Midterm 6

Question 1

What are the control elements of an expression vector?

Answer 1

Promoter and operator

Question 2

Promoter

Answer 2

Transcription starts here
RNA polymerase binds here

Question 3

Rbs - Ribosome binding site

Answer 3

Protein synthesis starts here
Used in translation

Question 4

Cleavage site

Answer 4

used to remove the ORF
Thrombin in the pet 29bvector

Question 5

MCS - Multiple cloning site

Answer 5

Contains restriction sites
Can place the ORF there

Question 6

STOP - Stop codon

Answer 6

for translation

Question 7

STP

Answer 7

Stop for transcription
terminator

Question 8

How is the ORF protein removed?

Answer 8

During purification the cleavage sites are used to remove the ORF protein
Cleaving is done after transcription.
Do not use this process in the present experiment due to cutting of the thrombin site.

Question 9

What is produced from the mRNA from expression vector?

Answer 9

Primary structure for protein

Question 10

What is larger, cloning or subcloning vector? Why?

Answer 10

Cloning vector is smaller than vector
this allows for quicker replication
Would need twice the amount of metabolic drain and time to clone an expression vector

Question 11

How large is the pet29b vector?

Answer 11

5.8kb/ 5800

Question 12

What direction does proofreading go in?

Answer 12

Proofreading goes in 3’ to 5’ direction

Question 13

What does proofreading activity do?

Answer 13

prevent and correct any errors

Question 14

Isolation (subcloning)

Answer 14

Objective: Join together insert and expression vector
Chemistry: Double digestion + ligation
Enzymes: Restriction Enzymes and Ligase
Experiment: PCR and double digestion
PCR: Use forward and reverse primers with restriction sites matching through restriction sites at the correct orientation of the vector, are not within the insert of the gene of interest (done through insilico), and cut sticky ends. The primers anneal creating restriction sites at the 5’ and 3’ end of the insert
Double digestion: Use two different enzymes to cut the vector and the insert. We use two different enzymes to prevent self ligation of the vector and to ensure the insert is placed in the correct orientation to promote directional cloning. The vector is cut to linearize it and the insert is cut at the restriction sites to produce sites that can bind to the vector. Both are cut with the same enzyme and create sticky ends.

Question 15

Insertion – Ligation

Answer 15

Ligation: Place the insert and the vector in a tube along with T4 DNA ligase. The goal is for the insert and the vector to join together.
Insert has stick ends with restriction sites corresponding to restriction sites in vector in the 5’ 3’ direction, cut by the same restriction sites.
We use T4 DNA ligase coming from T4 bacteriophage
Makes the phosphodiester bond- synthesis reaction
Need ATP, in the buffer, is in a buffer because all other processes occur in a buffer

Question 16

Amplification (subcloning)

Answer 16

Transformation

Question 17

Selection (subcloning)

Answer 17

Colony PCR
- Scrape colony off transformed plate
Number the colonies on a empty plate
- All reagents in PCR tube.
- Number tubes corresponding to the colonies on the plate
- Use forward and reverse primer corresponding to restriction sites on the insert so they only anneal to the insert.
- Pick up a colony and touch it to one of the numbered spaces on the empty plate and incubate the plate.
- Put colony in tube with PCR products and run PCR in thermocycler
- Then use the PCR product and run an agarose gel, one well for each tube.
- Wells with bands are the recombinant DNA
- The primers use will only bind to the recombinant DNA, not the self ligated
- Done in LB Agar + Kandimycin plate
- Might have self ligated vector

Question 18

Confirmation (subcloning)

Answer 18

Sequencing

Question 19

Components of Expression Vector

Answer 19

Promoter
Lac I gene
Control elements: Promoter and operator
RBS
TAG
Cleavage
MCS / Polylinker
Cleavage
TAG
his tag
STOP CODON
Stop for transcription (terminator)

Question 20

Blue white screening. Objective, enzymes, substrates, and chemistry.

Answer 20

Objective: Recombinant colony screening.
Chemistry/ mechanisms: Enzyme reaction
Enzyme: Beta galactosidase
Substrate: XGAL
Reaction: XGAL - Beta galactosidase - Galactose + Blue color
IPTG: Inducer for Lac Z gene to produce beta galactosidase
No blue colonies if IPTG is not added
Lac Z is the gene producing Beta galactosidase
Need IPTG because Lac Z is not constitutively expressed

Question 21

Subcloning Ligation: Objective, chemistry, enzymes, experiments.

Answer 21

Objective: Join together insert and expression vector
Chemistry: Double digestion + ligation
Enzymes: Restriction Enzymes and Ligase
Experiment: PCR and double digestion
PCR: Use forward and reverse primers with restriction sites matching through restriction sites at the correct orientation of the vector, are not within the insert of the gene of interest (done through insilico), and cut sticky ends. The primers anneal creating restriction sites at the 5’ and 3’ end of the insert
Double digestion: Use two different enzymes to cut the vector and the insert. We use two different enzymes to prevent self ligation of the vector and to ensure the insert is placed in the correct orientation to promote directional cloning. The vector is cut to linearize it and the insert is cut at the restriction sites to produce sites that can bind to the vector. Both are cut with the same enzyme and create sticky ends.
Ligation: Place the insert and the vector in a tube along with T4 DNA ligase. The goal is for the insert and the vector to join together.

Question 22

Practice dilution question. What is the default concentration of a working solution? When is the formula used? Want to make 1X solution in 50ul.

Answer 22

Used when you are taking from a stock solution
a solution with a concentration
The default working concentration is always 1X unless specified

Question 23

Polymerases in cloning and subcloning

Answer 23

Cloning: Taq Polymerase (no proofreading)
Subcloning: pfu taq polymerase (proofreading)
High fidelity - copies the strand with good accuracy
High processivity - once replication starts, the polymerase will continue replication without falling off until the end. Completes polymerization without falling off until completion. Results in no small fragments.

Question 24

Define DNA ligase

Answer 24

To join two fragments of DNA by creating a phosphodiester bond.

Question 25

Define DNA polymerase

Answer 25

It polymerization strands of DNA by synthesizing nucleotides complementary to a template strand.

Question 26

Define high copy number and state what is is dependent on

Answer 26

Definition: The bacteria has copies and the plasmid can replicate very fast due to underlying mechanism
dependent on the ORI

Question 27

What are the controls in transformation?

Answer 27

Positive control: No ligation, no insert
Negative control: ligation and insert

Question 28

Difference between cloning and subcloning primer.

Answer 28

FP has an ATG (start codon) in subcloning. This is because the goal of the insert is to be expressed into a protein and therefore needs a start codon to go through translation.
Primers in subcloning are designed in accordance with restriction sites on your expression vector