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LovTAP, blue promoter and Shipment
Thank you so much for staying in contact and sharing me your experiences in wet lab. They are very useful for me. ;D
About trpR-, we have the strain CY15001 that Charles Yanofsky kindly provided us, but I have to test it, I found that the mutation is a frameshift, so I have to do PCR and then sequence the products. It would be great if you could send us your mutant, in case that our mutant doesn’t work properly.
We tested the blue promoter using a GFP reporter genes, Jorge did the assay using a fluorimeter. Check this entry at his logbook
The time ranges units are minutes.
The reason to use different time intervals of light irradiance is that we want to obtain the curve of saturation of the time- light-dependent promoter activity.
We have a lot of crude data from this experiment, but the principal problem was that the negative control –the medium (LB)- emitted the highest fluorescence , with this unexpected background we can´t ensure that the reported activity of the blue promoter -very weak -is light dependent because the medium could be responsible of the fluorescence emission.
Besides, we have the temperature factor, so that we have to try different conditions. We haven´t been able to try another experiment because the fluorimeter was calibrated, and the responsible has been very busy. It seems a good idea if you also try to optimize our advances with the blue promoter.
We are working on make a more detailed explanation of this experiment at the logbook, you will understand much better.
On the other hand,I would like to ask you:
-How much time of light irradiance you are planning to use to activate LovTAP and at what wavelenght?
-What kind of luminometer do you have at your lab? It seems that the two that are available in our research center are very old.
-How your team is going with the genes pcyA and ho1 that synthesize the PCB chromophore of the red and green photoreceptors?, because we don´t have them and without them we won´t be able to test the green photoreceptor.
Up to now, we haven´t received yet the synthesis order from Mr. GENE.
I talked with the team and we wanted to ask you if your team would agree in provide us more parts- we would plan a list according to all the information that we have already received from your team-, if you accept the idea would be to plan the shipment as soon as posible, because if we do that later maybe we will receive them too late to work with them -considering our Customs problems-. As well, if you consider that some of our not synthesized parts are primordial for you, we could send them as soon as you tell us, thus avoiding to delay both team projects. What do you think?
We will be waiting for your answer.
Green Receptor & Shipment
Hi Lu, this is Hector from UNAM-Genomics Mexico.
Mariana is out of town for the weekend, so I'm replying for her.
CcaS did not arrive Friday unfortunately. We are expecting it on Monday (hopefully...), or maybe throughout the week.
Earlier this year we were in contact with Stefano Ferri (TokyoNokogen 2009 Instructor). He told us that they were going to characterize their iGEM 2009's parts this year, however we haven't heard from him recently. Since we require pcyA and ho1 for both red & green receptors, and I15008 & I15009 also have issues, we are planning on asking Mr Ferri for their K225001. We wrote to them last week and are still expecting a reply.
If you require further info, we will be happy to provide it.