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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
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 (K145013) was constructed with PCR. The template was the plasmid containing the luxI BioBrick (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)
Parts Registry:K145014 and Parts Registry:K145001
Construct
T7 RNA polymerase is an enzyme that will start transcription from a T7 promoter. We use this part in our filter and there it is necessary that this enzyme is degraded quickly. Therefore we also constructed a T7 polymerase with an UmuD tag. This tag will render T7 RNA polymerase susceptible to degradation by certain proteases. To test whether this tag really makes T7 polymerase degrade faster, one should build the following constructs:
| Construct 1 (no tag): | | + | .JPG){kind=small} |
| Construct 2 (UmuD tag): | | + | .JPG){kind=small} |
In these two constructs, TetR is constitutively produced and this will repress the production of T7 RNA polymerase from the tet promoter (R0040). Adding aTc will render TetR inactive and T7 RNA polymerase will be produced. So, in order to test the degradation of the enzymes, you should grow liquid cultures containing aTc of the cells with construct 1 or construct 2. These cells will start to produce T7 RNA polymerase or T7 RNA polymerase with UmuD. When these cells have been growing for a whole night, you can transfer them to another medium that does not contain aTc. The production of T7 polymerase will then stop and you can now measure the degradation rate of the T7 RNA polymerases. To do this, you should harvest some of the cells at several time intervals. Then you should isolate the proteins from the cells and determine the concentration of T7 RNA polymerase using anti-T7 antibodies. The tag works if the concentration of T7 RNA polymerase with UmuD tag decreases much faster in time than the concentration of normal T7 RNA polymerase.
Results
We constructed both T7 RNA polymerases with a PCR reaction. To built the T7 RNA polymerase without the tag (K145001), we did a PCR with pfx polymerase on the plasmid of I712022 as template and with the following primers:
To attach an UmuD tag to the T7 RNA polymerase (K145014) we did a two-step PCR. The first PCR was done with I712022 as a template and the following primers:
The second step of the PCR was performed on the unpurified PCR product of step one and with these two primers:
It took very long, but eventually the two PCRs succeeded. These PCR products were then cut with EcoRI and XbaI, and these digests were ligated into pSB1A2 plasmids. These plasmids were electroporated into TOP10 cells and the obtained colonies were miniprepped. Sequencing showed that both PCRs succeeded very well and that we made the correct constructs (K145001, K145014).
The first part of the test constructs (J23116+B0034+C0040+B0015) is actually our input and this was properly built, see here. We also tried to build the second part (R0040+B0032+T7polymerase+B0015), but various attempts to ligate the T7 polymerase genes to B0015 failed.
