IGEM:UNAM-Genomics Mexico/2009/Notebook/Collaborations/2010/08/25

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Shipment to Edinburgh team
Hello Mexico!

Thank you very much for the papers and your last e-mail with all the links- it's really useful.

I've recently tried to fix our LovTap by introducing missing base, subculturing, minipreping etc. I've submitted the newst clones to sequencing and hopefully tomorrow I'll get the results back, so we can retransform the cells and get to the characterization assay.

I didn't have a chance to work on the pcb promoter, I've passed your e-mail to Will and hopefully he will get back to you soon about this.

Also, as I'm going for a holidays on Friday, Maria will take over the LovTap and other experimetns from me. Will is mainly working with Luciferases. I'll forward emails to them if you send them to me,if I can't answer myself. But feel free to still contact me.

Once we have our parts ready we'll send them to you- the team will keep you updated. Thank you for your parts.

Good luck with all your work. Again- if you have any questions I'm happy to answer or you can contact Maria or Will- here are their e-mail addresses:

Maria- M.T.Kowal@sms.ed.ac.uk

Will - W.G.P.M.Rostain@sms.ed.ac.uk

Good luck with the lab work!

Marta

Answer quoting chernand@lcg.unam.mx
Hi Edinburgh Team!

We have been working during this week in order to have the shipment ready. As the process of restriction enzyme assays, DNA gel extraction and ligations of the synthesized constructions is very slow, taking into account the experience working with LovTAP. We considered that the best option will be to send you the PCR products from the parts synthesized.

We are planning to send you:

-CcaS&CcaR PCR product. -LuxY PCR product. -LovTAP PCR product.

All these sequences have the prefix and suffix sequences from the biobrick assembly protocol in the ends.

If LovTAP ligation with plasmid pSB1C3 is successful, we are going to send you it, too.

The experimental details of the PCR reactions and LovTAP gel extraction and ligation can be found in the next links:


 * CcaS&CcaR PCR / LovTAP gel extraction and ligations

http://openwetware.org/wiki/IGEM:UNAM/2009/Notebook/Modeling_logbook_Claudia/2010/0823#Working_on_CcaS_and_CcaR_synthesized_plasmid_from_Mr.Gene:_Edinburgh_Shipment


 * LovTAP and LuxY PCRs:

http://openwetware.org/wiki/IGEM:UNAM/2009/Notebook/Modeling_logbook_Claudia/2010/08/24#Working_on_LovTAP_and_LuxY_synthesized_plasmids_from_Mr.Gene:_Edinburgh_Shipment

Explore the logbook for more information and check it continuously for future updates.

Augusto had to repeat the LuxAB extraction from Vibrio fischeri genomic DNA, the primers that he is using already include the prefix and suffix sequences to assemble the LuxAB amplified product with the biobrick standard protocol. We could send you this PCR product if you were interested.
 * LuxAB

One strain harboring the luciferase mutant S284T, was analyzed in a preliminary test using luciferin and waiting 30 seconds of exposition, but there was no bioluminescence emission. The same happened with the WT luciferase. I don’t have all the details of the experiment, but those were the results. Mariana and Enrique will repeat the assay with other colonies and inside dark room. If we have the functional mutant, we will send it to you.
 * Luciferase

The blue promoter inside plasmid pSB1C3 is ready, maybe we could send you the promoter ligated to GFP, Jorge is checking the ligation.
 * Blue promoter

We are planning to send the parts on Thursday or Friday.

During the weekend we are going to update the information of the constructions that you will receive, thus you will get totally familiarized with them. We remain at your disposition for any question.

For us, it will be great to receive:

-LovTAP Read out system 1

-Cph8 PCR

-Mutated Luciferase 356K

-The plasmid harboring the genes to produce PCB in E.coli. (If you get it first)

If you have any suggestion or comment about the shipment, let us know. We are delighted to help you.

Best,
 * D

UNAM-Genomics-Mexico TEAM


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