BCH 5413 Exam 1
State 2 ways in which you can increase the transfer efficiency of DNA to a blot.
1. Soak the gel in dilute HCl to partially depurinate the DNA 2. Soak the gel in dilute NaOH solution so it can cleave at depurinated sites dsDNA now ssDNA
What are the steps to be taken if the protein is insoluble (3 points)
1. collect inclusion bodies and refold protein 2. reduce growth temp 3. use a low/moderate copy number plasmid vector 4.fuse periplasmic targeting sequence to N-terminus 5. Try a fusion protein (GST) 6. study a more soluble protein
Give 2 advantages of TaqMan probe over Sybr Green.
1. specific hybridization between probe and target is required = accuracy 2. different probes labeled with different dyes = 2 sequences in one reaction probe
Why would you do site directed mutagenesis? Give 2 reasons
1. study effects of point mutations found in diseases 2. investigate binding site of proteins 3. determine effects of a deletion within an enzyme
If you start with 10 copies of DNA, how many copies of DNA would you expect after 17 rounds of PCR (write out the values in formula, need not calculate)
10 copies of DNA = base number = 10 exponent = cycles = 17 10^17 copies
The core of DNA Pol III consists of __________.
3 subunits Alpha Epsilon Theta Responsible for polymerase and editing function, exonuclease
What is a common DNA probe?
32P-phosphate
Explain the steps of site directed mutagenesis
4 main types: 1. Ligation-mediated mutagenesis 2. PCR/Primer Extension mutagenesis 3. QuikChange mutagenesis 4. Inverse PCR mutagenesis STEPS 1. Need plasmid: most replicated by E coli 2. Need ORI, MCS, Ap^r 3. plasmid transformed into E. coli 4. isolate small amount of plasmid DNA 5. screen plasmid DNA for mutation by 1: restriction site or 2: southern hybridization 6. screen all mutated segments NEED to DESTROY RESTRICTION SITE EcoRI cuts at a site, digests, Klenow + dNTPs, T4 DNA ligase to put back together
What is a densitometer?
A device for measuring the radioactivity in a fragment of DNA the darker and denser the band, the more concentration, absorbance
What is autoradiography?
A process where radiation from a substance is captured on a camera. X-ray of gel electrophoresis
What is a contig?
A series of vectors that contain inserts that have overlapping regions of chromosomal DNA
What is southern blotting?
A technique that uses DNA probes to detect the presence of specific DNA in fragments separated by electrophoresis 1) DNA electrophoresed 2) denature DNA with base, transfer ssDNA to sheet of nitrocellulose/binding material either by diffusion or electrophoresis 3) hybridize the blot to labeled probe 4) detect with autoradiography or phosphorimaging
True or False a. Most biological DNA is positively supercoiled. b. Triplex DNA is pyrimidine rich c. Chargaff's rule: A=G & T=C d. A single zinc finger can recognize 10bp of DNA
A. False, negatively = Left-handed. Some is right handed = positive B. True C. False A=T C=G D. False, 3 bp, use multiple zinc fingers in a row for more sequence specificity/bp DNA is not a uniform structure, many localized variants can substitute protein domains, alpha helix/beta hairpin, in zinc fingers to make hybrids with different binding specificities (designated by alpha helix) for major grooves of DNA x-ray crystallography allows us to view and design custom designer DNA-binding proteins = alter gene fxn in intact cells
True or False: a. Blunt ends gives better ligation than sticky ends b. The general ratio of vector to insert in cloning is 1:3 c. If bacterial cells have taken up the plasmid, the colonies are white in blue/white selection d. E. coli methylate their own DNA to prevent infection by viruses
A. False, sticky ends can pair complementary overhangs b. False, insert:vector excess ratio 3x or 10x c. True, blue = re ligated, cleaves B-galac because intact. White = vector inserted, B-galac not intact d. True
Name a: Bacterial expression vector Mammalian expression vector
Bacterial expression vector: pET system vector = 40-100 COPIES OF A pET VECTOR PER ECOLI origin of rep, selectable marker Ap^r, polylinker MCS, lacZ blue-white screening, T7 promoter for expression, T7 terminator, ribosome binding site Eukaryotic: yeast, commonly used as a shuttle vector with Ecoli. Baculovirus. Tend to be folded more properly, less chance inclusion bodies. Modified = phosphorylated, glycosylated etc. Ti Plasmid = T-DNA, piece of DNA from Tumor Inducing plasmid, causes CROWN GALLS
What is the importance of blocking in a Southern Blot?
Blocking decreases non specific binding of probe to DNA consider copy number, high copy number decreases specificity
With reference to ChiP -How are cells cross-linked? How is the chromatin sheared? Which antibody is used as a control? Which two ways can the chromatin obtained from ChiP be quantified?
Chromatin Immunoprecipitation Assay cells cross-linked with chromatin by formaldehyde, preserves DNA-protein interaction, track DNA sequence for protein of interest chromatin sheared mechanically or enzymatically into smaller fragments incubate with added antibody for specific DNA binding protein sepharose beads to pull down antibody quantify chromatin: assay DNA by PCR? Densitometry to quantify gel band thickness
What is DAPA?
DNA Affinity Purification Assay Ex: Does aldosterone increase the interaction of MR & Per1 with the E-box response element in the alphaENaC promoter? Nuclear extracts treated with either vehicle or aldosterone Western blot performed mutated E-box probes as negative control Answer: YES, band color is much darker with aldosterone treated MR & Per1 than with vehicle
What is the function of DNA B and DNA G at E. coli replication?
DNA B = helicase, stimulates primase binding DNA G = primase
What is cDNA?
DNA copy from RNA
What does the Meselson and Stahl experiment confirm?
DNA replication is semi-conservative Heavy and light strands with nitrogen isotopes prove
What is DNA fingerprinting & DNA typing?
DNA typing = identifying individuals who have left behind DNA Fingerprinting = basically a southern blot, gets cut with restriction enzyme beforehand
Function of DNA Polymerase Gamma is ________.
Dimerizes core clamp loader
What is the assay in the figure below? What are (1), (2), (3)
EMSA electromobility shift assay 1) DNA bound to two proteins, heavier, didn't travel as far in the lane 2) DNA bound to one protein 3) Bare DNA polyacrylamide gel radioactive bc oligonucleotide probes lane 1 DNA alone lane 3 added nuclear extract to radioactive lableled oligonucleotide probe lane 4 added antibody to protein of interest sometimes negative control by adding anti IgG - shouldnt be sequence specific
Transfection vs Transformation?
Euks = transfection bc transformation refers to a normal cell converting to cancer-like cel
How would you use ligation mediated mutagenesis to replace an amino acid?
Ex: replace alanine in b subunit of ATP synthase find alanine oligo, replace single bp with many options result: 9 different oligos, all specifying a different missense mutation
What is a GST tag? How does it help in protein purification?
GST tag = fusion protein glutathione-S-transferase = strong affinity for immobilized glutathione-covered matrices since GST isoforms not found in bacteria, no competition for binding to purification resin enhances the solubility of euk proteins in bacteria solves problem of multiple bonds observed
What would be an ideal Loading control for a Western Blot experiment?
Housekeeping protein: Any protein that would not be expected to change with treatment ex. actin, GAPDH
What is in situ hybridization?
Hybridization of a nucleic acid probe to a nucleic acid that is still inside a cell if fluorescent = FISH
What is the function of IPTG in cloning?
IPTG = sugar, induces protein expression absence = lac promoter not active presence = lac promoter active = transcribe lacz mRNA produce B-galac IPTG allows us to add protein of interest
Name two methods to study protein-protein interactions
Immunoprecipitation: Antibody for a protein (A) produces two proteins (B) & (C), but no own affinity for (B), likely that (B) associates with (A) Yeast two-hybrid assay: demonstrates binding. Takes advantage of 1) transcription activators have 2 domains, DNA binding and transcription-activating, 2) domains have self-contained activities. Protein A fused to DNA-binding domain, B fused to transcription-activating. If proteins interact, domains = together = transcription of reporter gene
What is 2-D gel electrophoresis?
LESS POWER: use two different pH values to compare, pH affects net charge so different rates of mobility 2-D = 1) proteins electrophoresed through narrow tube gel with pH gradients by ampholytes. Once protein becomes neutral, no longer moves = isoelectric point 2) gel removed from tube, placed at top of regular gel for SDS-PAGE
What are probes?
Labeled nucleic acids, can be DNA or RNA
What is the relationship between MCS and lacZ and B-galactosidase?
MCS of pUC vectors lie in lacZ operon/sequence that code for partial B-galactosidase ex: when pUC18 transforms bacterial cell also carrying partial B-galactosidase, active enzyme is formed. If clones are then plated with B-galactosidase indicator X-gal, B-galac cleaves X-gal = indigo blue colonies BUT if this gene is interrupted, no cleavage = no blue colonies = white
Name one negative and positive control for PCR
Negative: for PCR, No template. For Ligation, no ligase. For DpnI, undigested plasmid Positive: For PCR, Control Plasmid with Primers. For Ligation, plasmid cut with 1 enzyme and ligase. For DpnI, control plasmid amplified with control primers
What is PAC?
P1 derived Artificial Chromosome max insert = ~100kb more difficult to make library than with cosmids or lambda recombination potential problem more difficult to purify and manipulate cloned DNA than lambda because of size
What kind of nucleotides are used in PacBio
Phospho-linked Fluorescence is linked to pyrophosphate so when it's cleaved, fluorescence isn't still holding onto nucleotide base
What is PAGE?
Polyacrylamide Gel Electrophoresis used for PROTEINS have to denature the protein = treat with detergent, SDS, sodium dodecyl sulfate SDS-PAGE SDS advantages: 1) coats polypeptides with (-) charge 2) masks natural charge so results are purely based on molecular weight
What is 454 sequencing?
Pyrosequencing - no DNA cloning needed reads up to 1 kb 1) primer hybridized to ssDNA template that has been PCR amplified, incubated with enzymes, DNA poly, STP sulfurylase, luciferase, apyrase, substrates, adenosine 5' phosphosulfate (APS) & luciferin 2) DNA(n) + dNTP plus DNA polymerase = DNA(n+1) + PPi (pyrophosphate) 3) since you have APS with PPi = ATP = drives luciferase conversion of luciferin to oxyluciferin = visible light 4) apyrase degrades nucleotides = degrades ATP and unincorporated dNTPs = light turns off. Reaction regenerates 5) Addition of dNTPs one at a time as process continues, complementary DNA strand is built up and the nucleotide sequence is determined from signal light peaks in Pyrogram
In the figure below, fill in the blanks: mRNA---->mRNA-DNA hybrid---->ssDNA---->dsDNA
Reverse Transcriptase-PCR mRNA to hybrid = first strand synthesis Oligo(dT) to bind polyA tail + reverse transcriptase hybrid to ssDNA via RNase H ssDNA to dsDNA via DNA polymerase
How is DNA copied at the replication fork - Continuous, Discontinuous, or Semi Discontinuous? Which experiment supports your answer?
Semi-Discontinuous Okazaki with T4 phage and E. coli, pulsed DNA for various lengths of time, separated fragments by size on gradient The longer time between pulses, the longer the fragments got so clearly Okazaki fragments were being stitched together but without ligase, stayed short
What is a Western blot?
Separation of proteins via electrophoresis blotted to a membrane proteins probed with specific antibodies antibodies detected with labeled secondary antibodies or protein A
What is inverse PCR mutagenesis?
Similar to QuikChange phosphorylated primers PCR with High Fidelity Polymerase ligate new PCR products with T4 DNA ligase
How does PCR work?
Start with dsDNA heat to SEPARATE 95C Add 5' primers at 3' end (since running both ways, need both forward and reverse primer) 50C so they can ANNEAL Add DNA polymerase (Taq polymerase that can withstand heat) 72C Add free dNTPs so Taq polymerase has something to build with Each round doubles previous amount of DNA
What is the function of T4 PNK?
T4 PNK is used during Site-Directed Mutagenesis - Ligation-Mediated ssDNA circular, from bacteriophage Heated to 55C, cool to room temp to anneal mutagenic oligo oligo must be PHOSPHORYLATED on 5' end T4 PNK (polynucleotide kinase) phosphorylates oligo 5' end
Give me a simple experimental way to detect if an animal is transgenic or not
Tail blot cut off portion of tail of each offspring, analyze southern blot with radioactive probe specific to fertilized egg Ask, what changed bc of expression of transgene
What is Affinity Chromatography?
Takes advantage of the affinity of different proteins to certain ligands. A ligand or other molecule that binds to a protein of interest is covalently attached to the beads in the column. protein of interest eluted from column with a solution that disrupts specific binding elute = remove supernatant = remaining solution
What is the function of the following: Topoisomerase Klenow fragment
Topoisomerase relieves torsional stress Klenow fragment is enzymatic product of DNA poly I from E. coli, retains polymerase action Lacks 5'—>3' exonuclease
True or False Okazaki fragments are 1-2kb long on prokaryotes RNA primers are 5-15kb long
True
How do you destroy a restriction site?
Use EcoRI to digest and make cuts for sticky ends Add Klenow & dNTPs, these fill in sticky ends so they are complementary Add T4 DNA ligase to seal back together ALSO FOR FRAME SHIFT
What is QuikChange Site-Directed Mutagenesis?
Use pfu Turbo DNA polymerase = thermostable, nonstrand displacing, 3'-->5' proofreading DpnI digests plasmid, preferentially nicks only methylated DNA strand of dam hemimethylated DNA transform into E coli cells pros: fast efficient cons: GC content is relevant
What is a cosmid?
Vector that can carry 100kB or more (compared to plasmid - 15kB)
Name two methods to study structure of proteins
X-Ray Crystallography rotate crystal and record diffraction patterns Immunoblots or Western Blots in order to quantify, but structure?
Give 2 disadvantages each for YAC BAC
YAC: Yeast Artificial Chromosome 1. difficult to make and maintain library 2. recombination significant problem 3. chimeric inserts a problem BAC: Bacterial Artificial Chromosome 1. more difficult to make library than lambda or cosmid 2. recombination potential problem 3. low copy number makes it difficult to purify significant quantities
What is chromosome walking?
a method of positional cloning used to find, isolate, and clone a particular allele in a gene library. Starting with the DNA sequence of a single clone, probes can be used to "screen" a genomic library for additional clones that contain fragments overlapping with the original clone. Repeat as necessary.
In A & Z DNA: a. Which is right handed helix? b. Which one has 12bp per helical turn? c. Which is the dehydrated form?
a. A b. Z c. A primary DNA is B
Answer the following questions with regard to CRISPR Cas9 tech: a. Expand CRISPR Cas9 b. Which organism was this system first discovered in? c. If you were to perform a gene knock out experiment against BCL11 using CRISPR Cas9, list the main components you need to perform the experiment. d. What is the importance of PAM sequence? e. What are the 2 possible repair mechanisms of a double strand break? Which one of the methods would you prefer if you wish to make a three nucleotide change in your target gene using CRISPR Cas9?
a. Clustered Regularly Interspersed Short Palindromic Repeats Cas = CRISPR Associated b. First discovered in E. coli c. d. Protospacer-Adjacent Motif In Phase 1: immunization, CRISPR system stores molecular signature of a previous infection by integrating fragments of invading phage or plasmid DNA into the CRISPR locus as "spacers" able to recognize when destroying foreign DNA sequences e. Homology-directed repair: provide donor template containing transgene(s) & Non-homologous End Joining: error-prone. NHEJ for 3 nucleotide change in target gene.
3. In the figure below, a. Which experiment is being done? b. Why is there a region of no bands? c. What type of interaction does this experiment determine?
a. DNase footprinting b. region of no bands = protein was bound to this sequence = footprint, couldn't be translated c. protein-DNA
In a plasmid, what are these and their functions? a. ORI b. Apr c. MCS
a. ORI = origin of replication, where replication begins b. Ap^r = selection marker c. MCS = multiple cloning site, large variety of restriction enzymes that cut plasmid once, allows you to put in own DNA of interest
True or false a. the ion torrent combines semi conductor technology along with DNA sequencing chemistry to sequence DNA b. The bridge amplification is the highlight of 454 sequence
a. True b. False?
If you want to identify the protein PTEN on a Western blot, what is: a. primary antibody? b. With reference to your primary antibody, what is your secondary antibody?
a. protein of interest is immobilized on membrane, primary antibody binds b. Secondary antibody is linked to primary antibody in order to detect it, and on the opposite side is linked to conjugate, HRP, horseradish peroxidase
What is Gel Filtration Chromatography?
after both anion and cation-exchange chromatography, can target different property of proteins = protein size pass a solution through a "whiffle ball" with varying size holes small proteins get stuck, large pass around and go further
What is a transcriptome?
all the transcripts an organism makes at any given time
Where do you look for sequence info on NCBI?
at ncbi website resources tab DNA & RNA in drop down menu BLAST = Basic Local Alignment Search Tool
Which of the following increases the stringency of Southern Blot? a. Decrease wash duration b. Decreasing ionic strength c. Increased hybridization d. Decreased wash temperature
b. Decreasing ionic strength c. Increased hybridization INCREASE wash duration and temp
Where in the genome are loxP sites located in a Cre/loxP model? a. Adjacent to the Cre locus expressed in a desired cell type b. On either side of the gene that is to be knocked out c. Random sites throughout the genome d. all of the above
b. On either side of the gene that is to be knocked out
Which of the following methods does not involve purifying protein: a. Ion exchange b. Ammonium sulfate precipitation c. Flow cytometry d. Gel filtration
c. Flow Cytometry
What is the difference between cDNA & genomic libraries? When do you need which?
cDNA: isolate mRNA, RT to ds cDNA, choose vector, insert cDNA, create phage or bacterial library with oligo-dT matrix as backbone compared to cDNA libraries, genomic libraries are "easy" 1)fragmentation to appropriate size for vector being used. For lambda = ~20kb same as cDNA
What is a DNA microarray?
circumventing size issue use inkjet printer to put tiny dots of DNA on chip after spotting, DNA air dried, covalently attached by UV radiation to a thin silane layer on top of the glass
Which of the following proteins have not been used in genome editing? a. ZFN b. TALENs c. CRISPR-Cas9 d. MHC
d. MHC
What do labeled tracers do?
detect tiny quantities of nucleic acids or proteins if tracer is radioactive, detect by: autoradiography x-ray or phosphorimaging liquid scintillation counting
What is F1 ori?
f1 is the name of the phage being used ori is the origin of replication in the plasmid if cell is infected by f1 helper phage for replication machinery, ss copies of the vector can be packaged into progeny phage particles basically, allows vector to be read
PFGE = Pulsed Field Gel Electrophoresis
for longer sequences as large as some chromosomes in yeast
What is the name of the map generated from microarray analysis?
heat map
How would I purify a protein if the protein has 'his-tags'?
his-tags have high affinity for divalent metal ions like nickel proteins with these regions can be purified with nickel affinity chromatography = after bacteria have made fusion protein, lyse them, add bacterial extract to nickel affinity column, wash out unbound proteins, release histadine with analog called imidazole add enterokinase to cleave
what is the purpose of reporter gene assays?
host cells should not have reporter gene activity B-galactosidase chloramphenicol acetyl transferase luciferase green fluorescent protein just downstream of enhancer/promoter you have a reporter gene in your vector to prove your gene of interest was cloned into your vector SHOULD BE EASY TO ASSAY
What is stringency?
how accurately a probe hybridizes, perfect stringency is not ideal you want your probe to tolerate some mismatches control stringency with: temperature ionic concentration wash duration
What does a Northern blot tell you?
how actively a gene is expressed using RNA & the size of the mRNA the gene produces 1) collect RNA from several tissues 2) electrophorese in agarose, blot to appropriate support 3) hybridize to labeled cDNA probe. Whenever an mRNA complementary to cDNA probe exists on the blot, hybridization occurs in a labeled band. 4) detect with x-ray 5) run RNA of KNOWN size to compare size of new unknown bands tells you quantity in how dark a band is = densitometer measure
Why would you use Primer Extension?
locate the 5' end of a transcript by hybridizing oligonucleotide primer to RNA of interest, RT to 5' end of transcript electrophorese reverse transcript to determine size
What are the key principles for primer design?
minimum base pairs required for single primer self-dimerization = 5bp minimum bp for hairpin: 4bp consider: hybridization to other sequences = check via BLAST secondary structure and Primer interactions Is it G:C or A:T at 3' end - depends on your application 3' deltaG Melting temperature differences
Gel electrophoresis
molecular separation technique can separate nucleic acids or proteins make AGAROSE gel with slots hot liquid poured with "teeth" put DNA in slot, run electric current at neutral pH DNA = negatively charged (phosphates) = migrates towards positive end key is friction DNA stained with fluorescent dye, looked at under UV unknown DNA electrophoresed parallel with known to determine size distance migrated = can plot on graph with log of molecular weights or number of bp ex: 20mm = 910bp
What cloning vectors were among the first generation of plasmids?
pBR322 pUC = ampicillin resistance & MCS that interrupts B-galactosidase
Explain library screening using plaque hybridization
plate with bacterial lawn lay nylon membrane over plate to lift off replica of plaques membrane acts like Southern blot membrane Blocking with nonspecific DNA, and using a transfer with a lumescent probe that will bind and light up sequences of interest detect with autoradiography
What do restriction endonucleases do?
recognize specific sequences in DNA & make cuts in both strands allows specificity with cutting creates sticky ends when staggered helps link 2 DNAs for recombinant DNA in vitro
How does PCR mutagenesis work?
requires 4 different primers for dsDNA one primer at beginning of each strand going in opposite directions, one primer at mutation going different directions result is 2 dsDNA, mutation is in the middle duplicated melt and anneal PCR products (cross-annealing) use flanking primers to amplify product Go to vector, digest T4 DNA ligase ligates oligonucleotides to vector pros: flanking restriction sites can be more than 100bp away cons: PCR makes mistakes complicated
What are the 2 advantages of PacBio sequencer?
requires minimum amounts of reagants and sample prep = cheaper Fast, 45-90mins vs an entire day needs no PCR, no amplification bias
What are SNPs?
single nucleotide polymorphisms differences within single nucleotides of a sequence sometimes associated with genetic disorders
What are the oligonucleotide requirements for Site-Directed Mutagenesis?
the longer the sequence the better = more specific and stable mutation should have 10-15 bp on either side remember melting temp. More GC's = higher temp
How do you make a cDNA library?
use mRNAs as templates + Reverse Transcriptase to make first strands first strands template for second strands via E. coli DNA poly I give cDNAs tails that base pair with complementary tails on cloning vector use recombinant DNA to transform bacteria
What is S1 mapping?
used to locate 5' or 3' ends of RNAs quantify amount of RNA in cells at a given time bc transcript is proportional to total label ssDNA probe hybridizes ONLY to transcript of interest apply S1 nuclease degrades ONLY ssDNA & RNA b/c probe will automatically form dsRNA-DNA hybrid = protected measure with electrophoresis
What is the main principle behind bisulfite PCR?
uses a combination of techniques to determine the methylation status of every C in a DNA sequence. when DNA is treated with bisulfite, C's converted to U's GC bp now = AT 5-methyl C is resistant to this treatment, so GC bp's remain Once DNA is treated PCR primers are chosen Avoid CpG in primer sequence primer design is critical
What is Ion-Exchange Chromatography?
uses a resin to separate substances according to charge (+) charged DEAE-Sephadex for anion-exchange (-) charged phosphocellulose for cation-exchange even though most proteins negative, center is positive so can still bind cation-exchange resin
What is the Sanger DNA sequencing method?
uses ddNTPs to terminate DNA synthesis yields series of DNA fragments measure with electrophoresis last base is known bc ddNTP was used to terminate each reaction each fragment one known base longer than the next = DNA sequence
What is the function of ddNTPs in Sanger Sequencing?
uses ddNTPs to terminate DNA synthesis yields series of DNA fragments measure with electrophoresis last base is known bc ddNTP was used to terminate each reaction each fragment one known base longer than the next = DNA sequence ex: fragment with ddATP might end at 22, 25, 27bp ddGTP ends at 24, 29, 30bp ddTTP ends at 23, 26, 31, 33 ddCTP ends at 28, 32 electrophorese can piece together sequence
How does RT-PCR work?
when you want to clone a cDNA from just one known mRNA sequence Add reverse primer 5'-->3' Add reverse transcriptase Denature mRNA Anneal forward primer from opposite end of new strand Add DNA polymerase do PCR with same 2 primers Cut with BamHI and HindIII (restriction sites) so ends can ligate into BamHI and HindIII sites of vector
Are there nonradioactive tracers?
yes chemiluminescence can also be detected by autoradiography or phosphorimaging, as if radioactive those that produce color can be observed directly
Name 3 features of an expression vector
yield the protein products of the cloned genes Bacterial expression vectors: 1) strong promoter 2) ribosome binding site near initiating codon = AUG (ATG in DNA) lac promoter induced by IPTG alternate method: T7 phage promoter controls plasmid, place plasmid in cell with another gene tightly regulating T7 RNA poly most expression vectors are inducible = avoids overproduction/inclusion bodies/foreign product most produce fusion proteins, extra AA help purify (histadines)
The number of bases per turn of DNA is___________
~10.5
