Lab 06 Assignment - Bacterial Transformartion PDF

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TRANSFORMING ARTIFICIALLY COMPETENT E CELLSGENERATED BY CHEMICAL MODIFICATION AND HEAT SHOCKWITH pPBR322 and pSF-CMV-Kan DNA PLASMIDS.BIOL 3150 A: Microbiology Name: Karishma Kaur ID: 215579378 TA: Saptarshi Sengupta Date: November 14, 20211a) What does competent mean with respect to cells about to ...

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TRANSFORMING ARTIFICIALLY COMPETENT E.COLI CELLS GENERATED BY CHEMICAL MODIFICATION AND HEAT SHOCK WITH pPBR322 and pSF-CMV-Kan DNA PLASMIDS.

BIOL 3150 A: Microbiology Name: Karishma Kaur ID: 215579378 TA: Saptarshi Sengupta Date: November 14, 2021

1a) What does competent mean with respect to cells about to be transformed? [1 mark] A cell is described as competent when it is able to take up DNA from the exogenous environment and thus becomes transformed (Madigan et al., 2021). 1b) How does natural competence differ from artificial competence? What might you expect if chemicompetent cells were used for electroporation? [2 marks] Natural competence is when a bacterial cell is able to take up DNA from the environment due to its genetic predisposition. It is genetically competent, allowing the cell to transcribe and translate genes and proteins that aid to facilitate the natural transformation of the cell. These cells can also regulate their competency through biochemical and environmental signals such as autoinducers produced through quorum sensing (Nicole, October 19, 2020). Whereas, artificial competence is when cells are modified in the lab through electroporation or chemical treatments in order to create temporary pores in the cell’s plasma membrane for DNA uptake. In electroporation, cells are made competent using an electrical pulse which causes gaps to be formed in the lipid bilayer. In a chemical modification, cells are made competent by suspending them in salt solutions such as CaCl2 or MgCl2 solutions. This neutralizes the negatively charged hydrophilic phosphate heads of the lipid bilayer and also the DNA plasmids (negative DNA phosphate backbone) that we want the bacterial cells to take in. This neutralization allows the plasmid to come in close proximity with the bacterial cell. Next, the cells are heat-shocked to allow the formation of temporary pores in the membrane for the plasmid to enter the cells (Martin, n.d.). If chemicompetent cells were used for electroporation, the cells would not be transformed with our plasmids. This is because the chemicompetent cells would have their membranes neutralized by salt and when an electric pulse would be given, the solution would be highly conductive. The electric current would run across the membrane due to the Ca+ charge and this would not disrupt the membrane and thus the plasmids would not enter the cells.

1c) Would you consider E. coli to be naturally competent? Explain why or why not by looking at the process at a molecular level. [2 marks] Although E.coli has homologues of competent genes used by other species for DNA uptake, it is not naturally competent. E.coli is not able to take up DNA and perform homologous recombination efficiently resulting in a limited number of transformed cells. This is due to the lack of gene expression for the competence activator protein, Sxy, which binds to motifs on promoters associated with the expression of competence specific genes (Sinha & Redfield, 2012). Also, E.coli does not have a homologue for the gene encoding for pilF2, which plays an important role in forming functional DNA uptake machinery during transformation. Specifically, pilF2 anchors the pilus of the donor cell to the membrane of the recipient cell for DNA transfer (Nicole, October 19, 2020). Thus, to make E.coli transformable, artificial competence must be induced through methods such as heat shock and electroporation. 2a) Prepare a data table with the number of transformants for the co-transformation similar to the ones set up on p.50 in this manual. Would you consider your experiment a success? Support your answer with direct references to your data (include an explanation of the controls you used and their purpose). [4 marks] Table1.0: Colony counts for undiluted and diluted samples of tubes 1, 2, and 3 plated on both LB-Amp+Kan+ and LB without antibiotic media. Tube 1 contained 25μl of DH5αTM cells and 12.5μl of each plasmid, tube 2 contained 25μl of DH5αTM cells and 25μl TE Buffer, and tube 3 contained 25μl of TE Buffer only with 12.5 μl each of pPBR322 and pSF-CMV-Kan DNA. Contents of tube 1, 2, and 3 were heat shocked for 45 seconds in a 42°C heat block, then placed in for 2 minutes. 250μl of 2LB medium was then added to each tube which were then incubated at 37°C for 45 minutes at 135rpm. Dilutions were only performed for tube 1 through 10-1 - 10-5, where only 100uL of 10 -3 - 10-5 dilutions were plated. LB-Amp+Kan+ Tube

Undiluted

#1 #2 #3

Undiluted 10-3 10-4 10-5 sample sample sample 0

0

LB without antibiotic

0

0

10-3 10-4 10-5 sample sample sample 27

300+ 0

Given the data in Table 1.0, I would not consider this experiment a success as no growth occurred in the LB-Amp+Kan+ plates inoculated with the 10-3, 10-4, 10-5 dilutions of tube #1 (25μl of DH5αTM cells and 12.5μl of each plasmid). If the E.coli cells had been transformed successfully with both plasmids, pBR322 and pSF-CMV-Kan, then the bacteria would have been able to grow on the antibiotic plates, however, they did not. Control was used in order to make sure that there was no error in dilution of the tube 1 sample, inoculation of 10-5 dilution on a plate without antibiotic was performed and growth was observed where 27 colonies formed Table 1.0. Thus, this confirmed that no growth was not due to a dilution error but because the bacteria were not transformed with the plasmids. Tubes 2 and 3 containing 25μl of DH5αTM cells and 25μl TE Buffer, and 25μl of TE Buffer only with 12.5 μl each of pPBR322 and pSF-CMV-Kan DNA respectively were used as a control in this experiment. 100 uL of undiluted tube 2 sample was inoculated on both LB-Amp+Kan+ and without antibiotic plates (Nivillac et al. 2021). The expected results occurred where no growth was observed on the antibiotic plates (since the bacterial cells were not transformed with either plasmid containing the resistant genes) whereas significant growth (300+ colonies) was observed on the plate without antibiotics. 100 uL of undiluted tube 3 sample was inoculated only on the LB medium without antibiotic to test if the plasmids themselves are capable of growth (without the bacterial cells) on the medium. This control was used to make sure that false colonies do not form which could be mistaken for transformed E.coli colonies. This control also confirms that no continuation has occurred in the experiment. 3) Which additional controls could be added to ensure the validity of this experiment? [3 marks] Undiluted tube 1 samples could be plated on both LB-Amp+Kan+ and LB without antibiotic media to ensure that the solution before the dilutions are performed indeed contain E.coli. Also, dilution samples 10-3 and 10-4 for tube 1 could be plated on LB without antibiotics to

ensure E.coli was present in the sample and therefore growth did not occur in the LB-Amp+Kan+ plates due to no transformation. Another control could be to create a 4th tube that contains E.coli and only pPBR322 plasmid and a 5th tube that contains E.coli and only pSF-CMV-Kan plasmid. These undiluted samples could then be plated on LB-Amp+ and LB-Kan+ (respectively) to ensure that both plasmids are functional. An additional control could include a 6th tube with cells that synthetic or identified natural bacterial cells which are highly guaranteed to be successfully transformed. In this tube, we would include our synthetic or natural cells and both plasmids. This sample would then be plated on LB-Amp+Kan+ media to ensure that cells being transformed with both plasmids does not inhibit growth (plasmids are not interfering with each other’s resistant genes). Moreover, control tube samples 4,5, and 6 can be plated on media without antibiotics to ensure E.coli presence.

REFERENCES 1. Madigan, M. T., Bender, K. S., Buckley, D. H., Sattley, W. M., Stahl, D. A., & Brock, T. D. (2021). 4.16 Oxygen and Microbial Growth. In Brock Biology of Microorganisms (pp. 135–136). essay, Pearson. 2. Martin, K. (n.d.). Introduction to competent cells. GoldBio. Retrieved November 14, 2021, from https://www.goldbio.com/articles/article/Introduction-to-Competent-Cells. 3. Nivillac, N. (2021, October 19). Horizontal Gene Transfer Part 1 [Lecture recording]. Moodle@YorkU. https://moodle.info.yorku.ca 4. Nivillac, N., Noel, T.C., Coukell, M.B. 2021. SC/BIOL 3150 4.0 Microbiology Lab Manual. York University. pp. 49-50. 5. Sinha, S., & Redfield, R. J. (2012). Natural DNA uptake by escherichia coli. PLoS ONE, 7(4). https://doi.org/10.1371/journal.pone.0035620...