Nathan Gong

AP bio

Period 7

Lab 6 : Bacterial Transformation

Introduction:

Bacterial transformation is when a bacterial inherits a new gene and expresses it.

Gene of interest is what gene you want the bacteria to express. The gene we will be

working with in lab six is the Green fluorescent protein, which would make the bacteria

glow under ultraviolet light. Gene of interest is the gene that we want in the bacteria to

express. Plasmids are rings of DNA that the bacteria can inherit with the help of some

chemical. CaC12 is going to help us make that happen by changing the membranes

permeability so the plasmids could enter the bacteria. Without the CaCI2, the bacteria

would kill the plasmid that enters the bacteria. Restriction enzymes are enzymes that can

cut out parts of DNA. This enzyme can cut out harmful DNA such as viruses that have

tried to combine their DNA with the bacteria's.

Glowing fluorescent protein is the coding we will try to incorporate within the bacteria. It

first used by Japanese scientist, Osamu Shimomura in 1960. He extracted it from a

jellyfish that had GDP and has been used to check if his bacterium has inherited the

plasmid.

The purpose of an experimental control is to determine if your experiment went

right or wrong. The controlled group is where everything should happen. The

experimental group is when you change one variant different to the controlled group.
 The questions whether performing transformation on different test subject is still

debated if its ethical or not. There are too many unknown facts on whether it is useful or

not. Scientist are using this way to cure many diseases tested in bacteria though.

Genetic engineering is used on many plants to show if diseased or not, for

example they used this protein in tobacco plants to tell farmers which section of their

crops has disease or not. It can also be used to tell if the bacteria have inherited the gene

of interest. Genetic engineering is also used in the medical field. Scientist use this to

produce vaccines by altering the genetic code to weaken the active virus so your body

can be immune to the virus with being so sick.

Hypothesis

If we put the bacteria in a heated environment with the gene of interest and let it

sit for a while, then we will see bacteria growth the next day with specific plasmid

growth, because the bacterium has acquired the plasmid and has adopted to its current

environment.

Purpose

To successfully transform bacteria

Materials

1. Cup with ice and water

2. Hot water bath and float rack

3. CaCl2

4. Plasmids

5. Sterile inoculating loops disposable pipettes

6. 2Microfuge tubes
7. 1 bacteria stab for streaking starter plate

8. Starter plate

9. Amp plates

10. LB plate

11. Tape

12. UV Lamp

13. Waste containers (10% bleach)

14. Micro centrifuge

15. Incubator

Procedures

1. Use sterile loop to spread bacteria around starter plate.

2. Place starter plate in incubator for 1 day

3. Take out starter plate and check for bacteria growth.

4. Retrieve 2 micro tubes and label one + and-

5. Use a pipette and drop 500 ul of CaCl2 in each tube then place both tubes in ice

6. Using a clean loop, transfer 5 colonies to each micro tubes

7. Mix each tube until it becomes a clear milky substance

8. using a pipette, add all of the plasmid you have acquired to the + micro tube

9. Add 100 ul of distilled water into the - tube.

10. Incubate the tubes for 10 minutes in the ice water

11. Take the ice water bath with the tubes in it to the hot water bath. Make sure the

water is about 42 degrees Celsius. Place the ice water bath with tubes in for 45

seconds.
12. put the micro tubes back into the ice for 2 minutes.

13. open the • tube and transfer half of the mixture to the LBIAMP· Petri using a

clean pipette. Add the rest of it to the LB plate.

14. Use the same pipette to transfer half of the mixture in the + tube to the LB/AMP+

plate.

15. using separate sterile loop, spread the bacteria in each of the plates

16. stack the plates up and place in incubator

17. wait 1-2 days for bacteria to change

18. use UV lamp to check if any bacteria colonies have inherited the plasmid for

GFP.

19. record data

Data

When our experiments were finished, we saw bacteria growth on LB/AMP+

plates and also 0 the LB plate. The LB/AMP+ plate that had the GFP grew bacteria, but

sadly did not inherit the gene to glow in UV light. The LB/Amp plate did not produce any

bacteria because the bacteria could not survive the environment it was put in

Conclusion

Our purpose for this lab was to transform the bacteria. Our group hypothesized if

the bacteria was put in with the plasmid and CaCI2, that the bacteria would inherit the

gene and glow. Our bacteria had failed to inherit the plasmid The bacteria had survived

the LBI AMP+ but show no sign of glowing at all. When we compared results to other
groups, many had same results but some had glowing bacteria. The bacteria from the

LB/AMP- had all died out because the amp had killed them all off.

Errors were most likely the main cause of the problem to our experiments. Some

of our lab partners were breathing directly into the bacteria when we were spreading the

bacteria in the plates and this might have killed the bacteria. Other factors could be,

everything wasn't sterile enough and could have killed the bacteria.

Questions

Could he bacteria have grown if the shoved he sterile loop into the LB?