IGEM:UNAM-Genomics Mexico/2009/Notebook/Collaborations/2010/09/08

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Shipment recieved and updating progress in each team projects
Hi guys,

We're sorry we haven't been in touch, we're down to three of us in the lab now so things are getting busier for each of us.

Thank you very much for sending the parts. We will start working with them as soon as possible. Just so we know, have all illegal restriction sites been removed from the synthesised products? We noticed one of you submissions (CcaS + CcaR) was incompatible with the registry's formats, but then we  figured you had left the biobrick standardised ends on the sequence :P. Can you link me to any information about the parts you've sent us? Our supervisor would like to read up on any known data, e.g. sequences and whether the PCR products have standardised biobrick ends (if not, is there enough room at either end to add them?).

You said you were working on a frameshift mutation in the trpR gene (which I assume you're combining with Lov-Tap). We've not come across anything so far as I know, but I will double check with Chris and Marta. We've been working a lot on trying to get a frameshift mutation out of Lov-Tap but with no success. I think we're giving up and looking at YcgEF instead - natural E.coli protein with no innate restriction sites - useful! :D However, if your version of lov-Tap is the correct one we may start working with that.

We are able to send you the lov-Tap readout system and the PCB production plasmid but unfortunately we are still unable to produce a registry-standard cph8 clone or firefly luciferase mutation. Would you like us to send you just the two? We have almost fixed the red light sensor we think (apart from cph8, obviously) so if you want us to continue with it while you work on the green light sensor (once we can  send you said cph8) we will.

We've had some success with the lux genes. We've managed to fuse luxAB with lumP and Chris got luxCDE to amplify out at last, so we'll be fusing that soon. Hoping to test and characterise the full construct at some point in the next few weeks. Would you like a copy of these? Pity you lost the Ppy stuff. We haven't attempted to mutate the RBS yet, but if you need more wild type we do also have some of that.

If, based on this, you want to send a revised shipment list, we can get that organised and send it to you either this week or next.

Sorry again for not replying sooner.

Hope you're all well.

Maria (and what's left of the Edinburgh iGEM team).

Quoting chernand@lcg.unam.mx:

Hi guys!

How is all going with your Project?

We are wondering if you have already received the shipment, in DHL webpage the status of the package is that it was delivered on Monday during this week.

We have started working on submit DNA sequences of the parts in the registry, we hope to have ready the annotations along this week. Working on LovTAP, Jorge Zepeda and I are still working with the regulatory region (trpL – mistakenly named trpR- promoter ligated to GFP and the CI inverter), besides I am trying to join LovTAP with the promoters, to plasmid psb3k3  for characterization.

I received the sequences of the trpR gene (from mutated cells and WT), I was looking for a frameshift inside the gene, according to the reports at the literature but I found a non synonymous mutation -alanine to proline- I don´t remember the position, I will check my notes. Dr. Miguel, our instructor told me that this change is very serious for the protein structure, so we are trusting in that the trpR gene is mutated. I would like to ask if you have more detailed information about your trpR mutant.

Mariana is now using Click beetle luciferases from Promega (they emit red and green bioluminescence) and is trying to get the Luciferase under the T7 promoter that is already in the registry. She lost the samples from the Photinus Pyralis luciferases with the RBS mutated.

LuxAB from Vibrio Fisheri has been very hard to ligate, because its PCR reaction seem to have a 600nt fragment that successfully ligate but It is not the correct one, thus Augusto purified the LuxAB band from gel and tried to join it to a plasmid. It seems that there are some transformed cells but now he has to test them.

We haven´t worked on CcaS photoreceptor neither with Cph8 because we haven´t receive any response about PCB synthesis genes.

Well, I think that is all for now.

Feel free to contact us for any doubts about the shipment.

Best,

Claudia


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