Midterm 3 Flashcards
What host organism has the greatest variety of cloning vectors?
E.coli
What are the simplest cloning vectors based on?
small bacterial plasmids
What are the 3 impotant qualities of a good vector?
less than 10kb in size, to avoid problems such as DNA breakdown during purification.
Carry sets of antibiotic resistance genes, to be used as selectable markers
High copy number.
What was the first cloning vector?
PBR322
What type of polymerase is needed for transcription?
RNA polymerase
What are the two common promoters for RNA polymerase?
T7 and SP6
What are the two groups of plasmid?
Conjugative: Can promote sexual exchange of genetic material, from one cell to another.
Define copy number
number of molecules of an individual plasmid that are normally found in a single bacterial cell.
What does centrifugation in purification do?
Remove cell debris.
What do you need to be careful about when centrifuging in purification?
To not break the DNA fragment
How is controlled lysis done?
Gently with treatment with EDTA and lysozyme with sucrose, preventing cells from bursting immediately.
Why is the cloning technique used called TA cloning?
The fragment contains A ends obtained from Taq polymerase while the PGEMT easy vector has T overhangs
In the gene obtained by PCR usable? Should it be called a gene?
It is not, a vector needs to be used. No because it does not have a promoter
Does Taq polymerase have proofreading activity? What does this mean and what is the result?
It means it cannot identify errors and the result is fragments have overhangs
What is a recombinant DNA molecule? Name the vector we use in this class when describing it. What process is it made through?
A recombinand DNA molecule is the insert and the vector, in this case the PGEMT easy vector. The process it is obtained through is ligation.
What are the 3 important parts in a cloning vector? Name an define them.
Origin of Replication (ORI). Allows for independent replication of the vector
Multiple cloning site (MCS) some call it a polylinker. Have unique restriction enzyme sites not present in any other place on the vector so cloning only occurs at that site. Unique presence of recognition of many restriction enzymes. The restriction sites are not present anywhere else on the vector. If it were present on multiple sites, the vector would be cut in multiple places rendering it useless since it will be separated into multiple fragments.
Selectable marker: genes for antibiotic resistance (we will be using AMP). Helps select the bacterial colony for growing. Presence of colony in an agar plate means success. The ampicillin help gets rid of the bacteria not containing ampicillin resistance indicating they do not have the recombinant DNA.
What are the 4 possibilities of ligation? What is the ideal?
Ideal hypothesis: Produce recombinant DNA molecule with insert and vector ligated (will grow in transformation)
Self ligated vector: The T makes two hydrogen bonds with another T. (unfavorable) (will grow in transformation)
Self ligated insert: The pcr product ligates to itself
Unligated vector and insert
What technique is used fro amplification?
Transformation
What cells are used in transformation?
bacterial competent cells
Define transformation
Exploit the natural tendency of bacteria to pick up foreign DNA.
What do bacteria do in transformation and why?
pick up DNA from their surroundings and replicate it because it allows for survival through production of proteins for antibiotic restistance.
Why is heat shock done in transformation?
increase the fluidity and permeability of the membrane
Why is ice shock done in transformation?
to constrict the membrane
What is done after ice shock in transformation? Why?
Give LB to competent cells and incubate at 37C with light shaking (aeration) to allow them to incubate and grow to assist them in surviving in LB + Agar + Antibiotic enviornment
What is plating done on in transformation? What are the components and what are they used for? Include b/w screening ccomponents
Plating (solid medium) on LB Agar + Ampicillin + XGAL + IPTG for blue white screening and selection. Agar is used because bacteria can grow without breaking the plate down.
Define self ligation
ends of the vector join together,
Why is self ligation a problem?
Makes it difficult to differentiate between bacteria with recombinant and non-recombinant colonies.
What does a recombinant colony contain? What does a non-recombinant colony contain?
Recombinant colony: bacteria containing the vector and the insert
Non recombinant colony: circular vector.
How is the issue created by self ligation resolved?
selective markers called LACZ’.
What is lac z’, what does it produce?
It is a part of the lac z gene in the lac operon, producing beta galactosidase which breaks down lactose to glucose and galactose.
What happens if XGAL and IPTG are not added?
Selection will not be done
Besides blue white screening, what can be done in selection? What are conditions of this method?
Can extract DNA from two different colonies and conduct two separate PCRs with primers for the gene of interest. Only the recombinant molecule will be amplified because primers can only bind to those vectors containing the gene of interest.
Can only be done with a few colonies
Conduct a miniprep to isolate the plasmid.
Define the selection step in cloning. How did we do it in this class?
Choosing the bacteria with the recombinant vector.
Blue white screening
What kind of host cells and vector need to be used in blue white screening?
cells compatible with blue white screening.
What will the plate in blue white screening look like? What color colonies are we interested in?
Blue white colonines; white
Why are we interested in only white colonies?
Because they have the recombinant DNA. They have no digested XGAL due to a insertional activation, breaking of the LacZ’ gene due to the insert, and therefore do not digest and subsequently release a blue color.
What are the blue colonies in the blue white screening plate?
They are colonies with the self ligated vector. We know this because the blue color indicates digestion of XGAL, which releases blue when digested. If this is digested, it means the cell has a vector with the LacZ’ intact and can produce beta galactosidase, through alpha complementation, to breakdown the lactose into glucose and galactose. Due to the presence of ampicillin, only those with anitbiotic resistance can grow namely the recombinant and self ligated vector since they contain the selectable marker. The self ligated molecule containg the selectable marker and intact lacz’ and therefore is able to survive and will turn blue when XGAL is digested.
How many AA sequences and nucleotides is Lac Z’
50AA, 150 nucleotides
What does antibiotic selection allow for?
the production of only cells in colonies with the self ligated vector and the recombinant DNA molecule.
What does soaking in cacl2 do to host cells?
soaking in CaCl2 allows the DNA to bind to the cell exterior.
Why is plating done?
To make it easier for selection and visualization. Also because due to limitations of space within the tube, oxygen and nutrients may become limited and bacteria may start to die.
Define transformation
The introduction of recombinant DNA molecules into host organisms
How is the DNA molecule added to host cells?
Bacterial cell membranes are disrupted to add DNA molecules.
Why are the cells called competent?
They are able to take up added DNA
Define electroporation
treat cells with an electrical current so DNA can enter the cell during a very brief period.
Define in vitro packaging
Introduce DNA into bacterial cells through phage particles made from extracts of phage infected cells.
Define gene guns
used to deliver DNA into cells as particles of heavy metals coated in DNA. Commonly used in plant cells
Define transgenic animals
animals and plants are permanently altered by inserting a foreign gene.
Define screening
process where cells carrying a trait can be distinguished from cell who do not.
Where are antibiotic resistance genes commonly obtained from?
transposable DNA elements known as transposons.
Define transposons
mobile segments of DNA, moving to different chromosomal or genomic locations
Define insertional inactivation
The inactivation of genes by insertion of DNA fragments.
how is alpha activity restored?
add the missing alpha fragment to restore activity to LacZ15 gene on a vector.
How does the insert affect alpha complementation?
Target DNA insertion renders the alpha complementation site inactive, therefore inactivating the lac Z gene. The lac Z gene will not be able to produce beta galactosidase, and therefore not be able to process lactose.
When does XGAL turn blue?
When it is cleaved by beta galactosidase, producing blue.
What does CaCl2 do to the competent cells
The calcium chloride treatments makes micro holes to increase permeability of the cell membrane of the bacterium. This increases the efficiency for picking up DNA. Allows for more uptake of DNA.
What temperature is heat shock during transformation done at? How long is it done?
about 42 C for 45 seconds
What does incubation during transformation allow?
allows for growth independent of ampicillin to increase chances of survival on the plate
What is produced to help break down ampicillin?
beta lactamase
What gene allows for the production of beta lacatamase?
AmpR; Constitutive
Define constitutive expression.
Expression done naturally. Always expressed without stimulation
Inducible expression: Expression done by stimulation
What is the size of beta galatosidase in AA, bp, and kb
1023 AA long, 3069 BP around 3kb
What type of gene is Lac Z
Tetramere
True or false, all E.coli possesses the Lac Z gene
True, they are all lac Z positive
How many fragments does beta galactosidase have>
Two
How big and where is the alpha peptide on the Lac Z gene? Say the same for the omega? What do the two coming together do?
alpha peptide. It is the first 50AA from the N-terminal.
omega peptide. 50AA from the N terminal to 1023 bp
A functional beta galactosidase should have an alpha peptide and an omega peptide.
What happens in a mutant Lac Z gene? Waht is the strain called?
only code from an omega peptide (no functional alpha)
Beta galactosidase is non function
Lac Z delta 15
How can you tetramarize a mutatnt Lac Z?
Can provide alpha peptide to tertramarize the gene and obtain a functional beta galactosidase.
How big is the PGEMT easy vector?
3kb
Why does the LacZ in the vector have a “ ‘ “
It is a portion of the Lac Z gene containing the first 150bp of the gene, only the alpha peptide. This is because the full lac Z gene is about 3kb.
What happens when the insert is placed into the vector to the peptide?
The alpha peptide is rendered non functional and therefore will not complement with the omega peptide to create a functional Lac Z gene and generate beta galactosidase.
Why is a self ligated vector able to produce a functional beta galactosidase?
The self ligated vector has an intact Lac Z’ resulting in a alpha peptide. The alpha peptide can complement with the omega peptide in the host to create a functional Lac Z’ and generate beta galactosidase.
What does XGAL break down into
Galactose and a blue color. It is a substrate of lactose
What must happen before the blue color of XGAL appears?
Oxidation
What cells will break down XGAL. Why?
Cells with a functional beta galactosidase will break down XGAL and turn a blue color. Self ligated vector.
What cells will not break down XGAL. Why?
Cells with recombinant molecules because they do not have functional beta galactosidase due to insertional inactivation and will not turn blue. Therefore they will be white. We are interested in white colonies.
What is the role of IPTG?
IPTG induces the gene expression to overcome the metabolic burden of not having glucose in the environment. XGAL is not enough to induce expression.
Why is there a metabolic burden associated with producing beta galactosidase?
In bacteria, the primary source of food is glucose. In an environment where lactose is present and glucose is not, beta galactosidase needs to be produced. It is a non constitutive expression meaning it is not always being produced. Therefore there is an energy loss due to producing something that is not always necessary.
What kind of sequencing was confirmation done through?
Sanger sequencing.
Who was sanger sequencing developed by> How many Noble prizes did they have?
Fredrick Sanger
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Define Lac Operon
Group of genes producing enzymes to digest lactose
Define Operon. Why are they in bacteria?
Operon: multiple genes working for a final product with one promoter
In bacteria due to limited genome
What is the role of Lac I
Lac I switches the Lac operon off by producing a repressor protein, later binding to the operator by preventing transcription by RNA polymerase
What happens to RNA polymerase if a repressor protein is present?
RNA polymerase is bound to the promoter, but cannot move forward if the repressor protein is present
What acts as an inducer in E.coli?
allolactose
How does allolactose work as an inducer?
It binds to the repressor protein resulting in change of confirmation of the repressor protein, changing it. The change leads to it “falling off”. The promoter can therefore work and transcription occurs, then translation leading to beta galactosidase.
