construction result
Input promoters construction:
In the AND gate, we used five different inducible promoters as inputs: pBad (respond to L-arabinose), pSal (respond to salicylate), pLac (respond to IPTG), pTet (respond to tetracycline), luxP (respond to homoserine lactone). When the inducing small molecule in presence, the promoter is activate, for example, IPTG can activate pLac.
We have constructed four of these inducible systems (including the activator or repressor protein and promoter) and used one directly from other parts. The general pattern is like this:
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 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
The tetR-pTet system (parts: K228820, mainly by Shuke Wu)
The colonies which contain the plasmid become very green without inducing and are not greener after inducing. The evidence without inducing is on Fig1a, and the data after inducing 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 which is a medium promoter (refer to parts BBa_I14033: http://partsregistry.org/wiki/index.php?title=Part:BBa_I14033), while the natural promoter of tetR is pTet itself (http://en.wikipedia.org/wiki/Tetracycline_controlled_transcriptional_activation). It is likely that the expression of tetR is insufficient to repress pTet. Another possible reason is the LVA tail of the tetR (refer to BBa_C0040 http://partsregistry.org/Part:BBa_C0040), and its function is for rapid degradation the protein. As a natural result, the amount of tetR may not enough to repress the pTet.
 Fig1. the result of tet & lac promoter systems. Fig 1a is tet system, the colonies are very green under blue light, without inducing, and the upper plate is for comparison. Fig 1b is lac system, the colonies are very green under blue light, without inducing, and the upper plate is for comparison. IPTG Sensor
HSL Sensor
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