Difference between revisions of "Part:BBa K3209006"

Revision as of 10:25, 20 October 2019

GFP generator driven by Plac, containing LacI gene

Overview:

Plac is one of the most common promoter in life science research field. It is mainly composed of Lac operon containing LacO site. LacI repressor, encoded by LacI gene, can bind with LacO site to inhibit the binding of RNA pol to the promoter, so the genes downstream expression are blocked. Serving as inducer, IPTG can bind with LacI inhibitor, making the latter’s conformation changes, so LacI is detached from LacO site, which enables the transcription of downstream genes. BBa_K741002 is a GFP generator driven by Plac promoter, however there is no LacI gene in it. Although the E.coli could express some LacI, it is not enough for inhibition GFP expression. So this GFP generator has some leakage expression, like the designer stated, even some GFP express without IPTG inducer presence.

We constructed a new GFP generator driven by Plac promoter and contains LacI gene, which can lower significantly the leakage expression. Both LacI and EGFP are linked to the downstream of Plac, which is regulated by LacI inhibitor and IPTG inducer. Using EGFP as a reporter, its fluorescence intensity appears a lower leakage expression. This new GFP generator could be self-regulated because LacI protein can inhibit its self expression, so that no excessive LacI expression which is considered as waste of resources. We detected the response of this generator to different concentration of IPTG, indicating that it could be inhibited by LacI, and induced well by IPTG.

Results:

First, we compared the inducing effect on the two GFP generators, using different concentration of IPTG. We set 4 groups: 2 experimental groups including old GFP generator (BBa_K741002) and new GFP generator (BBa_K3209006), one negative control without GFP expression and one positive control with constantly GFP expression. At 0h, all groups’ OD600 approximately reaches to 0.8, then certain concentration of IPTG was added to the culture medium, incubated cells at 22℃ overnight. Measure the fluorometric value at 510 nm and OD600 value for each group every 1h, using an automatic microplate reader. Sequence and Features