Biosynthetic Final

PCR Enzyme price

Wide range! Taw around 15 cents per reaction, Q5 is 6x higher

Are some recognition sites degenerate?

Yes

Should you choose right buffer for enzyme?

Yes, (mismatch buffer reduces efficiency and increases star activity

Should you include spacer in primer design?

Yes, before restriction enzyme site

Transformation recovery

cells are incubated with rich media for 1 hr without antibiotic to allow them to express the resistant protein

Is T4 ligase efficient?

efficient but finicky enzyme

Wallace rule

estimates Tm (other programs use nearest-neighbor thermodynamic theory to estimate) Tm (in C) = 2 (A+t) + 4 (G + C)

Star activity

(cutting at sequences that are similar but not exactly right) is enhanced by glycerol so keep total enzyme concentration low and by enzyme degradation so keep time short

PCR Enzyme thermostability

(half life at 97C) Limits number of cycles Not a major concern but some enzymes (PFU) can be super stable (5-20x more than Taq)

Binding sequence for sigma unit of RNA polymerase

-35 = TTGACA -10 = TATAAT

How fast does reaction happy for restriction enzymes?

1 unit will cut 1 ug of substrate DNA in 60 mins at 37C. HF enzymes work 2-5x faster

Size of recognition sites

4-8 nucleotides (commonly 6)

What is methylated in e coli?

A and C methylated (dam+/dcm+) Adenine methylation in N6 position GATC

flow cytometry transcriptional reporter

Advantages: fast, higher throughput, larger sample sizes Disadvantages: time series more difficult to collect, careful data analysis required, does not normalize by cell size, careful controls required, lower sensitivity than microscopy

Colonies transcriptional reporter

Advantages: fast, spatial information about growing colony Disadvantages: semi qualitative at best, typically requires special equipment to get even illumination, interpretation may be unclear

fluorescence microscopy transcriptional reporter

Advantages: full distribution of gene expression. Can be normalized by cell size. Often easier to troubleshoot. Highest sensitivity Disadvantages: more labor intensive, time series more difficult to collect, image analysis required

Electrocompetent

Cells briefly shocked with electric field of 10-20 kV/cm that creates holes in the cell membrane through which the plasmid DNA enters Commercial efficacy - 1010 CFU/ug DNA Easy preparation Requires clean DNA, better for supercoiled DNA and for linear DNA

Chemically competent

Chilling cells in presence of divalent cations makes cell membrane transiently permeable Cells incubated on ice with DNA and then briefly heat shocked Commercial efficacy - 109 CFU/ug DNA Multi-step preparation Commercially available but expensive No cleaning needed, better for reaction products

Plasmid copy number

Copy number is very broad from a few copies to many thousands Number of copies of given plasmid in the cell

Things used in gibson assembly

DNA inserts, 5' exonuclease, DNA polymerase, DNA ligase

Steps of PCR

Denaturation at 98 degrees (separates DNA strands) Annealing from 48-72 C (primers anneal to complementary strands (primer annealing temperature should be usually 3-5C below primer's ™) Primer extension (polymerase extends primer to form DNA strand) About 1 minute/kb for taq polymerase Much shorter amount of time for modern enzymes

What type of reaction is ligation

Energy hungry (ATP) reaction

Antibiotic resistance genes

Enzymes that deactivate or degrade the drug (bla - ampicillin, cat - CM, nptII - kan) Decrease drug permeability and/or increase active efflux (pumping out) of the drugs across the cell surface (tetA - tetracycline) Some of these give resistance to local environment (on plate, satellite colonies) or co-cultures

Why do synthetic biology?

Evolution has created diversity of biological systems with desired functions Often the functions in nature are not optimized for human use Major goals - greener agriculture, energy, mining and commercial goods, improvements in medicine, creating novel materials, enabling scientific discovery

PCR Enzyme properties

Fidelity (error rate) Processivity (product length) Thermostability (half life at 97C) Price

Why do molecular cloning?

Fluorescent reporters Mutations or domain deletion to study structural and functional properties Overexpression Complementation of a mutant coming up from a screen Enzymatic reporters Tag for IP, western Promoter bashing Shotgun cloning Yeast two-hybrid Metabolic engineering Synthetic biology

Steps of molecular cloning

Gene of interest Amplification Assembly Transformation Validation

PCR Enzyme fidelity

Inexpensive enzymes (Taq) have 1-7 x104 errors/base pair/cycle High fidelity enzymes (Platinum, Q5) can do 5-300x better

What should the length, GC, Tm, and other properties of PCR primer include?

Length = 18-24 bp GC = 40-60% Tm = between 55-60 C, difference between Tm of primers <2-3C Decrease secondary structures GC clamp - presence of G or C bases within last five bases from 3' end of primers helps promote specific binding Avoid runs fo 3 or more G/C in 3' end Avoid T at 3' end Around 20 bp of homology

Plasmid stability

Likelihood of a daughter cell to be plasmid free depends on toxicity, copy number, segregation mechanism

Primer design tools

Lots of primer design tools that say they will pick optimal primer for you but don't work that well Benchling works fairly well

Why do we need controls?

Most systems/experiments a fixed universal reference point does not exist

Lac Repressor

Only bind to DNA when not bound by lactose molecules Stops expression of enzymes that break lactose and a transporter that imports lactose (those are only needed in the presence of lactose)

Controls to include

Positive control - will give signal that you are looking for Negative control - will not give the signal you are looking for Background or reference sample for measurement system

Sanger Sequencing primer parameters

Primer length: ~20 bp GC content: 45-55% GC clamp on 3' end Tm = 50-65 Avoid secondary structure Check primer specificity Primer bind at least 70 bases upstream of your sequence

Thermocyclers

Programmable peltier blocks to precisely heat and cool (sometimes with single well precision)

What is needed to design inducible element

Promoter Regulatory element RBS RFP

Things used in site directed mutagenesis

Q5 master mix KLD enzyme mix

RT PCR

RNA is extracted, reverse transcriptase used to make cDNA, then cDNA is amplified by PCR

Plasmid size

Range from 1-100s kb

What is in PCR master mix?

Taq DNA polymerase, DNTPs, MgCl2, reaction buffers

PCR Enzyme processivity

Taq can do a few Kb, highly processive enzymes can go to 20 kb and more

What is needed for PCR?

Thermocycler DNA template Specific primers Nucleotides (ATP, CTP, GTP, TTP) DNA polymerase Buffer

How to choose a restriction enzyme

Unique sites on vector No sites inside your cloning sequence Know enzyme compatibility

Plasmid compatibility

ability of two plasmids to be maintained together

Culture transcriptional reporter

advantages: robust well mixed measurement of population average, repeated measurements give time series information, automated measurement systems exist (monochromator and filter based) Disadvantages: many different distributions of gene expression can have the same mean value (shape of distribution is lost)

Steps of site directed mutagenesis

amplification phosphorylation ligation template removal

Restriction enzyme

an enzyme that cuts DNA at specific recognition nucleotide sequences known as restriction sites

BF

bright field

Cloning workflow

extract dna PCR cut PCR and target vector purify fragments of correct size incubate fragments with ligase transform into bacteria verify success by phenotype and sequencing

RBS

generally 6-10 bases upstream of AUG in e coli AGGAGG (shine delgarno) Species specific and does not need to be a perfect match

Biological replicates

identically prepared biological samples (variability in biological system)

How to find molar ratio for ligation if DNA insert:vector

insert ng = ratio (insert length/vector length in bp) x vector mass in ng

Synthetic biology

is the art of constructing interactions to give rise to novel biological functions, building new biological systems with (or inspired by) existing biological parts

What will an excessively low Tm do?

lead to non-specific products but increase yield

Technical replicates

same biological sample measured multiple times (measurement error)

Multiple cloning site (MCS)

short polylinker sequence rich in unique restriction sequences

FISH

sinle molecule RNA FISH that attaches to RNA molecules and lights up

Systems biology

studies how functions arise from interactions among biological components

Molecular cloning

technique involving insertion of foreign DNA into a vector thus allowing its manipulation. Vector will be introduced into desired organism to study the structure or function of specific gene or its effect on other gene's function

Tm

temperature at which 50% of primer and its template are in duplex

Usually biological replicates have greater error than technical true or false

true

Polymerase chain reaction (PCR)

used to amplify single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence