Research Methods 2.4 Transformation

transformation

introducing the plasmid into living bacteria cells so that it can be replicated

competent cells

bacterial cells (E.coli HB101 - no resistance regular form) can be made competent by specific preparation methods such as using bacteria in the exponential growth phase, cold wash with gentle handling, and resuspension in CaCl2 solution

heat shock

plasmid DNA and heat shock competent cells in CaCl2 are mixed and incubated, mix plasmid with competent cells on ice, CaCl2 allows DNA to bind oto cell membrane, heat shocking creates pores in the membrane so bacteria can suck in DNA, and there is 1 hour recovery before plating

alternate heat shock method

heat shock is done directly on warm agar plate, mix plasmid with competent cells on ice, and spread directly onto warm plate - bacteria are in the log (exponential growth) phase in this method

CaCl2

causes DNA to bind to the bacterial cell wall, the cells are then subjected to brief heat shock by incubation at 42C for 30-45 seconds of 37C for 5 minutes, resulting in the uptake of DNA into the bacteria. cells must be in exponential growth phase to be highly competent. CaCl2 helps DNA bind to membrane for convenient entry

electroporation

when bacterial cells are subjected to brief electrical shock, small pores open in their cell wall allowing DNA to enter cells

what kind of bacteria can be used for transformation

since bacteria have defense mechanisms that use RE to degrade foreign DNA, only mutant strains that not longer have restriction activity can be used for transformation

what happens after transformation

cells are incubated in nutrient medium for up 1 hour to allow them to recover from the stress of transformation and bein to express the genes on the plasmid. cells are then plated on LB and AMP plates and only bactria that have been sucessfully transformed/carry the plasmid will be able to survive and divide on the plates. plasmid will replicate in the bacterial cells (using host cell machinery) and as bacteria divide, plasmids will be passed on.

why is IPTG added

to increase expression of ampr gene which is under control of lac operon and increases transformation efficiency

insertional inactivation

helps identify bacteria transformed with plasmids containing an insert versus plasmids that have religated without insert. plasmids are designed to have the cloning site for the insert lie between a promoter and a specific gene. if a fragment is inserted in between promoter and gene, the insert will prevent proper transcription of the gene. some property of the gene is then used to identify the bacteria in which the gene product is no longer made.

eco471

enzyme is lethal to the bacteria, so if its gene is not disrupted by an insert then the plasmid should express the gene and any bacteria contain religated plasmid should die. thus, there should be no growth of bacteria contain vector, only self ligated plasmids.

what does pJET enable

positive selection of plasmids with inserts due to disruption of the eco47IR allowing growth of successful transformants

pGAP plasmid

positive control to ensure cells are competent and healthy and transformation works

how is heat shock performed in this lab

by plating bacteria directly from uce onto warm agar plates at 37C.

what kind of bacteria will grow on LB amp plates

only bacteria expressing AMPr gene

explain the experimental samples

the ligation product which is the pJET and PCR product

explain positive control 1

no plasmid, growth on LB plate to test if competent cells are alive and healthy. this is just the E.coli without the pJET and PCR product to see if the E.coli cells are healthy and alive.

explain positive control 2

pGAP plasmid on an LB/amp/IPTG plate for growth to test if cells are competent. pGAP is known to have the ampr gene, so if the E.coli are competent then the pGAP ampr gene should be transferred to the E.coli and confer resistance so it grows on the plate

explain negative control plate

no plasmid on an LB/amp/IPTG plate should allow for no growth. this tests to see if ampicillin selection is successful and if the stock of ampicillin is viable. since there is no plasmid, it should just be regular E.coli (HB 101 strand w no resistance) so it shouldn’t grow bc without a plasmid the E.coli cells are not ampr