Beta Galactosidase Lab Report

The expression of lac operon in each tube equals the amount of beta-galactosidase produced. Therefore, by looking at the amount of beta-galactosidase under different conditions collectively is a good way to understand the function of inducers and repressors in supervising the expression of lac operon and the control of gene expression generally.

At the given time sets, CTAB was added to the tubes to kill the E. coli cells and lyse the cells to release its contents including galactosidase enzyme.

We can measure the amount of beta galactosidase produced in each tubes indirectly although it is difficult. ONPG is converted to galactose and o-nitrophenol by beta-galactosidase which has a yellow color with an absorbance at 414nm. The amount of ONPG

converted to galactose and nitrophenol by beta-galactosidase is represented by the absorbance reading in each tube. Knowing that, we can then determine the amount of beta-galactosidase enzyme produced in each tubes. For example, the lower the absorbance reading, the lesser the amount of beta galactosidase produced. In turn we can investigate the lax operon expression rate in E.coli in the presence of inducer, repressors and effectors.
Figure 1 shows the amount of beta-galactosidase produced in E.

coli after addition of inducer IPTG at different times. We can see from the graph that it does not cut the X axis at time 0. This is due to the fact that the lac operon was not induced by IPTG due to lack of time. The graph cuts the X axis at roughly 1 and a half minutes after adding IPTG. This indicates the time where lac operon was first induced because there was beta-galactosidase produced. From here we can deduce that production of beta-galactosidase does not take place immediately after adding IPTG but rather takes a while for all the expression staged to be passed by lac operon in order to produce beta galactosidase. From the graph we can see that for the control no beta galactosidase was produced. This is because the control contains water and the repressor was allowed to bind to the operator, causing the transcription process to initiate due to RNA polymerase II not binding to the operator. There is a positive linear relationship between the time of induction with IPTG and the amount of beta-galactosidase production in the tubes. IPTG acts as an inducer, stopping the repressor protein to bind to the operator region by binding to the repressor protein, This causes the lac operon to be

expressed.
From Figure 2 we can see that the amount of beta-galactosidase produced in the tube where lactose is added was quite low. When lactose is present in the tube, it is converted into products namely glucose and allolactose by beta-galactosidase. Allolactose functions similarly to IPTG and binds to the repressor, stopping it from binding to the operator region for lac operon to be expressed. However, allolactose is easily hydrolysed hence it does not induce as much compared to IPTG, causing the graph to appear flatter.
Under the presence of glucose, the graph appeared flat at first but increase a little as time went on. E.coli cells does not prefer lactose as a source of energy. If glucose is present, the cell will use up all the glucose before expression of lac operon. A second level of gene expression control exists to prevent lactose metabolism. Promoter of lac operon contains 2 binding sites, Rna polymerase binds to one while catabolite activator protein(CAP) and cycle AMP binds to another. In order for transcription of lac operon, CAP-cAMP complex has to bind to the promoter site. This complex is only present when glucose is available inside the bacterial cell. When there is low amount of glucose in the cell, the amount of cAMP increases and vice versa. But after cAMP level decreases, CAP+RNA Polymerase II complex inactivates. This causes the promoter to inactivate and shutting off of lac operon. As we can see for both glucose tubes, lac operon was repressed at first but after the glucose is used up lac operon can start to be expressed again.
Moving on to chloramphenicol tube, when it was added in after 10mins, the rate of production for beta galactosidase remained almost constant due to the fact that chloramphenicol is an antibiotic which inhibits bacterial ribosome peptidyl transferase activity blocking the elongation of polypeptide chains which is the beta-galactosidase enzyme.
Rifampicin also acts in a similar way where it inhibits DNA-dependant RNA polymerase in bacterial cells by binding to its beta subunit, preventing DNA transcription and protein synthesis. Streptomycin is also an antibiotic which inhibits protein synthesis. It works by binding to the 30S subunit of the bacterial ribosome, disturbing the binding of tRNA to the 30S subunit. This prevents translation of lac operon mRNA in E.coli and hence beta galactosidase is not produced. These 3 antibiotics keep the amount of beta galactosidase produced the same as before they were added by blocking the expression of lac operon. From these results, we can see that production of beta galactosidase is controlled by the expression of lac operon.