For this lab, Escherischia coli (E. coli), a strain of bacteria found in the human digestive system, will be 'transformed' with your DNA ligation reactions. You will utilize chemically treated E. coli that have been made "competent" to take up exogenously added DNA. Cells that are "transformed" by the pBS plasmid DNA (with or without the yeast DNA insert) will be selected for by their ability to grow in the presence of the antibiotic ampicillin, which normally inhibits the growth of E. coli. The overall goal is to isolate transformed bacteria which have taken up recombinant DNA molecules ("constructs") containing both the pBS and yeast DNAs joined together via the ligase enzyme. Note that linear DNA transforms into bacteria very poorly. What are the potential ligation reaction products that could be transformed into the cells, and which of these will be detected in the plating assay?

1) Aliquot competent cells into pre-chilled eppendorf tubes. Add each ligation reaction into one tube. Let incubate on ice.

2) Pour LB and LB/Amp plates.

3) Administer a 42°C heat shock to the cells. Add LB medium and incubate at 37°C.

4) Very briefly spin down cells in micro-centrifuge. Remove most of the LB medium.

5) Plate each set of cells onto LB and LB/Amp plates.

6) Let incubate overnight at 37°C.

Several types of bacteria naturally take up foreign DNA. However, other types, including gram-negative bacteria such as E. coli, require pre-treatments to facilitate uptake of exogenous DNA. This is typically done by growing cells in liquid medium and harvesting them in early logarithmic growth phase. These cells are then treated with calcium chloride and/or other salts which help permeablize their cell wall and cell membranes. It is important that the cells be kept on ice during these treatments to stabilize them and increase transformation efficiency.

DNA is added to the competent cells, a heat pulse is given, and non-selective media is added to permit the bacteria to recover and express the antibiotic resistance gene.The cells are then spread on petri plates that contain a solid agar medium. The plates are incubated at 37°C overnight to permit bacterial growth. An individual bacterium that takes up a plasmid DNA molecule will grow on the plate and give rise to small, cell-dense colonies. All of the cells in one colony are clones of the original transformant cell and so contain the same plasmid DNA. However different colonies may contain different DNAs, i.e. pBS only vs. pBS + yeast DNA insert.

Identification of the transformed bacteria is facilitated by the presence of an antibiotic resistance gene present on the plasmid. Only cells that have taken up plasmid DNA and express the antibiotic resistance gene will grow on plates containing the antibiotic. However, on the non-selective plates...... Ampicillin is a bacteriostatic antibiotic. It does not directly kill bacteria, but inhibits their growth. Cells containing the pBS plasmid express a secreted protein, ß-lactamase, which cleaves ampicillin and thus permits growth on the LB/Amp plates. If the transformed cells are grown at high densities, or for long periods of time, the accumulation of ß-lactamase can result in a local depletion of ampicillin and may then permit the growth of non-resistant cells. This is often detected via the presence of small "satellite" colonies surrounding a larger primary colony.

Note: it is important to keep the cells on ice at all times prior to the heat shock administration. It is also crucial to use sterile technique throughout this experiment.

2) Label 2 eppendorf tubes:

1- pBS

Place capped tubes on ice.

Re-cap tubes and keep them on ice.

4) Add ligation reaction 1 (pBS only) to tube 1 and ligation reaction 2 (pBS + yeast DNA) to tube 2.

Be sure to use a fresh pipetman tip for each transfer.

5) Flick tubes gently to mix.

6) Let sit on ice for 30 minutes.

7) Place eppendorf tubes in a 42°C water bath for 2 minutes.

8) Add 500 µl of LB media to each eppendorf tube using the P-1000.

9) Let incubate at 37°C for 30 minutes.

10) Spin down mixtures for 20 seconds in the microcentrifuge. Remove all but 100 µl of liquid using the P-1000.

11) Resuspend the cells via gentle flicking of the tube or via vortexing if necessary.

12) Transfer 50 µl of the cells in tube 1 onto an LB plate, and 50 µl onto an LB/AMP plate. Repeat for tube 2.

Be sure to label plates pBS or pBS/yeast.

13) Spread mixtures along the surface of the plates using the sterile plastic spreaders. Rotate the plate so that the liquid gets distributed as evenly as possible. All the liquid should soak into the surface of the agar.

14) Place plates in 37°C incubator.

15) For next week: Note the relative numbers of colonies that grew from the pBS ligation vs. the pBS/yeast ligation on the LB and LB/Amp plates.

1) Label the BOTTOM side of 4 sterile 100 mm plastic petri plates. 2 of these plates should be labeled LB and 2 should be labeled LB/AMP.

2) Pour the 2 LB plates with warm (liquefied) LB/Agar medium. The medium should rise approximately 1/3 of the way up the side of the plate (there may be a little line on the outside of the plate to help you estimate volumes).

3) Add 1/1000 volume of 50 mg/ml Ampicillin stock to the other LB/agar medium using the P-200 pipetman (the working concentration of ampicillin in the plates is 50 µg/ml). Pour 2 LB/Amp plates as before.

4) Let plates sit out at room temperature with the lids ON to solidify.

5) After plates have completely set, turn them upside down and slide the bottom plate onto the side of the top plate so that the surface can dry a bit before you plate your cells.

Note: if we have access to a sterile hood in the lab we will use this to help dry the plates.