7: Cloning Genes for Synthetic biology Flashcards
Cloning vector
Any molecule of DNA that can replicate itself within a cell and is used for carrying cloned genes or segments of DNA. Usually a small multicopy plasmid or a modified virus.
Chimera
Hybrid molecule of DNA that has DNA from more than one source or organism.
Synthetic biology
Discipline of biology that uses genetic engineering to redesign existing microorganisms for a new use, such as creating bacteria or algae that can make fuels or bacteria that can degrade toxic chemicals.
Properties of cloning vectors
The vector should be:
- Reasonably small and manageable
- Easy to move between organisms
- Easy to purify and generate large amounts.
Many vectors are designed to provide a means to perform:
- Mechanism to select hosts containing the vector.
- Ability to insert genes into the vector.
- Detect the presence of an inserted gene in the vector.
Plasmid
Extrachromosomal circular piece of of DNA that are found in various bacteria, archaea, and even some eukaryotes.
Often used as cloning vectors.
ColE1 plasmid of E.coli most common used.
- Remove E1 gene (encoding toxins that kill surrounding bacteria)
- Add antibiotic resistance gene (often ampicilin)
Elements of the plasmid:
Origin or replication
Origin or replication:
- DNA seq that are essential for the bacteria to make copies of the plasmid.
- Determines the number of copies of plasmids that are present in a cell and when the plasmids are replicated (anytime or during cell division).
- Important to choose plasmids with different origins in experiments, so that they aren’t rejected.
Elements of the plasmid:
Promoters
- Highly variable, as each host organism has different structural requirements in order to recognize it, and because it allows the researcher to control the expression of the GOI.
- Inducible: turns on a gene under specific conditions.
- Repressible: Turns off the expression of its adjacent gene under specific conditions, such as, in the presence of a small molecule or at a certain temperature.
Elements of the plasmid:
Terminator/PolyA sequences
- Positioned after the multiple cloning site (MCS).
- Majority of terminators rho-independent.
Series of A’s followed by a region that forms a stable hairpin structure. - Read-through: When RNA pol does not stop transcription at the terminator and continues to transcribe the DNA downstream of the gene.
Can be reduced by having two or more terminators in tandem. - PolyA-tail: series of A’s that are found on the 3’ end of a mRNA transcript that stabilizes the mRNA in the cell.
Adding inserts to a vector:
Restriction enzyme cloning
Cuts DNA fragment of interest and plasmid with the same restriction enzyme.
DNA ligase links the parts covalently together.
Blunt ends more difficult to ligate => often use T4 ligase as it can ligate blunt ends.
Most vectors have multiple cloning sites (MCSs)/polylinkers; length of DNA that contains several restriction enzyme sites in tandem.
Cut is specific for the MCSs.
Adding inserts to a vector:
TA cloning of Polymerase Chain Reaction (PCR) products
Uses Taq polymerase to generate single A overhangs on the ends of DNA segments that are used to clone DNA into a vector with matching T overhangs.
PCR amplified DNA inserts that are made with Taq polymerase have a single A overhang (extention) onto the 3’ end of each strand that can be cloned into a TA vector that has a single T overhang.
Don’t need to purify and cut the complementary vector.
Adding inserts to a vector:
Recombineering
Insert specific pieces of DNA into a vector or artificial chromosome by homologous recombination.
The red protein from bacteriophage lambda recognizes the ends of the insert with exact homology to the insertion site on the vector and recombines the DNA insert with the vector to make the two pieces one.
Advantages:
- Red protein only need small region of homology (45 bp)
- Quick and easy laboratory procedure.
- Recognizes short ss oligonucleotides and longer DNA fragments => can be used to create small mutations
Adding inserts to a vector:
Recombineering
Insert specific pieces of DNA into a vector or artificial chromosome by homologous recombination.
The red protein from bacteriophage lambda recognizes the ends of the insert with exact homology to the insertion site on the vector and recombines the DNA insert with the vector to make the two pieces one.
Advantages:
- Red protein only need small region of homology (45 bp)
- Quick and easy laboratory procedure.
- Recognizes short ss oligonucleotides and longer DNA fragments => can be used to create small mutations
Adding inserts to a vector:
Isothermal / Gibson DNA assembly
Uses 3 enzymes (exonuclease, DNA polymerase, and DNA ligase) to combine an insert and vector into one circular DNA.
Can be done on multiple fragments provided that the ends of every piece have overlapping sequence with the next.
Adding inserts to a vector:
Gateway cloning
Method of combining an insert and vector that employs lambda int and xis proteins and their recognition DNA sequences (attP, attB, attL and attR).
Entry vectors are used to add attL sites onto the insert/gene of interest.
In the LR reaction, lambda enzymes xis and int recognize the attL sites and induce recombination with a destination vector that has the attR sites, which transfers the GOI into another vector.
LR reversible with the BP reaction.
Transformation / transfection
Uptake of external DNA in bacteria and eukaryotes, respectively.
Transformation:
Procedure that uses calcium ions and temperature changes to get a plasmid DNA into the cytoplasm without killing the bacterium.
