Essay on Bacterial Transformation Report

Submitted By miriam7204
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Pages: 7

Bacterial Transformation Lab Report

I. Pre-lab
Bacterial transformation occurs when an organism receives DNA from a different species and expresses the implemented gene, thus becoming a transgenic organism. The practice proceeds as follows: first cut the plasmid from one organism and DNA from another, using the same restriction enzyme on a palindromic sequence. The recombinant plasmids then enter the host bacteria and asexually reproduce infinitely. We can identify that the bacteria has the new DNA by testing to see if it expresses the trait. We can see if it has the p-GREEN by putting it under a UV light to see if it glows, and we can see if it is ampicillin resistant by placing the bacteria on an LB plate with ampicillin. If bacteria are present on the plate, then they clearly are ampicillin resistant and express the DNA. The DNA we insert is placed in the plasmid. The plasmid carries genes in bacteria separate from those of the chromosome. In Genetic Engineering, plasmids are used to introduce the foreign gene into bacteria. Ampicillin is an antibiotic similar to penicillin and is used to kill bacterial infections.

The purpose of this lab is to genetically transform E. Coli bacteria by inserting an ampicillin resistant and p-GREEN containing plasmid.

II. Hypothesis: What do you expect to grow on each of the plates?

LB agar
LB agar + ampicillin
(Red stripe)
Do you expect to see any difference in bacterial growth on the two plates?

bacteria + DNA

Would expect to see an LB agar plate filled with many colonies of both transformed and non-transformed bacteria.
Some colonies, although not as many.
Yes, the LB agar plate with no ampicillin would be more plentiful with more variety, although both would have presence of bacteria, and the LB/amp plate would only have ampicillin resistant bacteria.

What do you think would have grown on these plates if no DNA had been added to these bacteria?

LB agar
LB agar + ampicillin
(Red stripe)
Do you expect to see any difference in bacterial growth on the two plates?

bacteria (without DNA)

We would see the same as the other LB agar plate without ampicillin. There would be a plate completely filled with bacteria.
No bacteria would be present, because if none of the bacteria are resistant to ampicillin, then in the presence of ampicillin, they would all die.
Yes, one would be plentiful while the other would be empty.

III. Materials
1 fresh E.coli starter culture (24hr old)
Permanent marker
Beaker of crushed ice
1 100 micropipetor and tips (preset to 50l)
1 1000 micropipetor and tips (preset to 250l)
1 .05-10 micropipetor and tips (preset to 10l)
For class:
2 42°C water baths
37°C incubator
Waste container for used tips and loops
10% chlorox/cloth
1 microcentrifuge freezer (freeze the plasmid)
Masking tape
2 LB plates
(LB/LB amp) CaCl2 solution
LB broth p-GREEN plasmid solution.
1 15-ml clear plastic culture tube
2 sterile inoculation loops

IV. Procedures

Pre-lab safety- wipe all surfaces with 10% chlorox before beginning your work with bacteria.

1. With a LARGE micropipetor, add 250 l of CaCL2 to a clear plastic culture tube.
2. Place the tube on ice.
3. Examine the starter plate of E. coli to see if there is any bioluminescence. None should be seen.
4. Using a sterile inoculating loop, transfer one or two large bacterial colonies to the tube.
5. Immediately re-suspend the bacterial cells in the tube by repeatedly pipetting in and in and out with the LARGE 1000-l micropipetor with a sterile tip.
6. Hold tube up to light to check that there are no visible clumps of cells. If you still see clumps, keep repeating step 5 until all clumps are gone.
7. Return the tube to ice.
8. Using the SMALL micropipetor, add 10 l of p-GREEN solution directly into the cell suspension. Tap the tube with a finger to mix. Avoid making bubbles or splashing suspension up on the sides of the tube. (your teacher may have you centrifuge the p-GREEN solution to move all of