GFP was measured -had round 6-7 fold change when comparing GFP HeLa with cells without GFP Previously described Glucose Sensor -Using the same HeLa cells but contain sensor for glucose inside (detect presence of glucose) -authors took HeLa cells with Glucose Sensor, freeze-dried them, and put them on paper -resuspended them by adding liquid and did assay -added different amounts of glucose, which fluoresced at 528nm -did get response, but was not very robust -error bars were low, so results were still statistically significant

Cas Associated Transposase

Transposon elements found near CRISPR elements on genomes -Transposons = jumping genes that randomly insert into DNA -brought up the idea of CRISPR Guided Transposases Functionally demonstrated to provide targeted transposon insertion in E. coli -2 papers showed that this was the case, where CRISPR system helped transposons jump from one bacteria/genome and insert into another -showed you could repurpose CRISPR System located next to transposons to site specifically insert DNA -there are sgRNA dependent proteins that also associate with transposases, and the transposon DNA is inserted into the genome -turns transposon that should be random into something that is site specific

Golden Gate Cloning

Uses *Type IIs Restriction Enzyme and Ligase* -One-pot Assembly Process: -primers are added to each end of the gene of interest on PCR product -at end of each primer, SapI recognition site is added -SapI is 7bp recognition and 3bp overhang -produces actual cut site for gene -on plasmid backbone, 2 different restriction sites flank the portion we wish to cut out -after addition of restriction enzymes, there is cleavage for both desired gene and in plasmid backbone -restriction sites removed and sticky ends left -sticky ends can then anneal and ligate together -makes one continuous piece of DNA *No restriction sites are in final construct* -Digestion and ligation can happen in same reaction -possible because restriction site is located outside of the recognition site -*greatly increases efficiency* Problem with Golden Gate Cloning -need a plasmid backbone that has restriction sites in place that you want them, in orientation that you want them -Restriction Sites can be made easily in Ligation Independent Cloning

Gibson Cloning

Uses 3 Enzymes -Exonuclease = chews back -Polymerase = fill in -Ligase = seals Isothermal -1 temp for all enzymes *Cons* -special enzyme mix needed -moderately expensive Process: -starting with 2 dsDNA that have homology to each other -all enzymes are added together -Exonuclease chews back 5' ends to leave free 3' ends -Free 3' ends are complimentary and can form dsDNA -Polymerase fills in big gaps -Ligase seals nicks Use Gibson Cloning to make larger constructs - unique sticky ends are the overhangs made by exonucleases -unique ends will align in particular way that makes one continuous construct (efficient if designed well) *Multiplex type assembly can be done with CPEC, SLIC, and Gibson* -CANNOT be done with Restriction Digest Cloning because its not efficient enough -reason why Golden Gate Cloning is good because it made ordering cloning of constructs easy

Video: Synthetic Biology - Programming Living Bacteria (Cell program online)

We want a computer system that allows us to design DNA circuits and plasmids *because computation underlies everything we see in biology* -if we want to exploit products that biology produces, have to control what types of computations cells are doing and how they are thinking and processing their environment Reason we can't access all components of Biotechnology is 2 reasons: 1) All of the functions require many genes -all products we currently get out of biology is made up of a few genes -more complex products require 100s or 1,000s of genes 2) It's not just important to control the genes -must tell each gene what to turn on -timing and location are important (particular environmental conditions) Purpose of programming is to *allow a person to go into a cell that they're trying to build, and tell exactly what genes to turn off at what time* -part of building these products Cells are doing computation all the time -thinking and processing about their environment -figuring out where hey are -figuring out what genes need to be turned on to survive Cells do computation by using large sophisticated regulatory network -Regulatory Network = where you have proteins, DNA, and RNA all interact with each other -it is within these interactions that computations arises One of the first "Simple" Regulatory Networks Discovered: -encoded very simple decision that a virus makes when entering bacteria -virus must decide it it will immediately kill bacteria and escape, or hide in the genome -Yes/No decision gets encoded by different interactions Language of electrical engineering started to be used in these networks in 1990s -Gate in images = logic operation Natural Regulatory Networks in bacteria involve hundreds of regulators -many "synthetic" circuits have been built, but their size is limited and each one has error -building a "simple" circuit takes tears of effort, and still long way to go before we ca build larger circuits What inhibits building synthetic regulatory networks? Design -number of well-characterized regulatory proteins -sensitive to genetic an environmental context -toxicity Construction -require large DNA fragments with many parts -expensive and slow Debugging -cells change as they grow -can only observe output using "reporter" -circuits are defined by many states Creating a software program to program a cell: 1) Write desired circuit on computer -write textual program that software compiles into circuit diagram/genetic circuit -made up of individual gates connected to created desired circuit function 2) Convert to DNA sequence -software then creates DNA sequence associated with circuit function -DNA sequence is synthesized and put into cell One of the *most challenging* parts of building circuit is *building the gates* -are one of the fundamental units of computation that work so robustly you could put them together in different configurations to allow user to make any program they want *Sensor* = piece of DNA that allows a cell to respond to signal and then control the expression of a gene -in sensor, there is piece of DNA that encodes a gene that makes a protein, that can bind to the signal -when protein binds to signal, it can bind to DNA at a promoter -causes flux RNA Pol to turn gene that it is connected to on ~ presence pf molecule = high flux of RNA Pol ~ absence of molecule, slow flux of RNA Pol *Black Box* = simplifying something -covering the mechanisms of a sensor and not thinking about what is inside -just think that it is a sensor and has an output of RNA Pol Flux according to stimulus Gene Circuits are different from Sensor -has BOTH input and output as RNA Pol Fluxes (like high RNA Pol flux input converts to low RNA Pol Flux output) -*key about this design is that both inputs and outputs are the same (RNA Pol Flux) and is easy to connect them to sensors and to each other* *Theory from Electrical Engineering* -Some logic functions are known as "Boolean Complete" Boolean Complete =anything that you can imagine on computer can be broken down into simple Boolean Complete Logic Gates without additional computational functions -*one of the basic principles that allows digital computing* NOR Gate =2 input 1 output logic function -when both input signals are off, output is one -if either is on, or both on, output is off -basics to build a computer NOT Gate = function of NOR Gate that can be encoded into DNA -input on, output off -input off, output on -in DNA, have repressor that turns off promoter -input is RNA flux going into gate -produces repressor protein, which turns off output promoter -*as you turn on the input promoter, you turn off the output promoter* NOR Gate =2 input promoters connected to each other in DNA -if either input promoter is on, RNA Pol flux produces repressor protein, which turns off output -when both input promoters are off, output promoter is on -Basic NOR function can be built upon for any other circuit function desired -*critical about the design is that both inputs and outputs of gate are promoters* -means you can take output promoter of one gate and feed that as input promoter of next gate -can be built up in series Non-interfering gates -*only promoters that do not interfere with each other can be used in a logic gate* -around 16 promoters that don't interfere exists, so 16 gates can be used together for circuit design To prevent interference, *insulators* were made -allowed gates to be moved around in different combinations/contexts to build various circuits One Gate Library was made, could develop software that would put the gates together into circuits -used software language Verilog -hacked it to compile into DNA, not silicon Cello "Cellular Logic" Website made: -user writes circuit they want on website with Verilog commands -when they hit compile, program figures out circuit diagram -then goes into library of insulated gates and figures out how to put all the gates together to get good circuit function -output of program is DNA sequence of circuit that can be synthesized and put into cells Multiplexor =circuit that has 3 inputs -one of the inputs selects between other 2 and figures out what the output should be -3 input 1 output logic function Priority =inputs have defined priority order -each output corresponds to input -priority circuit assigns priority to one of the 3 inputs, and the 3 outputs are determination of priority

Part Mining (Paper - Genetic programs)

When trying to build the circuit, authors looked through literature to find appropriate parts Found example of Native AND Gate -Virulence Excretion System ~need to different proteins to come together to activate transcription from a promoter *Chaperone* and Transcription Factor/TF* needed to turn promoter on -the 2 proteins in Virulence Excretion System Chaperon and TF bind together and activate promoter -AND Gate because you need BOTH proteins to get output -Promoter allows transcription of an effector, which chaperone binds to -effector then transported from cell and causes sickness Authors mined genomes to find putative orthogonal parts -looked for closely related proteins, and tested examples of those proteins -need the biology to underlie the synthetic biology system (can't produce a circuit without first understanding the system) -shows how necessary biology is to enable synthetic biology

Homology Directed Repair/ HDR

Where DNA is cut with Cas9, but DNA has been supplied that has homology to cut site -Cas9 cuts target strand into 2 pieces -Repair Template has homology to upstream and downstream region that was cut -in between the 2, desired insert can be used to fill space -cell then has mechanism that repairs cut by taking the homologous template and combining it into chromosome Technical detail of how you provide repair template and nuclease varies with cell type -efficiency also varied between cells (is large field of research) How could you make the repair template? -need to add up and downstream homology to some piece of DNA -Ligation Independent Cloning Techniques -Ligation Techniques -putting into plasmid and constructing that way -Sewing PCR with overlapping sequences HDR Includes: 1) dsDNA Insertion -Donor Template with up and downstream homology -what is between Donor Template's areas of homology are incorporated into chromosome 2) ssDNA Insertion -cell uses ssDNA as Repair Template -in the end, left with scarless target alteration Efficiency of HDR varies widely between organisms -Often <1% of surviving cells have the insert you are trying to put in -Cas9 is efficient at cutting chromosomes, but difference between NHEJ and HDR is less clear -if Cas9 cutes and inserts is not put in, NHEJ repairs instead of HDR -most cells have indels that change the target site

Colony PCR (variation of PCR)

Whole cells used as DNA template -like normal PCR< but whole cell is used as DNA rather than specific DNA Initial Denaturing step in PCR lyses cells which release DNA -Gram+ cells are tough to lyse Taq DNA Pol performs best -not high fidelity, but is robust in conditions it can amplify in Saves time -no DNA prep needed Steps: -Pick colony and put onto second plate -Replicate colony so you have more bacteria to recover later -Add colony directly to PCR tube that has PCR reaction -Do PCR and analyze

Nucleases (Paper - Genome-scale Engineering)

ZFNs and TALEN Tethered to FokI -can cut chromosome and insert some type of gene with HR -HR is not efficient in bacteria *Are proteins targeting DNA* Difficult to assemble for targeted DSB In bacteria, DSB does not always (rarely) cause indels or HDR -rather than NHEJ or HDR, has DSB -DSB is lethal -killing the cell can be used as counter selectable marker

Dead cas9/ dCas9

dCas9 is a nuclease null variant -no longer able to cut DNA, but has site specificity dCas9 can be used for Gene Silencing or blocking transcription -if you target dCas9 to promoter or ORF, it will site on DNA and prevent RNA Pol from making transcript -if RNA Pol can't move, gene is silences because no transcription or translation to protein -can be used in eukaryotic cells and bacteria -generally called *CRISPR Interference* No additional domains needed to be fused to dCas9 to be used in bacteria *Not genome editing* - but useful for *dynamic control* -Essential Genes can be targeted with dCas9 -you cannot delete an essential gene from bacteria/any organism or it will lead to death -can, however, do transcriptional depletion where cell grow happily, and then you can induce dCas9 to block transcription of essential gene, which depletes the essential gene -can study how the cells change after essential gene is blocked Can also use CRISPR Interference tool for Nonessential Genes -in many cases, making gene deletion is more laborious than targeting the sgRNA to specific sequence -using dCas9 to deplete gene and study what happens without it -easier and faster than actual gene deletion