Difference between revisions of "Part:BBa K568003"

Revision as of 13:41, 21 September 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 25th of September 2011. For this purpose, a Miller Assay was conducted. It was expected that a transformed culture induced with IPTG should yield higher Miller Units than an uninduced one. 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 strain was not able to produce T7 polymerase.

It shows that the cultures induced by isopropyl-β-D-1-thiogalactopyranoside (IPTG) yield higher Miller Units than the uninduced control after 60 min. Therefore, a higher amount of β-galactosidase was produced. This shows that the part is working 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 due to a too high concentration of IPTG. It can also be observed that the uninduced control yields higher Miller Units than the β-galactosidase negative, induced control. This can be explained by leaky biosynthesis of the genome-coded T7 polymerase. If the production of T7 polymerase is more tightly controlled, the level of expressed β-galactosidase should go down to the minimum of the negative, induced control.

The experiment was conducted as follows: Overnight cultures of BL21(DE3) transformed with BBa_K568003 and DH5 alpha transformed with [1](as negative control) were set to an OD_600 of about 0.7 and incubated for another 30 min. A first data-set was taken. The OD_600 of alle cultures was measured. 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 and 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 derived using the following formula: Then, the BBa_K568003 strain was 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.

Sequence and Features