Bilka 1

Bacterial Transformation

Anthony Bilka

AP BIO

Mr. Bess

27, Feb. 2017

Abstract:
The purpose of this lab was to get the highest bacterial transformation rate possible by adding plasmids.

Heat shock is then applied to each of the microtubes (one with plasmids and one without) in order to see

the effects that adding plasmids has on a solution. By adding more plasmids the rate of transformation

was able to go up, due to the fact that with more plasmids being present, transcription is more easily

replicated.

Introduction:

A plasmid is a tiny DNA molecule within a cell that is typically separated from a chromosomal DNA, and

is then able to replicate independently. In most instances, the DNA is circular, double stranded, and

located in extremely small bacteria. However, plasmids can also be located in eukaryotic organisms.

Plasmids utilize the enzymes and proteins for replication, which is encoded from the host chromosomal

DNA. In many cases, plasmids can be transmitted from one bacterium to the next by the way of

horizontal gene transfer. This lab had a goal of showing the effects of adding more plasmid to the bacteria

DNA, so that the highest rate of transformation could possibly be reached. Plasmids often enter bacteria

and shield it from ampicillin, adding more plasmids, with a steady rate of bacteria, could in fact lead to

more bacteria being present and transformed. Overall, with the information presented, the hypothesis was

that by adding more plasmid to bacteria, the rate of transformation will be higher than if the amount

of plasmids stayed the same at 0.005 g/l of plasmids.

Procedure

Materials:

Safety goggles
Lab apron
 Latex gloves
Plastic Cup
Ice
Pipets
Micro tubes
Inoculating loops
Dish
Glass beads
Tape
Incubator
Water bath
Ice
Luria Broth
Plasmids
E. coli
Calcium Chloride
Ampicillin

The experiment must begin with a control. For the control we take two sterile micro tubes

in which one will be marked + and the other will be marked - (this represents whether or not

they have plasmids in them). A sterile pipet will be used to add 250 microLiters of calcium

chloride (ice cold) to each tube. Both micro tubes will be put into a plastic cup filled with ice.

Next, using an inoculating loop one loop of E. coli for each tube will be transferred into the them

and then placed back on the ice. Then, a sterile inoculating loop will be used to add one loop of

plasmid DNA to the + plasmid tube then it will be placed back into the cup of ice for 15

minutes. During this time the plates were labeled LB/AMP + Plasmid, LB/Amp -

Plasmid, LB + Plasmid, and LB - Plasmid. Once the 15 minutes of the ice bath have

ended the tubes were placed in a 42 degree celsius water bath for 90 seconds to commence the

heat shock. After the 90 seconds has ended the tubes are put back into the cup of ice for 1

minute. When the minute was up the tubes were removed from the ice and 100 microLiters of

Luria Broth was added to each of them with a sterile pipet and gently mixed. They were then

placed on a test tube rack to be at room temperature for 10 minutes. Using a sterile pipet, transfer
100 microLiters of the + plasmid solution to each of the + plasmid plates and then transfer

100 microLiters of - plasmid solution to each of the - plasmid plates. Next, place 4 glass

beads on each of the plates using the clam shell method. Once the beads are on the plates

shake them to mix the solution around. Once mixed, let sit for 5 minutes, then remove the beads.

After all the beads are removed tape the plates together, upside down, and place them in an

incubator at 37 degrees celsius for 24 hours. After 24 hours all evidence should be recorded.

For the next part of the experiment, the control will be followed except there will be 2

microtubes; one will have 2 loops of plasmid and the other has 3 loops of plasmid compared to

the 1 loop that was used in the control. There will be two plates with Ampicillin and one of the

plates will hold 100 microLiters of the 2 loops of plasmid solution and the other will hold 100

microLiters of the 3 loops of plasmid solution. The plates should be treated otherwise the same,

they will be incubated for the same amount of time and under the same conditions. All other

steps should follow the control lab procedures. Measurements and observations should be

recorded in a data table and then equations for transformation efficiency will be used to analyze

and compare the transformation rates of the bacteria with different amounts of plasmid.

Results

A control of 1 loop of plasmid was established, so this information was available for

observation. However, to remain philosophically consistent, and reach a true conclusion, the one

dish that contained no plasmid and no ampicillin needed to show no colonies. In dish one, one

can see that this does indeed take place, but in the others there is much different results. This is

due to the fact that when more loops are added, as they were in figure two, more colonies are
present. Lastly, three loops were added to the final plate, which amassed an immense of amount

of colonies. While putting plasmid into the control dishes, (1 loop of plasmid) when it is added,

the amount present can be dependant on the loop, which causes different amount of transformed

bacteria.

Conclusion:

Originally, the hypothesis was stated that by adding more plasmid to bacteria, it would impact

the rate of transformation in a high matter. In analyzing the results, all evidence leads to the fact

that the control is indeed consistent with the results one acquired. Adding more plasmid led to

more colonies being produced, as supported by the 137 colonies that 3 loops created. However, it

would be extremely beneficial to have a more precise way of measures the plasmid, as the loops

is rather inconsistent. This can be satisfied by loops being reengineered or loops somehow being

prepackaged with plasmid, is that is possible.