Final 1 Flashcards
What can low peaks on a chromatogram be attributed to and what can happen?
not enough PCR product
Cant guess what the nucleotide is
Cannot do anything about overlapping waves
Should control the experiments from the beginning to have a good PCR product.
How do you find the forward primer in an alignment?
Go from the start codon
What is histidine of the FP and the RP?
FP: CAC; RP: GTG
What does a translation vector have allowing for overexpression?
It has a ribosome binding site.
In transformation, what is the most important step? What is it for?
1.5 hr of incubation for antibiotic resistance.
What are used as the material and host for transformation in overexpression?
Use recombinant DNA plasmid - pet29b-CR (vector)
Competent cells - BL21DE3 (is an ecoli strain)
What 3 things are required for expressing a
recombinant protein?
need good design
Vector-host combination
Control of Basal Expression (Leaky expression)
What are the features of a good expression vector?
Strong promoter Ex: t7 promoter
Controllable promoter - Operator
Plasmid copy number
Plasmid stability
What is the confirmation of the vector?
Lac I - T7 promoter - operator - rbs - ATG- ORF insert- hist tag- stop codon
Where does RNA polymerase bind on the vector?
RNA polymerase binds to the promoter
What is the stop codon for?
Stop codon is for translation
True or False: The RNA polymerase will not transcribe the entire vector
False
What is the role of Lac I?
Lac I gives the repressor protein. Has a high affinity for the operator. It binds to the operator arresting it.
What can the happen between the operator and repressor protein, allowing for translation without a confirmational change of the protein?
Operator and repressor protein relationship can be leaky allowing for translation. RNA polymerase can transcribe
Where does the repressor protein bind?
Repressor protein always binds to the operator
What is added to express the gene? Is it the only way the gene can be expressed? What happens
Add inducer IPTG
The only way the gene can be expressed is IPTG
The repressor protein binds to IPTG. Repression protein has a greater affinity to bind to IPTG than the operator.
The combination of IPTG and repressor protein creates a conformational change, and cannot bind to the operator.
Where to where does the vector translate
Rbs on mRNA, then start to stop codon
What is on one end and the other end after translation? What will it give>
one end has amino group, other end has cooh like a protein structure
A functional Cr enzyme
What is the relationship between the plasmid copy number and expression?
The more plasmids the more expression
Does e.coli always recognize the plasmid transferred?
No
What happens if E.coli rejects the recombinant DNA molecule?
When the E.colIi divides, the plasmid can be excluded and only the chromosomal DNA will be passed on
What is needed for the plasmid to be passed on? What happens if it is not?
Need a stable plasmid
If the plasmid is not passed on, the antibiotic resistance is not passed on, and colonies will die
The plasmid will be divided only if there is selection pressure such as growing the cells on a plate with antibiotic..
Without antibiotic, no selection pressure, no carry over of plasmid DNA
What is the promoter is the vector? What is its origin?
T7 promoter has a viral origin
What is the host promoter? Where does it come from and what is its name?
Lac promoter is from bacteria
Ecoli: LacUV5 promoter (weak promoter)
How big is LacU5? What are the promoter regions? What are the sequences at the -10 and -35? Where is the promoter located? What is the structure?
40 base pair sequence (promoter length)
Region -10 BP and -35 BP, counted from the left of the start codon.
The regions are necessary for promoter activity.
-10 (5’TATAAT) is called Pribnow Box
-35 (5’TTGACA) is called
The promoter is present upstream/ left of the start codon
5’ TTGACA (-35) - - - - - TATAAT (-10) - ATG (+1)
How were the regions is LacUV5 decided? What happens if tehre is a mutation in the region?
These regions were decided because they are the most conserved. Must have these regions.
If there is a mutation in these regions (10bp or -35bp, the promoter strength will be affected)
Mutations will make the promoter unusable
What kind of promoter is T7? What does that result in? What is its origin? What are the promoter regions? What areas of mutation kills promoter activity?
Phage origin
Will have more mRNA transcripts
Highly conserved -23bp region
Bases necessary for promoter activity, are -7 & -11, within the 23bp region
Mutations at -7, -8, -9, -11 kills the promoter activity, within the 23bp region
Why are the lacUV5 and T7 promoters different sizes?
The reason for differences between weak and stronger promoter is due to the RNA polymerase size
A bigger space between bp is necessary for binding of larger enzymes
What is the structure of T7 RNA polymerase? What is its size?
Monomer (1 polypeptide)
Size 99kDa
Why do problems in expression occur in translationin the current experiment?
Problem occurs due to T7 promoter on pet29b.
E.coli RNA polymerase cannot be recognized the T7 promoter
T7 RNA polymerase cannot be recognized by the Lac Uv5 promoter
The promoters are very specific.
What is the procedure for translation for overexpression?
Grow colonies on plates (LB Agar + Kan)
Transfer colony to liquid medium (LB Agar + Kan) to grow
Grow colony overnight
Take 250 ml of liquid culture and add to 25ml Lb + kan and grow for 2-3hrs
After growing, add IPTG to the 25ml solution and decrease temperature to 22C and grow
after growing, harvest the cells and store at -20 deg
What type of combination is the host cell we are using? What is it called?
Bl21De3, a E.coli and virus combination
What should a host cell be?
Compatible for the specific vector
What wavelength is bacterial growth measured at?
600nm
What should the concentration be around when IPTG is added?
0.6-0.8
What does a bacterial growth curve look like? What are the axes?
Y-axis : Growth, X axis: Time
What are the phases of bacterial? What should be taken at each process? At what OD is bacteria growing and what does it mean?
Starts at lag phase, then log phase, and then stationary phase at flattening of curve
Take OD at each process When the OD is 0.6-0.8 the bacteria is growing.
Means the culture is active and ideal for protein growth
What enzyme inactivates the promoter?
introduce T7 lysozyme. T7 lysozyme has a high affinity to the T7 RNA polymerase.
Where does the enzyme inactivating the promoter come from? How can it be done? Do we need to to do this?
T7 lysozyme is produced by the T7 lysozyme gene.
Introduce the plasmid using BL21De3PLS S/E host rather than BL21De3
No because the CR protein is not toxic to the E.coli if it is need to use BL21De3PLS S/E.
What happens in transformation for overexpression after IPTG is added? What is the entire system called?
When IPTG is added, the IPTG binds to the repressor protein resulting in a conformational change of the repressor protein, and makes the operator site free from the repressor.
The T7 gene is transcribed into mRNA to be translated into T7 RNA polymerase
The T7 RNA polymerase will bind to the PET29bCR at the T7 promoter
IPTG is already bound to the repressor protein PET29b so the operator is active
Transcription will occur resulting in mRNA resulting in the CT protein
Entire system is called the PET system (Host(BL21de3) + Vector(Pet29bCR)
What are the steps of subcloning. Name the steps and experiment.
Restriction digestion (in silico)
Double digestion
Sub cloning PCR
PCR purification
Gel electrophoresis
Transformation
Colony PCR
gel electrophoresis
Isolation
Sequencing
2000bp DNA (double stranded), polymerase can only extend 500bp. How many bands in a gel and what size?
Principle is DNA synthesize. DNA polymerase extends the primer. One band. 520bp (primer and template)
Why is PCR an important technique? Name the steps and principles behind it?
Good technique to amplify DNA. Denaturing: Break down double stranded to single strand, annealing: Primers anneal to the strand, and extension: polymerase extends the primer.
What is TM?
A: melting temperature is the temperature the primer will separate from the template. Important to know the melting temperature to determine the Annealing temperature. Should be within 5 degrees C from the annealing temperature.
What information do you get from running an agarose gel? What property allows it to run through the gel
Size of the DNA. Topology of the DNA. Negative charge of the DNA.
Primer design with restriction sites.
Design the primer and add the restriction sites.
What is alpha complementation? Why is it important for selection? Why do you use XGAL?
Alpha complementation is a when an alpha peptide and omega peptide come together to create a functional beta galactosidase. It is important for selection in sub cloning. XGAL is used to give blue color.
What is the natural substrate of beta galactosidase?
Lactose
What is the biochemical function of carbonyl reductase?
Oxidative reaction
Draw dNTP and ddNTP? Explain basic principle of chain termination?
Using a ddNTP, no phosphodiester bond will form and the extension will terminate and no synthesis will occur.
Calculate the amount needed to make 20ml or 25mM glucose?
Answer: .09g
What is gene cloning? What are the strategies? What are the steps in order?
Making identical copies of a gene. Strategies are PCR based and Library based. Steps are: Isolation, Insertion, Amplification, Selection, and Confirmation.
The DNA concentration in your plasmid is 400ng/ul. You have 50ul. An enzyme is 10U. How much enzyme do you need?
1 unit digests 1ug per 1hr
2 U
Pet29b is a translation vector and there is a frameshift at BamH site
True
An ideal vector should have a low molecular weight and a MCS
True
ddNTP lacks a OH in the 3’ position
True
orientation of expression vector (control elements)
Goal of colony PCR
to identify the recombinant DNA molecule
If you have: If you have DNA polymerase, dNTP, and template DNA, what is needed to conduct a PCR
Need primer, sequence of nucloetides 18-20bp.
What kind of overhangs does PGEMT have?
T overhangs
Define ORF.
sequence of nucleotides transcribed into mRNA
How do restriction enzymes make it possible to make a recombinant DNA molecules?
Recombinant DNA molecule means you are joining two pieces of DNA from two different sources. Restriction enzymes make this possible because you can cut different sources with the same restriction enzyme if they have the same restriction sites, and join them together. Restriction enzymes cut nucleotide as specific sequences so the cutting can be specific.
What are the two types of expression vectors?
A: translation and transcription. Major difference is rbs in translation.
