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3.Response Concentration:</br> | 3.Response Concentration:</br> | ||
Next, we designed experiments to verify the response concentration. Through these experiments, we can also conclude under which concentration can the AND gate have the best effect. We designed a series of gradient about how many inducers should be added. The concentration of IPTG and arabinose varied from 10^-3, 10^-4, 10^-5, 10^-6, to 0. Following is the result.</br> | Next, we designed experiments to verify the response concentration. Through these experiments, we can also conclude under which concentration can the AND gate have the best effect. We designed a series of gradient about how many inducers should be added. The concentration of IPTG and arabinose varied from 10^-3, 10^-4, 10^-5, 10^-6, to 0. Following is the result.</br> | ||
− | <div align="center"> | + | <div class="row"> |
− | <img src="https://static.igem.org/mediawiki/2016/ | + | <div class="col-md-6"> |
+ | <div align="center"> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/6/61/Logic_gate_absorbance.png" width="75%"> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | <div class="col-md-6"> | ||
+ | <div align="center"> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/9/9b/Response_concentration.png" width="75%"> | ||
+ | </div> | ||
+ | </div> | ||
</div> | </div> | ||
Seeing from the figure, with the increase of the inducers’ concentration, the value of fluorescence also increases. The figure has met our expectation. However, there has a peak when adding IPTG of 10^-7mol/L and arabinose of 10^-6mol/L, and we think this is because inaccuracy caused by measurement. | Seeing from the figure, with the increase of the inducers’ concentration, the value of fluorescence also increases. The figure has met our expectation. However, there has a peak when adding IPTG of 10^-7mol/L and arabinose of 10^-6mol/L, and we think this is because inaccuracy caused by measurement. |
Revision as of 15:14, 19 October 2016
Light Control
We have successfully constructed plasmid used in the light-induced system and achieved co-transformation in E.coli JT2. After then, we have tested the light sensitivity and the response time.
Construction of Plasmid
(1) The construction of Part
(2) The green light system
(3) The red light system
(4) The result of co-transformation
About response
Delay
We observed that the sharp increase of fluorescence mainly happens after 3 hours. And this sharp increase is really a helpful index for us to test the system. Thus, we think the response time of TCS is between 3 to 4 hours.
In general, we have verified that these two systems are feasible. However, they did not match up to our expectations, and the results seemed far different from our references. Following are the possible reasons:
1、The results can be affected by the copy number of plasmid and the replicon’s intensity, especially for the red light TCS.
2、Maybe this is because the LED intensity was not strong enough. Also, we used LB medium and it absorbed light of different wavelength, making light attenuate too much and could not activate TCS effectively.
3、The antibiotics we used may affect the system’s expression.
Future plan
1、Decrease the leak level. In the light-induced system, the reporter gene’s leak will affect our measurement. What’s more, it may even lead to unexpected errors for users to read the information. In order to avoid such errors, we have several alternative plans:Use a new light-induced system - we have already found a system using blue light. Keep balance between every components by changing the intensity of rbs. Keep balance between every components by changing the copy number of plasmid and the replicon’s intensity.
2、Future measurement of the red light system after inserting NOT gate.
3、Mix bacteria of the green light and red light system together, and measure their response towards light.
4、Analyze the response curve, to find the optimum light intensity.
5、Replace LB medium as M9 medium and compare their results.
6、Explore the optimum quantity of antibiotics.
The key points for repeating experiment
1.When constructing plasmid, with the existence of double terminator, it is prone to appear disorder when using overlap PCR to join two segments together. Using seamless connection to construct plasmid is also prone to appear deletions of segments, and we can consider to use the method of enzyme-digestion and enzyme-connection.
2.When measuring the fluorescence expression of bacteria after co-transformation, the results are affected by medium. Thus, we recommend to centrifuge first, then use PBS buffer solution to clear the remaining medium. These two steps can be repeated by 2 times in order to preclude the effects caused by medium.
Logic Gate
Overview
We simplified the three-plasmid system based on our reference by constructing all the segments in one plasmid. And we tested in E.coli MG1655. We used arabinose promoter (PBAD) and lactose promoter (Plac) as inducers and we have verified the corresponding concentration and time.
1. Construction of Plasmid:
Part:
First, we constructed the input plasmid and output plasmid.
Input:
Oscillation
To describe the single-period oscillation system in a better and more precise way, we measured pluxR promoter’s response states towards AHL of different concentration. By using PCR and one step cloning, we removed luxl gene (fig1a, fig1b), and thus the plasmid, pTD103luxI-sfGFP, was mutant. In this way, we excluded its interference to our experiment.