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Bacterial Transformation of E-Coli

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Bacterial Transformation

  • Guiadem Ntoukam

 

Abstract:

In biology, transformation is the genetic alteration of a cell resulting from the direct intake of the genetic material called DNA. Bacterial transformation is a really easy way to transform due to the fact that it is single- cell. In this lab experiment, E. coli bacteria is used because it is singled-cell. The pGLO plasmid will be inserted into E. coli bacteria, and it contains the gene for green fluorescence protein (GFP). According to Bacterial Transformation, with Special References to Recombination Process, GFP is a protein that allows bacteria to Florence. The hypothesis was supported by this experiment because in the presence of the GFP gene the bacteria grew and glowed and bacteria that contain the GFP gene will survive and will also be able to grow and glow in the presence of ampicillin, due to the fact that the green fluorescence protein gene will resist the ampicillin; however if the sugar component is absent then, there will not be any glowing under the UV light. From this experiment it is determined that in the presence of an inducer will induce the process of transcription.

Introduction:

Bacterial transformation is the process by which bacterial cells take up naked DNA molecules. If the DNA has an origin of replication recognized by the host cell DNA polymerases, the bacteria will replicate the DNA along with their own DNA. Bacterial transformation occurs when bacteria take a fragment of DNA that codes for a gene, to transform the bacteria and give it a new trait or phenotype. In this lab bacterial transformation was done using the calcium chloride and heat-shock technique. Calcium chloride solution neutralizes the repulsion between the plasmid DNA and the bacteria cellular membrane, since both are have a negative charge. Then the quick change in temperature makes the bacteria create pores on its membrane to allow the plasmid DNA to enter the bacteria cell, accepting the new gene (BIO281, 2015).

In this experiment bacterium Escherichia coli (E. coli) will be used since it is a single cell organism and a single celled organism should be more easily transformed due to the fact that there is only one cell to transform and it contains all of the DNA that is needed and it needs to take up the new gene. An organism which reproduces quickly is a better candidate for this experiment, because fast production of offspring or new progeny will definitely make it easy to asses if a new trait has been passed on (Kimbal 1). To ensure that the organism doesn’t harm an individual or the environment, the organism should grow vigorously in the lab environment. It should not have the ability to infect plant as or other organisms. E. coli is made of only one cell, and it also reproduces every twenty minutes, E. coli doesn’t make people sick, and this bacterium cannot survive outside the laboratory. It is hypnotized in this experiment that the E. coli of the protein can be transformed. In this experiment ampicillin, which is an antibiotic that can kill this bacteria was also used. There are genes on the plasmid that encode for proteins that have some effect on the ampicillin. The antibiotic resistance gene was only used to find out if antibiotics are actually effective.

The genes for the ampicillin resistance is found on the pGLO plasmid, meanwhile, the gene that will be inserted into the bacteria codes for green fluorescent protein (GFP). The most important fact about the insertion of this plasmid is that it contains a special gene regulation system; known as the operon that can be used to control the expression of the fluorescent protein under the UV light in transformed cells. An inducer known as arabinose is sugar component that allows transcription to occur, by binding the Ara C protein near the GFP gene, which is the repressor and then RNA polymerase is binds to the promoter to start transcription.

It is hypnotized that bacteria that contain the GFP gene will survive be able to grow and glow in the presence of ampicillin; however, if arabinose is not present, then there fluorescence will not be observed under the UV light. In this experiment, it was predicted that if bacteria with +pGLO plasmids are resistant to the antibiotic ampicillin and have the gene for GFP, colonies will survive and grow on the petri dishes that have the LB and ampicillin; however, the bacteria in the petri dish with the +pGLO, LB, ampicillin, and arabinose will grow and glow green in the presence of UV light because of the arabinose. The negative control is the pGLO with LB, this will determine if the bacteria has been contaminated if nothing grows because bacteria should grow in this petri dish.

Methods:

The first step in this experiment was to label two micro test tubes with your group name, and then write +pGLO on one and the –pGLO on the other micro test tube and the place these test tubes in the foam tube rack. The + one indicates the presence of the +pGLO gene, and the –pGLO represents the absence of the gene. Then use a new transfer pipet to insert 250 microliters of calcium chloride into each of the micro test tubes; these micro test tubes should then be sealed and placed in an ice bucket. Meanwhile the test tubes are in the bucket of ice, colonies of e coli should be added to the test tubes using a sterile loop to pick up a single colony of bacteria; once a colony is scooped make sure not to press on the Agar, and make sure that you spin the loop between your finger and thumb until the entire colony is in the solution. To avoid contamination, a new loop should be used for the next solution and the place the test tubes on the rack then back in the ice bucket. Once all the micro test tubes contain the bacteria, the +pGLO solution should be added to the test tube that indicates it should receive the gene. To add the gene; use a loop tool to by dipping it in the +pGLO solution, and make sure that there is clear solution around the loop then adding it to the test tubes, an analogy to for this instruction would be like blowing soap bubbles with the exception of blowing on the loop tool. As the test tubes sit in ice, you should label the petri dishes with the proper label. You should label two plates that contain LB, two that contain LB and ampicillin, and one that contains LB, ampicillin, and arabinose; these labels should be located on the bottom of the petri dishes. Then once the 10 minutes have passed place the test tubes in a foam rack so they can be in one place, then drop them into a water bath that is set at 42’C for 50 seconds. After the 50 seconds quickly place them back into the ice where they should incubate for 2 minutes. One the 2 minutes have passed use a transfer pipet to add 250 microliters of LB nutrient to both of the test tubes. They should be incubated at room temperature for 20 minutes; a different pipet should be used every time new LB is added. With your fingers you could tap on the test tube to mix the solution, then 100 microliter of the appropriate solution either ampicillin or arabinose or both should be added to the appropriate test tube, using a different transfer pipet every time. Then use a new loop tool each time to avoid contamination, spread the appropriate solution to the appropriate petri dish, around the surface; back and forth strides across the dish, so it’s not everywhere. Make sure not to dig deep on the agar. After the plates are ready stack them upside down, seal or tape them together and then incubate them in a 37’ C incubator for a 24 hour period.

Results: The table displays growth of bacteria and glow under UV light, indicated with a plus

Sign or a minus sign.

More than 300 colonies: +++

5-300 colonies: ++

1-4 colonies: +

No colonies: -

Plate 1: -pGLO LB

Plate 2: -pGLO LB + Amp

Plate 3: +pGLO LB + Amp

Plate 4: +pGLO LB + Ara

Plate 5: +pGLO LB + Amp + Ara

(Table 1)

 

pGLO LB

pGLO

LB+ Amp

+pGLO

LB+ Amp

+pGLO

LB+ Ara

+pGLO

LB+ Amp+ Ara

Relative

Growth (+++ or + or -)

(+) Growth

(-) There was no growth clear dish

(+) Growth

(+) There was Growth

(+) There was only one visible colony

Fluorescent under UV

(-) Nonfluorescent

(-) None was visible

(-) None that was visible

(+) Large colony that was visible

(+) Small colony that was visible

Figure 1.

C:\Users\Owner\AppData\Local\Packages\microsoft.windowscommunicationsapps_8wekyb3d8bbwe\LocalState\LiveComm\b93b1a45d4f3ee31\120712-0049\Att\20007909\20150317_142441.jpg

Figure 1. This is a picture of the results from transformation of bacteria experiment.

Graph 1.

Graph 1. The Major findings: While the petri dish that did contain the +pGLO plasmid, and the GFP gene, they could not express the GFP gene because they did not grow in the presence of arabinose, due to the fact that the RNA polymerase cannot attach itself to the promoter. In the petri dish that did not contain the -­pGLO but had LB, and ampicillin; the bacteria were wiped out since there was nothing to make them resistant to the antibiotic.

Discussion:

The purpose of this experiment was to incorporate a gene into an organism in order to change the organism’s trait. This lab was also conducted to verify if antibiotics such as ampicillin are actually effective. In this lab experiment there were four different outcomes. (See graph 1). In the LB, ampicillin plate containing the +pGLO, a few colonies of, nonfluorescent bacteria were detected, and in the pGLO that contained LB, and arabinose there seemed to be a very large colony that appeared to be fluorescent. While the petri dish that did contain the +pGLO plasmid, and the GFP gene, they could not show the GFP gene due to the fact that they did not grow or reproduce in the presence of arabinose, because without an inducer the RNA polymerase cannot attach itself to the promoter (Bickle 95). The one that contained the arabinose, had a large colony that was fluorescent. In the petri dish that did not contain the –pGLO, but had LB, and ampicillin the bacteria seemed to be gone. Assuming that the bacteria was wiped out due to the fact that it wasn’t resistant to the ampicillin, when compared to the other petri dishes that contained colonies of bacteria, this one did not show any colonies. The dish that contained the LB without the pGLO just to confirmed that bacteria was present.

In the petri dish containing LB, ampicillin, arabinose and agarose plate containing the +pGLO, there seemed to be only one fluorescent green colony that developed (see figure 1). This is because the +pGLO plasmid which codes for GFP, green fluorescence and ampicillin resistance which makes that bacteria resist the antibiotic ampicillin. This petri dish also contained arabinose, which is an inducer. Arabinose made the gene to be transcribed by binding RNA polymerase to the promoter.

The hypothesis was supported by this experiment because in the presence of the GFP gene the bacteria grew and glowed. According to AP Lab #6: pGLO Transformation Lab, it states that the –pGLO bacteria that didn’t have the plasmid couldn’t survive on the ampicillin plates, which eventually resulted in no bacterial growth. This results from this other experiments supports the result in this lab because it was also hypothesized that the ampicillin would have wiped out the bacteria. Based on the predictions that were made, the results agree with the prediction that when arabinose is present there is a green fluorescence, it grew and glowed in the presence of arabinose. Which concludes that in the absence of arabinose there is no glow in the bacteria, or no green fluorescence in the bacteria because of the absence of the inducer the bacteria will not be able to bind to the RNA to bind to the promoter and begin transcription for the green fluorescence gene. In this lab, there might have been some sources of errors that could have affected the results of this experiment. There might have been sources of error such as, cross contamination, the pipettes may have been used more than once for different solutions, the measurements of the solutions in the pipettes, or the timing during the heat shock process.

In conclusion, bacteria will code for the GFP gene if there is an inducer such as, arabinose available to bind the RNA polymerase to the promoter and begin transcription, RNA will not be able to bind to the promoter without an inducer present. This experiment is important because other experiments can be furthered to understand the need for an inducer in other researches. The findings of this lab experiment are very important, because genetic transformation doesn’t only involve making bacteria glow in UV light, but it involves a lot more such as, making fish glow under fluorescent light, or making crops resistant to air borne diseases. Future research that can be considered would be changing the process in which the plasmid is inserted into bacteria or maybe even changing the inducer.

Work Cited

Bickle, T. (1982) inNucleaseseds Linn, S.M. and Roberts, R.G. (CSH, NY) p. 95-100.

Biology 281 Conceptual Approach Bio Majors 1. “Laboratory Exercises for General Biology 1.” Plymouth: Hayden-McNeil Publishing, 2015. Print.

"AP Lab #6: PGLO Transformation Lab."Scribd. PointWeb, Web. 31 Mar. 2015. <http://www.scribd.com/doc/83020743/AP-Lab-6-pGLO-Transformation-Lab#scribd>.

"Bacterial Transformation, with Special Reference to Recombination Process."- Annual Review of Genetics, 4(1):193. N.p., n.d. Web. 31 Mar. 2015. <http://www.annualreviews.org/doi/pdf/10.1146/annurev.ge.04.120170.001205>.

Kimball, J. (2014, April 25). Recombinant DNA and Gene Cloning. Retrieved March 17, 2015, from http://users.rcn.com/jkimball.ma.ultranet /BiologyPages/R/ RecombinantDNA.html


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