Part:BBa_K1960000
Sensing device with switch for gram-negative bacterium
This part can sense the signaling molecules AHL, then the promoter Lux pR will be activated, CFP and LacI will also be expressed. CFP's product can tested by the fluorescene detection, for respresenting the responder gene's expression. And the Lacl's product can inhibit the expression of λcI gene.
In this schematic diagram (Figure 2), the sensor part is able to sense the signaling molecules.Then the responder expresses. The toggle switch is used to control the product of the lysis gene can make the engineered bacteria cracked. When the switch is in the "ON" state, the expression of the lysis gene is repressed. When the switch is in the "OFF" state, the inhibition of the lysis gene is counteracted. According to our circuit model, we chose the Gram-negative bacteria's quorum Lux sensing system as our sensor part. The toggle switch is consisted of lacI gene and cI gene.
Here is the concrete gene circuit.
In our experiment, to test our circuit more conveniently, we used ecfp (BBa_E0020) and constructed another reporter, BBa_K1960001 using GFP (BBa_E0040) 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 gene’s 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 expresses at the wild-type levels, while lacI-2 and GFP are repressed since the cI protein is high. When AHL around the engineered bacteria reach a certain concentration, it can be sensed by the sensor part so the promoter Lux-pR will be activated, CFP and lacI-1 can also express. Then the CFP can be measured by fluorescence measurement, and the lacI-1's product-LacR protein can inhibit the expression of cI gene. As a result, lacI-2 and GFP can express. The LacI-2's product can further repress cI's expression, then GFP can be measured by fluorescene detection to represent the lysis gene's expression. The time interval of firstly detecting CFP's fluorescence and firstly detecting GFP's fluorescence can represent the delay time which the switch give for responder's expression.
Usage and Biology
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 OD600 of the E.coli and the fluorescence intensity of CFP and GFP for 900 minutes. The OD600 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.
Figure 1.1 The OD-600 of E.coli and its variety with time on the AHL of 20ng/μL and 0ng/μL.
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.
Figure 1.2 The mean cyan and green fluorescence intensity in E.coli and its variety with time on the AHL of 20ng/μL and 0ng/μL.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 985
Experiments
We found that the concentration of AHL could have an effect on the expression of this device. In order to find out the relation, we decided to make a AHL gradient induction for this device. 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 work of this device.
Figure 1 The mean cyan fluorescence intensity in E.coli and its variety with time on the AHL of 1ng/μL, 10ng/μL, 20ng/μL and 0ng/μL.
AHL would influenced the expression of CFP directly. The higher the concentration of AHL is, 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.
Figure 2 The mean green fluorescence intensity in E.coli and its variety with time on the AHL of 1ng/μL, 10ng/μL, 20ng/μL and 0ng/μL.
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.
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