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Team:KULeuven/Data/Other Parts
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Contents |
Introduction
As Dr.Coli is a very extensive project, so there was not enough time to test all the new parts that we created. This page contains a summary of how our new basic parts could be tested (the construct that have to be made and the actual testing protocol). It will also give an overview of the results we had so far. We worked as much parallel as possible, building different parts and subsystems at the same time. This way we could still have some results, even when some of the parts didn't succeed. All the new parts introduced in our project were properly built. The actual testing of these parts will have to be done be next year's teams, our advisors or some other students.
Antisense LuxI
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K145013 Parts Registry:K145013]
Construct and testing protocol
Antisense LuxI (asLuxI) is an RNA molecule that can bind to the mRNA of luxI, thus repressing the translation of the LuxI enzyme. Testing this new part would not be very easy. One would need two rather complex systems :
| System 1: |  | + | .JPG){kind=small} | + | .JPG){kind=small} | + | .JPG){kind=small} | ||
| System 2: | | + | | + | | + | | + | .JPG){kind=small} |
In system 1 the Lux repressor (LuxR) is produced constitutively. On its own LuxR will not do much, but when it is bound to HSL it can activate the transcription of GFP from the lux promoter (R0062). HSL is a molecule that can be generated by LuxI, and the production of LuxI depends on the presence or absence of aTc. So, when no aTc is present, there will be no LuxI and therefore no HSL. This means no production of GFP. When aTc is added to the system, LuxI will be produced and so will HSL. HSL can then bind to LuxR and start the transcription of GFP (fluorescence should become detectable).
System 2 works very much in the same way, but now we also have the constitutive production of antisense LuxI. This means that, when aTc is added to the system, transcription of luxI will occur. However, the protein LuxI will not be produced because asLuxI will bind the mRNA of luxI. No LuxI means no HSL and this means no GFP. In system 2 the production of GFP will never occur, with or without aTc.
To test this, one should built these 2 systems and transform them into E.coli cells. After that you can prepare liquid cultures of the 2 systems. Then you should add aTc (100 ng/ml) and follow the fluorescence of the liquid cultures in time (e.g. with FACS). If all goes well, you should see the fluorescence of system 1 increase, while the fluorescence of system 2 should remain low.
Results
Antisense LuxI ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K145013 K145013]) was constructed with PCR. The template was the plasmid containing the luxI BioBrick ([http://partsregistry.org/wiki/index.php?title=Part:BBa_C0061 C0061]). We used the following primers :
This PCR succeeded very well and we then cut the PCR product with EcoRI and SpeI. This digest was consecutively ligated into a pSB1A2 plasmid with T4 DNA ligase. This plasmid was then electroporated into TOP10 cells and these cells were grown on agar plates containing ampicillin. Everything went well and also sequencing showed that part K145013 was built correctly !
T7 RNA polymerase (with and without UmuD)
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K145014 Parts Registry:K145014] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K145001 Parts Registry:K145001]
Construct
Results
Hybrid promoter
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K145151 Parts Registry:K145151]
Construct
Results
ccdB
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K145150 Parts Registry:K145150]
