Metabolism of Free Guanidine in Bacteria †Free Samples to Students

Question: Discuss about the Metabolism of Free Guanidine in Bacteria. Answer: Introduction To control the beginning and the end of transcription activities an induction process is carried out.Lac operon, a special enzyme aids in the process of transcription. It consists of three genes which are lacZ, lacA, and lacY. LacZ encodes enzymes which split lactose to monosaccharide. LacY, on the other hand, encodes a membrane-embedded transporter.IPTG acts management as an inducer. It, therefore, stimulates the beginning of transcription process. Ortho-Nitrophenyl--galactosidase (ONPG) was used in measuring the enzymatic activities of the galactosidase in this experiment to indicate a yellow color when galactosidase is present. Therefore when the intensity of the yellow color increases the enzymatic activities also increases. -Galactosidase helps in the initiation of metabolism of lactose in the bacterial cell which further hydrolyses the lactose to galactose and glucose. Explain how the expression of the lac operon (which includes the lacZ gene) is regulated. Show a diagram of the operon, including all regulatory DNA sections, chemicals, and proteins/enzymes, to help with your explanation. The expression of lac operon is regulated using three genes which are lacA, lacZ, and lacY. The genes are combined into a single mRNA which is under the control of a single promoter. lacZ main function is to encode enzymes which splits lactose into monosaccharaides which are fed into glycolysis. On the other hand, lacY is tasked with the encoding of a membrane-embedded transporter which aids in bringing of lactose into the cell. Besides the three genes, lac opera also has regulatory DNA sequences which regulate proteins thus controlling the transcription of the operon (Witte et al., 2013 p.13). The operator in the operon is a negative regulatory site that is bounded by lac repressor protein. In a case where the operator binds the lac repressor, RNA polymerase is not able to bind to the promoter for transcription to start. Catabolite activator protein (CAP) acts as the positive regulatory site. It aids in transcription by binding the RNA polymerase to the promoter. The promoter, on the other hand, is the binding site for RNA polymerase (Runti et al., 2013 p.5351). The diagram below shows the operon including the regulatory DNA sections. What is IPTG? IPTG is Isopropyl -D-1-thiogalactopyranoside which stimulates the transcription of lac opera. What is chloramphenicol, how does it work, and what is its purpose here? Chloramphenicol is an antibiotic used to treat some bacterial functions. It works by killing the bacteria which causes an infection majorly eye infections. The purpose of using Chloramphenicol in this experiment tests and study its effects on the growth of plants since it prevents bacterial protein synthesis. How is -galactosidase activity being measured here? The activity of -galactosidase was measured by production of an intense colour of some compound which was produced in conjunction with galactose resulting from the action of an enzyme whose action is stimulated by ortho-nitrophenyl--D-galactoside (Lima et al., 2013 p.380). Write an aim for the experiment This experiment aims to study the effects of lactose, glucose management, IPTG and chloramphenicol induction on beta-galactoside of Escherichia Coli. Induction of galactosidase enzyme. The investigation dwells on two sets of culture conditions. One was set in the condition of adding water which is the control experiment and another in the condition with IPTG.Microfuge tubes labelled 15 containing 75 l of the CTAB solution are prepared and put in the ice bath.5ml Escherichia Coli K14 was taken into two separate 50 ml conical flasks. Later the flasks are covered using a foil and then immersed in a shaking water bath whose temperature is 36 C .250 ml of water was then added in the control flasks. This then transferred to the microfuge tube labelled A tube. The same procedure is then repeated for other samples used in the experiment for up to 40 minutes (Singh et al., 2014). Comment and Explain Your Actual Results From the experiment, it shows a positive result when IPTG is used in the induction of the units of beta-galactosidase. On the other hand, when water is used instead, there is no response observed. Besides, when more time is taken in the IPTG induction, more units of beta-galactosidase bacterial culture are produced. It is therefore concluded that when the IPTG is exposed to culture environment, a lot of units of beta-galactosidase is produced (Campbell et al., 2013 p.5545). Summarise and discuss the trends of values you obtained of TEA for various conditions: lactose, IPTG, Glucose, Glycerol, and chloramphenicol. An increasing trend is also witnessed when glucose is used together with IPTG. For both Glycerol and chloramphenicol, an increasing trend is observed, but after some time about 10 minutes later, a constant level is maintained. What was the effect of the added substance on the -galactosidase activity? When chloramphenicol was added, the units of beta-galactosidase remained constant since chloramphenicol is an antibiotic which inhibits the protein synthesis process and growth. The expectation was that the same amount of units of beta-galactosidase would be produced irrespective of the time allowed for the experiment using different substances. The results failed to agree as the different quantity of units of beta-galactosidase were produced depending to on time allowed for every experiment (Nelson et al., 2017 p.230). Explain the biochemical principles behind your results. IPTG is an inducer. Therefore, it induces the activity of the beta-galactosidase enzyme after some time. Comment on the potential applications of this enzyme Beta-galactosidase is applied in food and dairy industries in the hydrolysis of lactose in milk. It is also used in the production of galactosylated products. Conclusions In conclusion, the production of beta-galactosidase is increased with the addition of IPTG and lactose irrespective of the time. On the other hand, the production of beta-galactosidase is slowed down when substances such as glucose, glycerol, and chloraphelonic are used depending on the time allowed for the experiment. References Singh, A.K., Pluvinage, B., Higgins, M.A., Dalia, A.B., Woodiga, S.A., Flynn, M., Lloyd, A.R., Weiser, J.N., Stubbs, K.A., Boraston, A.B. and King, S.J., 2014. Unravelling the multiple functions of the architecturally intricate Streptococcus pneumoniae -galactosidase, BgaA.PLoS pathogens,10(9), p.e1004364. Nelson, J.W., Atilho, R.M., Sherlock, M.E., Stockbridge, R.B. and Breaker, R.R., 2017. Metabolism of free guanidine in bacteria is regulated by a widespread riboswitch class.Molecular cell,65(2), pp.220-230. Lima, S., Guo, M.S., Chaba, R., Gross, C.A. and Sauer, R.T., 2013. Dual molecular signals mediate the bacterial response to outer-membrane stress.Science,340(6134), pp.837-841. Campbell, J.H., ODonoghue, P., Campbell, A.G., Schwientek, P., Sczyrba, A., Woyke, T., Sll, D. and Podar, M., 2013. UGA is an additional glycine codon in uncultured SR1 bacteria from the human microbiota.Proceedings of the National Academy of Sciences,110(14), pp.5540-5545. Witte, C.E., Whiteley, A.T., Burke, T.P., Sauer, J.D., Portnoy, D.A. and Woodward, J.J., 2013. Cyclic di-AMP is critical for Listeria monocytogenes growth management, cell wall homeostasis, and establishment of infection.MBio,4(3), pp.e00282-13. Runti, G., Ruiz, M.D.C.L., Stoilova, T., Hussain, R., Jennions, M., Choudhury, H.G., Benincasa, M., Gennaro, R., Beis, K. and Scocchi, M., 2013. Functional characterization of SbmA, a bacterial inner membrane transporter required for importing the antimicrobial peptide Bac7 (1-35).Journal of bacteriology,195(23), pp.5343-5351.