Team:PKU Beijing/Project/AND Gate 1/Inducible System Result
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==='''IPTG Sensor'''=== | ==='''IPTG Sensor'''=== | ||
The lacI-pLac system (Part: <partinfo>K228819</partinfo>, mainly by Shuke Wu) | The lacI-pLac system (Part: <partinfo>K228819</partinfo>, mainly by Shuke Wu) | ||
- | The result is similar to the tetR system. The colonies which contain the plasmid | + | The result is similar to the tetR system. The colonies which contain the IPTG sensor - GFP plasmid was leaky and expresses GFP even without induction and flourescence didn't increase either after induction. The evidence that this construct is leaky without induction is on Fig2, and the data after induction are not showed here. |
- | Nevertheless, we found another way to bypass the problem. That is making use of the lacIq mutation on the F plasmid of ''E. coli'' strain [http://ecoliwiki.net/colipedia/index.php/JM109 JM109]. Because the F plasmid is one copy per cell, | + | Nevertheless, we found another way to bypass the problem. That is making use of the lacIq mutation on the F plasmid of ''E. coli'' strain [http://ecoliwiki.net/colipedia/index.php/JM109 JM109]. Because the F plasmid is one copy per cell, so that pLac (<partinfo>BBa_R0010</partinfo>) should be on a low copy plasmid such as pSB4K5. The pLac can be repressed by endogenous lacI even without its represssor coexpressed on the same plasmid. Thus we constructed the simplified system (Part: <partinfo>K228821</partinfo>) and induced it by different concentration of IPTG. The result is shown in Fig3, judging from the induction curve, pLac promoter may be qualified in constructing the AND Gate. |
- | [[Image:PKU_Lac_1.png|500px|center|thumb| | + | [[Image:PKU_Lac_1.png|500px|center|thumb|Fig3. lac promoter induction curve.]] |
- | However, there is still | + | However, there is still another problem in this simplified system: the loss of F plasmid of JM109 may lead to activation of promoter pLac without induction. From the plate (without induction), we found that part(esspecially the central part) of a singal colony turned green, while they are supposed to stop express GFP in the presence of lacIq. A stronger evidence is the data from flowcytometry: The strength of GFP fluorescence obviously shows double peaks. All these suggest that some of the cell retains the F plasmid while the the others lost it. |
- | [[Image:PKU_lac_1.png|500px|center|thumb| | + | [[Image:PKU_lac_1.png|500px|center|thumb|Fig4. the JM109 colonies that contain low copy pLac-GFP plasmid. Center of the colonies turns green, while in the other area GFP expression is suppressed. It suggests that some E.coli lost their F plasmids]] |
- | [[Image:PKU_lac_2.png|600px|center|thumb| | + | [[Image:PKU_lac_2.png|600px|center|thumb|Fig5a, b & c. The fluorescence was measured by flowcytometry to see the fluorescence of each cell in a population. <br>Fig5a illustrates that there are leaky cells and normal cells without induction. Fig4b shows the data from the cells induced by 10^-6M IPTG, and Fig4c show those induced by 10^-5M IPTG. Double peaks can be observed on both 4a and 4b, which means there are two groups of E.coli, but as the induction saturates, two peaks merge into one peak.]] |
- | + | In a word, the lac promoter is not a perfect promoter to construct AND Gate, however, if a more stable source of lacI can be supplied, we believe it would be better. | |
==='''HSL Sensor'''=== | ==='''HSL Sensor'''=== |
Revision as of 06:45, 20 October 2009
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