Team:PKU Beijing/Project/AND Gate 1/Inducible System Result

Construction result

Input promoters construction: Before the construction of AND gates, five different inducible promoters were chosen as inputs: pBad (respond to L-arabinose), pSal (respond to salicylate), pLac (respond to IPTG), pTet (respond to tetracycline) and luxP (respond to homoserine lactone). When the inducing small molecules are in presence, the corresponding promoter is activated, for example, IPTG can induce pLac. We have constructed four of these inducible systems (including the activator or repressor protein and promoter), from basic parts, and used one directly from other composite parts. The general pattern is like this:

fig1. Result of construction

They are: BBa_K228004: NahR-pSal system; BBa_K228009: AraC-pBad system; BBa_K228817: lacI-pLac system; BBa_K228818: tetR-pTet system. They are not only composite parts which can be used in our project, but also valuable devices which can be employed in other designs or circuits. For the luxR-luxP system, we simply utilized the parts: BBa_J09855, which is designed and constructed by iGEM2006_Pennstate.

Input promoters test: In order to characterize the AND gate (e.g. the transfer function), the activities of these promoters should be measured independently by constructing GFP (with strong RBS B0034) downstream the promoters and testing the output in response to the small-molecular inducers.

Therefore we successfully fused these promoters to GFP and test results are following:

aTc Sensor

Fig1. the result of tet promoter system.
This is a picture of tet system, the colonies are green under blue light, even without induction, the upper plate is for comparison.

The tetR-pTet system (parts: , mainly by Shuke Wu)
The colonies which contain the aTc sensor - GFP plasmid become very green without induction and showed no induction after aTc is added to the base. The evidence that it is green even without induction is on Fig1, and the data after induction are not showed here.

Discussion: the leakage is obvious and the reasons may lie in the constitutive promoter we used to drive the expression of tetR and the LVA tag of tetR coding sequence. The constitutive promoter we used is pCat([http://partsregistry.org/wiki/index.php?title=Part:BBa_I14033 BBa_I14033]) which is a medium promoter, while the natural promoter of tetR is [http://en.wikipedia.org/wiki/Tetracycline_controlled_transcriptional_activation pTet itself]. It is likely that the expression of tetR is insufficient to repress pTet. Another possible reason is the LVA tail of the tetR(For detail see [http://partsregistry.org/Part:BBa_C0040 BBa_C0040]), and its function is for rapid degradation the protein. As a consequence, there are not enough TetR in presence to repress the pTet.

IPTG Sensor

The lacI-pLac system (Part: , mainly by Shuke Wu) The result is similar to the tetR system. The colonies which contain the IPTG sensor - GFP plasmid expressed GFP even without induction, and the GFP flourescence didn't increase even after IPTG 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, so that pLac () 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: ) 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.

Fig3. lac promoter induction curve.

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.

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

Fig5a, b & c. The fluorescence was measured by flowcytometry to see the fluorescence of each cell in a population.
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

The luxR-luxP system (Parts: K228010, mainly by Min Lin)

We have constructed the plasmid (based on the part:, constructed by iGEM2006_Pennstate. ), and used different concentration of homoserine lactone to induce (See Protocols). The fluorescence of individual cell is tested by flowcytometry. The result shows that this HSL Sensor can be induced to 8 fold with HSL(Fig 5).

However, the basal of this promoter is quite high. And it is later that we found this promoter is not qualified enough to construct our AND Gate(See the AND Gate 1 Result Section).

^Top