Elena Knudsen

Mrs. Handest

12 December, 2016

E. Coli Lab

Background

1. What is the source of the GFP gene? Include a labeled diagram of the pGreen plasmid

that contains the gene. Briefly describe how this plasmid was engineered.

The GFP gene is from bioluminescent jellyfish called Aequorea victoria.

The pGREEN plasmid is a mutant version of green fluorescent protein. Scientists used the GFP

gene from the jellyfish and derived a different version of the gene, pGreen.

2. Describe bacterial transformation. Is this considered asexual or sexual reproduction in

bacteria?

Bacterial transformation is when a bacterial cell incorporates a plasmid DNA and incorporates it.

Bacterial transformation is sexual because it involves the transfer of DNA.

3. What is the connection between bacterial transformation and recombinant DNA? Give

at least one example of how these two processes would work together for medical,

pharmaceutical or agricultural applications.

Recombinant DNA is the use of restriction enzymes to add specific strands of DNA to a bacterial

plasmid. It is very similar to bacterial transformation because both processes involve the

incorporation of foreign DNA into a bacterial cell. Scientists can insert their preferred plasmid

into a cell, recombining DNA through transformation. This can be used if a scientist wished to

produce a high number of a protein, like insulin for diabetic patients, and they could use this

method to accomplish this.

Procedure

1. Describe the parts of the procedure that were used to make the E.coli bacteria

“competent” for the transformation.

Both the temperature changes and the calcium chloride helped the bacteria become competent.

The temperature shock was supposed to make the bacteria more able to incorporate the plasmid.

2. What process for spreading bacteria onto the growth medium did your group select.

Why did you choose this method and what were difficulties or drawbacks?

Our group spread the bacteria by using the beads, which was easier than an inoculating loop

would have been, but the method was not as precise. Two of the plates had lawn growth instead

of colony growth, and that may have been partially due to the overspreading of the bacteria on

the beads.

Data

Growth Medium Type of Bacteria (- Prediction of Actual Growth

or + plasmid) Growth

Luria Broth LB + plasmid Lawn growth; Lawn growth; not

 glowing glowing

Luria Broth LB - plasmid Lawn growth; not Lawn growth; not

glowing glowing

Luria Broth LB/AMP + plasmid Colony; glowing Colony growth;

glowing

Luria Broth LB/AMP - plasmid No growth; not No growth; not

glowing glowing

Data Analysis

1. What does the growth (or lack of) mean about your transformation experiment? Did the

transformation process work? How do you know?

The transformation process only worked in the LB/AMP + plasmid plate, because it was the only

plate that glowed. The LB + plasmid did not glow under UV light, which was either a result of

incompetence or slow protein production, but either way, the results were contradictory to the

expected results. If the plasmid was not incorporated in the bacteria in the LB/AMP + plasmid

plate, there should have been no growth, but the plates did show growth and glow, which means

that the plasmid was incorporated.

Conclusion

1.What plate would be used to determine if the E.coli strain was viable?

The LB - plasmid plate would be used to determine if the E. Coli strain was viable, because

nothing is affecting the bacteria, and the Luria broth provided the nutrients it would need to

survive.
2. What plate would be used to determine if the Ampicillin that we used was viable?

The LB/AMP - plasmid plate would be used to determine whether or not the ampicillin was

viable, because if there was no growth, it would prove that the ampicillin was effective.

3. What is your transformation efficiency? What factors might influence transformation

efficiency? Explain the effect of each factor.

a. Determine the total mass (in 𝝁g) of plasmid used.

mass of plasmid=10𝝁L x 0.005𝝁g/𝝁L

mass of plasmid= 0.05𝝁g

b. Calculate the total volume of cell suspension prepared.

volume of cell suspension= 250𝝁L+250𝝁L+10𝝁L

volume of cell suspension=510𝝁L

c. Calculate the fraction of the total cell suspension that was spread on the plate.

fraction spread=100𝝁L/510𝝁L

fraction spread=0.196𝝁L

d. Determine the mass of the plasmid in the cell suspension spread.

mass of plasmid in spread=0.05𝝁L x 0.196𝝁L

mass of plasmid in spread=0.0098𝝁g

e. Determine the number of colonies per mass of plasmid in spread plasmid DNA.

3 colonies/0.0098𝝁g=transformation efficiency

3 colonies/.0098𝝁g=306.12 (and in scientific notation, 30,612 x 10^-2).

Factor that may affect transformation Effect of factor

efficiency

Competence of the bacteria If a higher percentage of bacteria were more

 competent, many would have been able to
incorporate the plasmid and produce the
proteins with more efficiency.

Health of the E. Coli If the bacteria were kept unhealthily, their

ability to perform transformation would have
been impaired as well as their ability to
reproduce.

Growth medium If the growth medium did not provide

adequate nutrients for the E. Coli, then they
would have starved and the experiment would
have unreliable results.

4. Were the ampicillin and GFP both necessary to determine if our transformation was

successful?

They were not both necessary. The experiment could have been deemed successful or

unsuccessful with only one of these factors. If the experiment had been performed without

ampicillin, observers would still observe the plate with the plasmid, that was glowing, and the

plate without the plasmid, which would show normal growth without the glow. This would have

worked in the same way if GFP was not used, and instead ampicillin was the only assessed

factor, because the lab would be assessing bacterial survival instead of glow. Either way, it

would be evident whether or not the bacteria incorporated the DNA.

5. List possible sources of error for your group.

The group may not have collected enough bacteria, or it may not have been spread

properly. Also, the timing may not have been accurate enough, and the bacteria may not have

been temperature shocked enough. When the bacteria was gathered, it is possible that more

nutrient agar was gathered than bacteria.

6. Research ‘next steps’. What procedures would be used once we have the transgenic

bacteria with copies of our donor gene? Explain desired outcomes.

Once a donor gene has become incorporated into bacteria, they can yield a variety of uses. They

can become ‘protein factories’(biotechlearn.org), and they will produce the protein that the

plasmid instructs the bacteria to build. Scientists may even attempt to make bacteria incorporate

a plasmid that would cause the bacteria to perform a specific function, such as producing fuels or

becoming indicators of bacterial infections.

Work Cited

"Bacteria in Biotech | Biotech Learning Hub." Biotechnology Learning Hub RSS. N.p.,

n.d. Web. 12 Dec. 2016.