IGEM:UNAM-Genomics Mexico/2009/Notebook/Collaborations/2010/08/05

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Working on LovTAP:Communication by E-mail

Hi Claudia,

thanks for your e-mail. We did visit your collaboration web site, in case of any doubts I'll let you know- it looks fine now :)

About LovTap- there is a Trp deletion strain in our lab and we will use that with LovTap construct as well. Read out syst. 1 (K191004)alone grows red in the dark on normal medium, which suggests that it is not repressed by the trp from the medium. It might be a case of trp concentration, or there is a different repressor in the original biobrick construct.

Right now I've obtained double transformants- with LovTap and read out system 1. We are trying to confirm that they are really double mutants, as so far they haven't provided expected results- not all of them are red in the dark :/ We are not considering temperature sensitivity yet, we'll deal with the degradation tag later as well- we are trying to rule out simple reasons first.

Once we get this parts to work, we will characterise it along in the TrpR deletion strain. Do you have problems with obtaining it? Originally it was bought commercially for some other project in the lab, so we are not sure if it's legal for us to send it over to you; we can find out if you have any problems with obtaining it.

We would be grateful for LuxY, promoter for the inducible blue light sensor and LovTap - in case our won't be working properly by then. About Lausanne- I didn't contact them initially, but I asked my colleague- Will- to send an e-mail, asking if we can pass their e-mail addresses we used. It took us a while to get a response as well and our e-mail was passed along few times, so they still might get back to you within few days. We also asked about the sequences of the plasmids that the parts were supplied in- as soon as we hear back from them I'll let you know.

About E. coli blue light sensor- it would be good if you could send alone the promote together with the synthesised parts- I guess you will focus your work on the new parts, so we could help you in optimising the promoter and running some experiments if it's ok for you.

Hope you will get your parts soon and your work will be successful! :)

Marta

Quoting chernand@lcg.unam.mx:

¡Hola! (Hi!) Marta

I would like to know if you have visited the Collaborations Log that we link in our wiki, I´ve been updating it, with the aim to document the transfer of information between our teams. I would like that you visited to know if your team agree whit the content that we are reporting, in order to establish the collaboration under the best terms.Any comment you have, just let me know. I think that there won’t be any problem. :D

I will scan the documents that came with the package that we recieved from you, and I will submit them to the collaboration log.

Answering to your mail, I think that at high expression levels of LovTAP you probably will lose the specific regulation under dark-state and light-state according to Devin´s advices. I´ve been updating my OWW personal page, there is a section of Details to be considered to work with LovTAP, check it. We are planning to use weak and medium strenght promoters.

Jorge, other member of the team will send you information about the experimental design that used to probe the native E.coli blue- light inducible promoter. We plan to use the same to characterize LovTAP. There you will notice that as negative controls we are stimulating the promoter under red and green Leds, so besides probing if daylight stimulate LovTAP, I think that it would be a good idea to include those negative controls.

Another condition to take into account is the possibility that temperature could also regulate LovTAP and the blue promoter. I am attaching the reference paper. Check the last paragraph on page 7.

By the way, how are you avoiding the crosstalk of native trpR repressor with LovTAP response, with a strain E. coli mutant or with the tryptophan concentrations in the medium?

During the weekend , I tried to contact a member of Lausanne 2009 team to ask if they could send us the two read out systems that they used, but I hadn’t receive response yet, do you have any mail address to contact Lausanne team?.

As I understood, with the reporter system 1 you want to obtain a repression response once LovTAP is activated. In our case we want an activation response, so that we designed a new reporter system 2, using a repressor from lambda phage instead of TetR. And we also need to add a degradation tag (LVA) on the lambda phage repressor. It seems a good idea to characterize both team’s particular systems with tag and without it.

Returning to the Parts shipment that we have received from your team, we could send you our LovTAP and LuxY synthesized, as well the blue light inducible promoter inside the plasmid PSB4A5 (up to now we can´t ensure that is working well. In the only experiment that we have done, the induction seems to be really very weak, we have to repeat the experiment more times). What do you think?

We hope that our synthesized Parts will arrive next week, actually they are at Customs Office.

The weather in Cuernavaca, the city where is my University, is so crazy!

Yesterday were really raining cats an dogs but other days are very sunny. Some times both stages are possible in the same day.

Well, see you! ;D

Claudia

CcaS and parts to synthesize (UNAM-genomics MEXICO)


Dear Mariana,
I am Lu, part of Edinburgh iGEM team. How is your project going? We are now designing the green light sensor based on the priciple of fusion protein between CcaS and PhoR.
Have you got the order of the CcaS? If so, could you please send its DNA to us? We tried to contact with the last year's Japanese team which designed a green light sensor, but they didnot reply.
If anything you would like to know about our work, we are quite willing to share information with you. Thank you very much!
Best wishes!

Lu and Edinburgh iGEM 2010 team
-- The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336.



Dear Lu,
It is really nice to get in touch with you too. Hope everything is going out well with your project.
As you should know by now, our team is waiting for the arrival of the synthesized parts. They should be here by tomorrow (or at least they are supposed to be); however, we will upload the information about the design of the parts this weekend, so you get the sequences and specific information of your interest. These are the parts that we are expecting:

  • LuxY
  • CcaS-CcaR
  • Regulated region of CcaR
  • LRE
  • LovTap (modified)
  • BBa_K23803 (modified/reduced)
  • BBa_K23803 (modified/reduced and with RBS and GFP: BBa_E0240)


About my part on the project, I'm working with the mutation on luciferase. After a few complications, I assume to have them fully transformed and proved by next week. How are you doing with luciferase? Did you get to have a better signal than the previously measured?
Please let us know if there are any parts of your interest besides CcaS and LuxY so that we could send them to you during next week.
The best of luck!
Mariana and the UNAM-genomics Mexico team.




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