Experiment

Entire ciruct test

In our experiment, for testing our circuit more conveniently, we use the cfp gene and gfp gene to replace the responder and lysis respectly when constructing our gene circuit. Because the fluorescence is easier to be measured. Also, the strength of the fluorescence can stand for the strength of the corresponding genes' expression.

When AHL around the engineered bacteria is at low concentration, AHL can't be sensed by the Lux sensor part, and the cI gene is expressed at the wild-type levels. While lacI-2 and YFP are repressed since the CI protein is high. When AHL around the engineered bacteria reach a certain concentration, AHL can be sensed by the sensor part,. So the promoter LuxpR is activated, CFP and lacI-1 are expressed. So the CFP protein can be tested by fluorescence measurement. And the lacI-1' product-LacR protein can inhibit the expression of cI gene. As a result, lacI-2 and GFP can express. The LacI-2' product can further repress cI gene' expression, and GFP protein will be measured by fluorescene detection to represent the lysis gene's expression. The time interval of firstly detecting CFP' fluorescence and firstly detecting GFP' fluorescence can represent the delay time which the switch give for responder' expression.

Result

From the Last September to October, nearly one year of learning and working, we made so many of mistakes in lab work but eventually obtained ideal data and results. From the results we had, we made following analysis of our system.

1. The expression of our gene circuits

The expression of our gene circuits could be induced by AHL. When AHL is sensed by the sensor part, the promoter LuxpR is activated, CFP and lacI-1 are expressed.

The E. coli containing our gene circuits were divided into experimental group and blank control group. We added AHL into experimental group and measured OD-600 of the E. coli and the fluorescence intensity of CFP and GFP for 900 minutes. The OD-600 showed the growth trend of the E. coli. The fluorescence intensity data and time showed the relationship of the expression of CFP and GFP to time.

The growth curves were similar. It was suggested that they had the same growth trend and the difference of fluorescence intensity was mainly from the expression of circuits.

The experimental group and control group all were significantly different. The fluorescence intensity between the two groups were compared with independent t-test. It showed that the cyan and green fluorescence intensity in experimental group started to be different from that in control group in the 240th minute and 420th minute separately. The expression time of GFP was delayed to 180mins post CFP.

2. AHL Gradient Induction

We found that the concentration of AHL could have an effect on the expression of our circuits, in order to find out the relation, we decided to make a AHL gradient induction for our gene circuits. As shown in the graph, the fluorescence intensity data and time on the AHL of different concentrations show the the relationship of AHL to the expression of our circuits.

AHL would influenced the expression of CFP directly. The higher the concentration of AHL was, the more CFP expressed in a range from 1ng/μL to 10ng/μL. In addition, the expression time of CFP was different. T-test showed that CFP in 10ng/μL and 20ng/μL group started to express in the 240th minute and it started to express in the 480th minute in 1ng/μL group.

The concentration of AHL didn't influence the expression of GFP directly. The GFP in 10ng/μL and 20ng/μL group started to express in the 420th minute and it had no significant difference in expression level. The GFP didn’t express in 1ng/μL group.

Responder test

Reference