Difference between revisions of "Part:BBa K568003"

Latest revision as of 21:34, 28 October 2011

T7 promoter lacZ reporter part

beta-galactosidase expression upon T7 polymerase binding

Usage and Biology

This part can be used as a reporter plasmid. It expresses beta-galactosidase if there is a T7 polymerase (or a mutant variant) nearby or transcribed through another part. The polymerase binds to the T7 promoter and transcription of the lacZ-gene can occur, leading to beta-galactosidase expression.

Experimental Testing

The part was tested by the 2011 team of the TU_Munich on the 15th of September 2011. For this purpose, a Miller Assay was conducted using E. coli BL21 (DE3). This strain expresses T7 polymerase upon induction with IPTG.

The experiment was conducted as follows: Overnight cultures of BL21(DE3) transformed with BBa_K568003 and DH5 alpha transformed with Part:BBa_I732017 (as negative control) were set to an OD_600 of about 0.7 and incubated for another 30 min.

A first data-set was recorded. The OD_600 of all cultures was measured as follows:

10 µl of each culture was mixed with 500 µl Z-Buffer, 40 µl chloroform, 20 µl 0.1 % SDS and 430 µl LB medium. After vortexing for 10 sec, the samples were centrifuged for 1 min at 13000 rpm. 20 µl ONPG (4 mg/ml) were added to 100 µl sample. Immediately after this, the reaction was stopped by adding 50 µl Na2CO3 (1 M). The start- and end-time of the reaction was measured. The OD_420 and OD_550 were measured and the Miller Units were calculated using the following formula:

Then, the culture of BL21 (DE3) containing BBa_K568003 was devided into equal portions which were then induced with 0.01 mM, 0.1 mM, 1.0 mM, 1.5 mM or no IPTG. The negative control was incubated with 1.0 mM or no IPTG. After every 30 min, a new data-set was taken.

It was expected that IPTG-induced E. coli BL21 (DE3) transformed with BBA_K568003 should yield higher Miller Units than uninduced ones. The results can be seen below.

Results of the Miller Assay of BBa_K568003. The part was transcribed by genome-coded T7 polymerase, which was induced by addition of varying concentrations of IPTG. The control group contained Part:BBa_I732017 in DH5 alpha cells.

The data shows that the cultures transformed with BBa_K568003 and induced by isopropyl-β-D-1-thiogalactopyranoside (IPTG) yield higher Miller Units than the uninduced culture after 60 min. Therefore, a higher amount of β-galactosidase was produced. This shows that the part works as expected. Furthermore a slight quantitative dependence of the Miller Units on the concentration of IPTG can be seen. After 120 min the Miller Units of the culture induced with 1.0 mM IPTG go down again, which might be evidence for increasing of proteolysis caused by death of bacteria. The reason for this might be a too high concentration of IPTG which results in extreme over expression of protein which might "exhaust" the cells and thus kill them. It can also be observed that the uninduced culture yields higher Miller Units than the negative, induced control. The control should not be able to express β-galactosidase, as it lacks a necessary promotor on the plasmid. The significantly higher Miller Units of the uninduced cultures with BBa_K568003 can be explained by leaky biosynthesis of the genome-coded T7 polymerase in the BL21(DE3) cells. If the production of T7 polymerase would be more tightly controlled, the level of expressed β-galactosidase should go down to the minimum of the negative, induced control.

The experiment was repeated, this time using a positive control consisting of BBa_J23100 + BBa_I732017. However, as it did not work as expected, the data of the positive control was not used.

Results of the Miller Assay of BBa_K568003. The part was transcribed by genome-coded T7 polymerase, which was induced by addition of varying concentrations of IPTG. The control group contained Part:BBa_I712074 in BL21 (DE3) cells.

Again, the experiment worked as expected. It shows that the level of β-galactosidase activity increases strongly after 30 min of incubation with IPTG. After this induction, the level seems to stay at this level. There is also an increase of β-galactosidase activity by the un-induced control after 30 min. This might be explained by a leaky expression of T7 polymerase. However, the value decreases again after 90 min, which cannot be satisfyingly explained. We can also see that BBa_K568003 works in a quantitative manner, as increasing concentrations of IPTG yield more Miller Units. In general, the measured activity is much higher than in the first experiment. This is most likely due to a slightly different experimental approach, as we started the experiment with significantly lower OD_600 values. Thus, the bacteria had more IPTG available to start expression and suffered from less stress due to overgrowth.

Sequence and Features