The Chemical Reactions of Living Cells - Assignment Example

Name Course Date Formal report: Blue-white selection of pCC8 vector with insert and galactosidase assay. This experiment analysis the transformation of bacteria by plasmid DNA which provides a chance to study a physical trait acquired by transformed bacteria cells. There are various plasmids which are significant in molecular biology. An example of the plasmid is pUC8 with 2700 base pairs which can be modified through genetic engineering. It replicates in the cell independently. Map of pUC8 In this experiment, the bacterial cells were transformed by a plasmid DNA. The transformants displayed blue colour and thus gain the antibiotic resistance, because of the integration of ampicillin and β-galactosidase. The number of transformants was used to calculate the transformation efficiency. The E. coli gene presents in plasmid pUC8 codes for the β-galactosidase enzyme. The cells transformed by pUC8 create an active β-galactosidase. With the help of fabricated galactose (X-Gal), the colonies containing pUC8 appeared blue when X-Gal cleaved on the product. The transformation of the cell was shown by the resistance to ampicillin. Although the E. coli cells in the experiment are very sensitive to the ampicillin, pUC8 possesses genes which encode for the enzyme β-lactamase spread around the cells and thus making the ampicillin inactive. As time goes by, the white colonies may surround blue colonies. The cells in the colonies are resistant to ampicillin, but they are growing in the region where β-lactamase form the transformation has made the antibiotic inactive. The number of colonies may increase if the plates incubate for a long time. This experiment had three sections. These are: the ligation of DNA fragment in a linearised vector; the transformation by introducing DNA into E. coli cells; and the plating out the cells. Aims The aims of this practical were as follows: a) To reinforce the principles of cloning using the Lac Z system, including alpha complementation b) To study the effects of ligation of a DNA fragment to a vector and its transformation into competent E. coli host cells. c) To study differences between the above transformation and transformation by a supercoiled form of the same vector plasmid Methods Part A. Ligation of linearised vector and DNA fragment The experiment begun with whirling T4 Ligase/ Buffer pellet/ ATP in L3 tube before taping it on the bench for the pellet to settle at the bottom. 35 μl of sterile TE Buffer in L4 was then poured into L3 tube and allowed to hydrate for 5 mins. Two tubes were also labeled ‘Ligation’ and ‘Control’ respectively and the mixture in L3 were stirred carefully using the tip of the pipette before pipetting them up and down so as to mix ligase and buffer, before dividing 15μl of the mixture into the tubes labeled ‘Control’ and ‘Ligation’. Then 20 µl of linear DNA vector of the plasmid pUC8 and DNA fragments in L1 were poured into ‘Ligation’ tube, and whirled briefly. 20 µl of plasmid pUC8 in L2 were also added into the tube labeled ‘Control’ and stirred briefly. They were then incubated for 1 hr at room temperature while whirling periodically every 15 minutes. Part B. Transformation of E. coli cells The second part of the experiment began with labeling one of the microcentrifuge tube which contain ligation DNA with ‘T-ligation’ and the second one with superhelical plasmid DNA labeled ‘T-Control’. 250 μl of ice cold CaCl2 solution were then added to each tube through sterile 1 ml pipette. 4 colonies of E. coli cells with 3mm long were also picked carefully and placed in each of the tubes before suspending the cells by taping the tubes. 5μl of ligation reaction from part A were also added to the tube labeled ‘T-ligation’ and 5μl of control reaction were added to the tube labeled ‘T-control’ before incubating them on ice for 15mins. Both tubes were then placed in a water bath with a temperature of 420C for 90 secs to enhance the entry of DNA into E. coli bacterial cells. Thereafter, both tubes were returned to the ice bucket and incubated for 2 mins and 250 μl of Recovery Broth were added using sterile pipette into each tube and then mixed before incubating the cells for 30mins in 370C water to allow bacterial cells to recover from taking up the exogenous pUC8 DNA before removing and placing in a microcentrifuge. Then the mixture were spun for 5 mins to form the cells. Finally, 0.30 ml of supernatant were removed and discarded and 0.2ml remained in the tube before resuspending the pellet in it by gently pipetting them up and down for few minutes. Part C. Plating out the Transformed Cells The last part of the experiment began with labeling two plates of agar with initials and date. These plates contained ampicillin, IPTG and X-Gal with one plate being labeled Control and the other litigation. All of the recovered transformed cells were pipetted into the tube labeled T-ligation. Then the cells were spread evenly over the whole surface using a sterile loop. The plate was turned at an angle of 900 before spreading again as shown below. It was also repeated using a fresh pipette using control plate and T-control sample, before covering them and allowing them to absorb completely into the media. Finally, the plates were staked together inverted, taped and placed in 370C incubator overnight. Results: Control plate Ligation plate Control plate had 200 blue colonies and 400 white colonies. Ligation plate 1: had 1 blue colony and 20 white colonies Ligation plate 2: had 1 blue colony and 200 white colonies The transformation efficiencies for total transformants are obtained from the formula: Since the final recovery volume of the cells was 0.50 ml and after the cells were centrifuged and volume reduced, the volume plated was 0.10 ml. Also the quantity of DNA used was approximately 25 ng. Ligate plate 1 Ligate plate 2 The percentage of colonies with vectors inserts are: Control plate Ligate plate 1 Ligate plate 2 The ratios of total transformants on the plates with ligated vectors vs. transformants in the plate with superhelical vector are: Legate plate 1 The ratio = 21/600 ≈ 1:30 Lagate plate 2 The ratio = 201/600 ≈ 1:3 There are more colonies in the control plate than in the litigation plates. Discussion: 1. The number of counted colonies was marked with a marker as it provided a convenient way for counting the quantity of viable cells in the sample. This will also mark better arrangement so that there will be no confusion and reduce errors. 2. All of the above colonies are transformants since they grew on agar medium. All the colonies that grew on agar medium are made up of transformed cells because only transformants are ampicillin resistant. Both blue and white colonies contain either pUC8 plasmid or recombination which has DNA fragments in MCR (Multiple Cloning Region). The blue colonies contain cells with β-galactosidase enzymes and also lac Z genes. The colonies are non-recombinants. On the other hand, the white colonies are cells which do not have β-galactosidase activity and disrupted lac Z genes. They form recombinants. The E. coli remained untransformed grew into colonies in the presence of incubation. The ampicillin in the plates is destroyed by β-lactamase produced after subsequent transformants. The bacterial cells is transformed by a plasmid DNA, the transformants displayed blue colour and thus gain the antibiotic resistance, because of the integration of ampicillin and β-galactosidase. The number of transformants was used to calculate the transformation efficiency (Metzler, 2003). 3. The transformation efficiencies indicate the number of cells that are transformed in one µg mass. The transformation efficiencies for control plate was 1.2x105 transformance/µg, in the first ligation plate the efficiency was 21 transformance/µg, and in the second litigation plate the efficiency was 20100 transformance/µg. The transformation is high in control plate as compared to the first plate, which shows the efficiency was better in the second plate (Radolf, & Samuels, 2010). 4. The percentage of colonies with vectors that have inserts out of the total number of transformants on the plates varies as shown in the results. The results were not as expected because the numbers of blue colonies in the Ligation plates were very low. Some of the reasons include introduction of errors during the experiment. For example, the problem of self-ligation in the control plate due to the restriction enzymes not working. Another reason may be that the digest vector being isolated such that the cohesive ends cycled the vector. Other sources are the quality of the sample, incubation conditions and human errors (Casali, & Preston, 2003; Radolf, & Samuels, 2010). 5. The ratio of the total transformants on the plates with ligated vectors vs. transformants in the plate with superhelical vector for ligate plate 1 is high and it was not expected. This can be due to errors incurred in the course of the experiment. 6. The superhelical DNA is over and under winding of DNA strand, resulting in a compact strand. The other forms of plasmids are linear plasmid and circular strand plasmid (Watson, et al., 2014). 7. The self-ligation of the vector in the experiment can be prevented using different strategies. It can be by use of alkaline phosphate, double digestion or by transforming the insert or the vector. In the first case, the DNA molecule is treated with enzymes like calf-intestine phosphate leading to dephosphorylated vectors that prevent self-ligation. The process also ends the blunt end conversions, by trimming the overhang and therefore getting rid of unpaired sequence (Chaudhuri, 2012). Conclusions 1. The experiment has shown how the transformation of bacteria occurred by plasmid DNA. The antibiotic-resistance markers enabled the transformants to be selected from large number of untransformed cells. Transformants are the colonies which had taken up the plasmid. Not all of the cells are transformed, due to inefficiencies a given portion of the cell takes up the plasmid. The transformation efficiencies for the colonies in the second ligation plate had 20100 transformance/µg, which is higher compared to transformation efficiencies in the first ligation plate which had 21 transformance/µg. The number of blue colonies in the Ligation plates was very low. The transformation was not efficient due to errors introduced during the experiment. The sources of errors are self-ligation in the control plate due to the restriction enzymes not working. Other sources are the pour quality of the sample, incubation conditions and human errors. 2. The information learnt from this experiment. a) Better understanding of the principles of cloning using the Lac Z system and alpha complementation in a practical way. b) Studied and learned the effects of ligation of a DNA fragment to a vector and its transformation into competent E. coli host cells. c) Understood the differences between the above transformation and transformation by a supercoiled form of the same vector plasmid 3. This experiment has developed my skills that are significant when performing scientific research. I have also learned new techniques while using the biotechnological equipments and the standard procedure used in transformation. I hope to use this knowledge in my future academic research. References Chaudhuri, K. (2012). Recombinant DNA technology. [S.l.], The Energy And Resources. Casali, N., & Preston, A. (2003). E. coli plasmid vectors: methods and applications. Totowa, N.J., Humana Press. Shukla, A. N., & Shukla, A. N. (2009). Elements of enzymology. New Delhi, Discovery Pub. House Pvt. Ltd. Watson, J. D., Baker, T. A., Bell, S. P., Gann, A., Levine, M. S., Losick, R., & Harrison, S. C. (2014). Molecular biology of the gene. Metzler, D. E., & Metzler, C. M. (2001). Biochemistry: the chemical reactions of living cells. San Diego, Calif, Harcourt/Academic Press Palmer, T., & Bonner, P. L., 2007. Enzymes biochemistry, biotechnology and clinical chemistry. Oxford, Woodhead Publishing. Schweizer M. (2003). Methods in Biotechnology CRC Press Metzler, D. E. (2003). Biochemistry (2 volume set) the Chemical Reactions of Living Cells. Burlington, Elsevier. http://public.eblib.com/EBLPublic/PublicView.do?ptiID=535320. International Conference on Advances In Biological Sciences, Sabu, A., & Augustine, A. (2013). Prospects in bioscience addressing the issues. New Delhi, Springer India. http://dx.doi.org/10.1007/978-81-322-0810-5. Radolf, J. D., & Samuels, D. S. (2010). Borrelia: molecular biology, host interaction, and pathogenesis. Norfolk, UK, Caister Academic Press. Read More

Then the mixture were spun for 5 mins to form the cells. Finally, 0.30 ml of supernatant were removed and discarded and 0.2ml remained in the tube before resuspending the pellet in it by gently pipetting them up and down for few minutes. Part C. Plating out the Transformed Cells The last part of the experiment began with labeling two plates of agar with initials and date. These plates contained ampicillin, IPTG and X-Gal with one plate being labeled Control and the other litigation. All of the recovered transformed cells were pipetted into the tube labeled T-ligation.

Then the cells were spread evenly over the whole surface using a sterile loop. The plate was turned at an angle of 900 before spreading again as shown below. It was also repeated using a fresh pipette using control plate and T-control sample, before covering them and allowing them to absorb completely into the media. Finally, the plates were staked together inverted, taped and placed in 370C incubator overnight. Results: Control plate Ligation plate Control plate had 200 blue colonies and 400 white colonies.

Ligation plate 1: had 1 blue colony and 20 white colonies Ligation plate 2: had 1 blue colony and 200 white colonies The transformation efficiencies for total transformants are obtained from the formula: Since the final recovery volume of the cells was 0.50 ml and after the cells were centrifuged and volume reduced, the volume plated was 0.10 ml. Also the quantity of DNA used was approximately 25 ng. Ligate plate 1 Ligate plate 2 The percentage of colonies with vectors inserts are: Control plate Ligate plate 1 Ligate plate 2 The ratios of total transformants on the plates with ligated vectors vs.

transformants in the plate with superhelical vector are: Legate plate 1 The ratio = 21/600 ≈ 1:30 Lagate plate 2 The ratio = 201/600 ≈ 1:3 There are more colonies in the control plate than in the litigation plates. Discussion: 1. The number of counted colonies was marked with a marker as it provided a convenient way for counting the quantity of viable cells in the sample. This will also mark better arrangement so that there will be no confusion and reduce errors. 2. All of the above colonies are transformants since they grew on agar medium.

All the colonies that grew on agar medium are made up of transformed cells because only transformants are ampicillin resistant. Both blue and white colonies contain either pUC8 plasmid or recombination which has DNA fragments in MCR (Multiple Cloning Region). The blue colonies contain cells with β-galactosidase enzymes and also lac Z genes. The colonies are non-recombinants. On the other hand, the white colonies are cells which do not have β-galactosidase activity and disrupted lac Z genes.

They form recombinants. The E. coli remained untransformed grew into colonies in the presence of incubation. The ampicillin in the plates is destroyed by β-lactamase produced after subsequent transformants. The bacterial cells is transformed by a plasmid DNA, the transformants displayed blue colour and thus gain the antibiotic resistance, because of the integration of ampicillin and β-galactosidase. The number of transformants was used to calculate the transformation efficiency (Metzler, 2003). 3. The transformation efficiencies indicate the number of cells that are transformed in one µg mass.

The transformation efficiencies for control plate was 1.2x105 transformance/µg, in the first ligation plate the efficiency was 21 transformance/µg, and in the second litigation plate the efficiency was 20100 transformance/µg. The transformation is high in control plate as compared to the first plate, which shows the efficiency was better in the second plate (Radolf, & Samuels, 2010). 4. The percentage of colonies with vectors that have inserts out of the total number of transformants on the plates varies as shown in the results.

The results were not as expected because the numbers of blue colonies in the Ligation plates were very low.

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