By the conclusion of this laboratory investigation, the student will be able to:
Define and properly use synthetic biology terms:
Define and properly use molecular genetics terms:
Introduction
Data Table
In your lab notebook, you will need to construct a data table as shown below. (These may be provided)
Procedure
Part 1: Culturing Bacteria
We will be receiving two strains of bacteria to transform. These strains will come in the form of a "stab" or "slant", a test tube with a small amount of bacteria on a slanted media. To continue the experiment we will have to further culture the bacteria.
Day 1:
Using a sterile toothpick or inoculating loop, gather a small amount of bacteria from the stab and transfer it to a petri dish containing Luria Broth (LB) agar medium.
Repeat with the remaining stab samples, streaking out each onto a different petri dish.
Place these cultures in a 37°C incubator overnight.
LB agar petri dish showing 3 patches of bacterial strain "4-1." This petri dish would provide enough cells for 3 students (or group of students) to transform 2 plasmids
Using a sterile inoculating loop, transfer a bacterial colony from one of the petri dishes to a new LB agar petri dish, drawing a 1 cm x 1 cm square of each strain. Each square you draw this way will yield enough cells to transform with 2 plasmids.
Repeat for each strain you will need for the transformation lab.
Place petri dishes in the incubator at 37°C overnight.
Part 2: Transforming bacterial strains with color-generating plasmids
You will be preparing 200 ul of competent cells for each strain you want to transform. "Competent" refers to the cell's state when it can take plasmid DNA in from the environment. The strains are not normally competent but can be artificially put into a competent state by gently treating the cells with a cold salt solution. The salts introduce tiny holes in the cells membrane and help carry the DNA into the cell's cytoplasm. Using a sterile dowel to scrape bacterial patch into "TB" transformation solution
For each strain you will be transforming, pipet 200 ul of "TB" transformation solution into an eppendorf tube and place the tube on ice.
With a sterile dowel scrape a patch of the bacterial strain 4-1 off the LB agar plate and into one of the TB aliquots, swirling the dowel to remove the cells from the dowel into the solution. Discard the dowel in the contaminated waste bin.
Repeat the scraping and swirling of cells 4-2 and 4-3 into their own eppendorf tubes of 200 ul cold TB.
Leave the cells on ice while you set up 2 new eppendorf tubes for each strain (so you will need a total of 6 new tubes if you are transforming all three strains with 2 plasmids each).
Aliquot 5 ul of pPL002 DNA into three eppendorf tubes (label these 1, 3, and 5)
Aliquot 5 ul of pGR004 DNA into three eppendorf tubes (label these 2, 4, and 6).
Keeping all the tubes on ice, move 75 ul of competent 4-1 cells into tubes 1 and 2. Flick to mix the cells with the DNA. Save the remainder of competent 4-1 cells to serve as your "No Plasmid" control transformation.
Repeat the addition of cells to DNA, aliquoting 75 ul of 4-2 cells into tubes 3 and 4, and 75 ul of 4-3 into tubes 5 and 6. Be sure to flick to mix the DNA with the cells and to keep the tubes on ice whenever you can.
After all the additions have been completed, heat shock the cells and DNA at 42° for 90 seconds exactly.
Move the tubes to a rack on your bench and use sterile technique to add 0.5 ml of LB broth to each tube. This addition will help the cells recover from the transformation procedure.
Label the LB and LB + Kan petri dishes *on the bottom* (not the lids). Be sure to include the strain, plasmid, media and member names.
Using sterile technique, pipet 250 ul of each transformation solution onto an LB agar petri dish and an LB + Kan petri dish. Spread the liquid over the surface of the petri dishes using sterile glass beads or a sterile spreader.
Incubate the petri dishes overnight at 37°C, agar side up.
The following day: count the number of transformants on each petri dish and make notes about the size, shape and color of the bacterial cells you see. If the cells are growing uniformly on the surface of the agar, you can call this a "lawn" of cells. If the cells are growing as colonies but are too numerous to count, you can subdivide the area into quarters or eighths and count one sector then multiply by the number of sectors.
Calculations
transformation efficiency. The DNA stocks are at 1.8 ug/ul. Remember that you added 5 ul to each transformation but counted the colonies that arose after plating only 1/2 of the final transformation mix.
