V. Lab | 79. Analysis and generation of a recombinant DNA Flashcards
I. BACKGROUND INFORMATION
1. What is the flow of genetic information?
I. BACKGROUND INFORMATION
2. What is the aim of “ Analysis and generation of a recombinant DNA construct” lab?
Aim of this lab is to demonstrate the steps of cell-free protein synthesis (in vitro).
- First, a recombinant vector encoding the GFP will be generated by molecular cloning.
- Secondly, the gene will be transcribed into the corresponding mRNA using the RNA polymerase of a bacteriophage under in vitro circumstances.
- Finally, the mRNA will be translated using a wheat germ based in vitro translation system, and the GFP produced here will be detected by induced light emission.
I. BACKGROUND INFORMATION
2. What is Recombinant DNA?
Recombinant DNA = insert DNA + vector DNA
=> make another cell produce the protein
II. Molecular cloning
1. What are the 5 steps of molecular cloning?
1) Find appropriate vector
2) Prepare insert -> code for GFP (green fluorescent protein)
3) Ligation: restriction endonuclease used to make their ends compatible
4) Transformation: put recombinant DNA into a bacterial cell
5) Colony screening
II. Molecular cloning - Vectors
2. What are Vectors of molecular cloning?
- The majority of vectors used today are modified versions of plasmids (naturally occurring bacterial, extrachromosomal, self-replicating, circular, double-stranded DNA molecules).
- Available vectors are usually used for recombinant protein production (expression of vectors)
=> production of GFP.
II. Molecular cloning - Vectors
3. What is the expression vector?
An expression vector will be implemented that harbors regulatory elements essential to drive transcription of the protein-coding insert in the corresponding express system of eukaryotic origin.
II. Molecular cloning
4A. What are the 4 properties of expression vector?
- Origin of replication (ORI)
- Promoter sequence (SP6)
- Multiple cloning site (MCS)
- Selection marker (ampR)
II. Molecular cloning - Properties of the expression vector
4B. Why is Origin of replication (ORI) a property of the expression vector?
Origin of replication (ORI): ensures the replication of the vector in the host cell
II. Molecular cloning - Properties of the expression vector
4C. Why is Promoter sequence (SP6) a property of the expression vector?
Promoter sequence (SP6): site of assembly of the pre-initiation complex that
contains the RNA polymerase
=> operator region
II. Molecular cloning - Properties of the expression vector
4D. Why is Multiple cloning site (MCS) a property of the expression vector?
Multiple cloning site (MCS): contains restriction enzyme recognition sequences to help open the plasmid
II. Molecular cloning - Properties of the expression vector
4E. Why is Selection marker (ampR) a property of the expression vector?
Selection marker (ampR): selection marker gene that allows bacteria to be resistant to antibiotics. Ampicillin (beta-lactam antibiotics -> destroy translation machine)
II. Molecular cloning - Insert
5. How does “insert preparation” step work?
- The sequence of interest to be inserted into the vector, insert (gene), codes for the protein of interest = GFP.
- The insert is usually amplified using PCR from cDNA (copy DNA) / gDNA (genomic DNA). Primers are designed to have restriction endonuclease recognition sites in their sequences + start/stop codons.
- It is cleaved using the same restriction endonucleases as for the vector.
=> Primers: amplify gene of interest
II. Molecular cloning -
Restriction digestion and ligation
6A. How does “Restriction digestion and ligation” step work?
Restriction endonucleases recognize short, 4-8 nucleotide long palindromic sequences, cleave both strands which results in the formation of a free 3’ OH and a 5’ OH group.
II. Molecular cloning -
Restriction digestion and ligation
6B. What is the role of Ligases?
Ligases join the ends of DNA fragment together by the formation of phosphodiester bond between the 3’- hydroxyl of one DNA terminus with the 5’- phosphoryl of another (this reaction requires ATP).
II. Molecular cloning -
Transformation
7A. What is Transformation?
Transformation is the uptake of recombinant DNA vectors into competent bacterial cells.
II. Molecular cloning -
Transformation
7B. What is Horizontal (lateral) gene transfer?
plasmids taken up by the cell get incorporated into the genomic DNA of the bacteria
II. Molecular cloning -
Transformation
7C. What are Competent cells?
Chemically competent + electrocompetent cells take up DNA
II. Molecular cloning -
Transformation
7D. What are Artificial competent cells?
Artificial competent cells are cells that are treated by chemicals (CaCl2) in order to take up the DNA from their environment.
- The mechanism is that DNA enters the bacteria through tiny pores opening up in the outer membrane of the cells. cells.
II. Molecular cloning -
Transformation
7C. How does “Transformation” step work in molecular cloning?
- Treatment of bacterial cells with CaCl2 will eliminate the repulsive forces between the DNA and the bilayer
-> recombinant DNA gets closer to cell membrane. Cells are heated during heat shock
-> permeabilization of cell membrane and formation of pores within the lipid membrane
-> cells can take up plasmids. - Next, the cells will be cooled down abruptly by placing them on ice to allow the bacterial membrane to regenerate.
II. Molecular cloning - Selection of clones
8A. What is the purpose of Selection of clones in molecular cloning?
To check if the vector constructs, as the insert may not have been inserted in the correct orientation or the vector can close on its own (= empty vector)
II. Molecular cloning - Selection of clones
8B. What is Colony PCR?
- Colony PCR: bacterial cells are lysed and a portion of their DNA content is amplified with insert- or vector-specific primers.
- The amplified DNA segments are visualized by agarose gel electrophoresis
II. Molecular cloning - Selection of clones
8C. What is Positive selection?
- Positive selection vectors comprise a gene, the product of which is lethal to bacterial cells, such as ccdB (a potent inhibitor of the DNA gyrase).
- Successful insertion of the insert results in disruption of the reading frame of the toxic gene, making the recombinant clone workable
II. Molecular cloning - Selection of clones
8D. What is Negative selection?
- A genetically modified E.coli strain lacking a part of the beta-galactosidase (lac operon) coding gene is used here.
+) The beta-galactosidase is the missing subunit expressed using the cloning vector - Beta-galactosidase activity is only present in cells containing empty vectors.
+) This activity can be followed up using the X-gal dye, an artificial lactose analogue, which yields a blue product upon enzymatic hydrolysis.
+) Insert containing colonies appear colorless on the agar plate
II. Molecular cloning - Selection of clones
8D. What is X-gal dye?
It is an artificial lactose analogue, which yields a blue product upon enzymatic hydrolysis
