IGEM:UNAM-Genomics Mexico/2009/Notebook/Collaborations/2010/10/27

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Communication during the last days: Wiki Freeze is today
Hey guys,

How's it going? Do you think you'll have everything up before the wiki freeze?

We never got any decent results from our LovTap readout, nor from the red light sensor. Did you ever get anywhere?

We did manage to get a decent-ish spectrum for our red luciferase and we've built but not submitted or tested the green light sensor.

Our side project, the genomic insertion stuff, also hasn't produced anything in the way of actual results, although we've gotten a couple of decent experimental write-ups out of it.

There's a girl in our class who will be taking over the light stuff as her final year project in January. We feel quite sorry for her.

We're going to keep working for the next week just in case we get results before the Jamboree. Are you planning to do the same or will you just work on your presentation and poster?

Hope you guys are all well and not as stressed as I am...

See you soon :)

Maria.

E-mail response: Communication during the last days: Wiki Freeze is today
Hi Maria!

Great to hear news from you in this crazy day when wiki freeze.

My team is also working during this last part of the iGEM, even we have finished the construction of some devices (luciferase+LRE, Lumazine and LuxY co-transformations with LuxABCDE operon, blue light inducible promoter and LovTAP co-transformations with RFP read out system) and tried to characterise them, our data don't seem to be very good.

Lumazine and LuxY co-transformations with LuxABCDE operon, don't show the expected wavelenght shift. Temperature and the usage of a correct measurement device are the principal issues.

Luciferase+LRE. We are not very sure that the ligations were performed correctly, as well our luminometer is not the best device to make a good kinetic analysis, besides that one guy of my team spend 4 hours yesterday trying to get some good data, such a crazy experience and quantity of data in a large strip (it will remain in my memory as the most crazy characterisation attemp working in the iGEM). But unfortunately the data are very rare.

Blue light inducible promoter, after repeating the ligations with GFP, Jorge did an assay yesterday but he will analyze the data today.

I've been working with LovTAP and I've testing different procedures to characterise it, following some literature and my personal thoughts.

Doing that I have noticed qualitatively that the mutant show more repression than the wild type (something not expected), and even I am not yet totally sure that the LovTAP repression is light dependent it seems that in cells co-transformed with LovTAP versus those transformed only with the trpL-RFP system, there are lower levels of RFP. I did the experiments with LovTAP under the constitutive promoters J23102/J23105/J23114/J23117).

Using the stronger J23102, there is considerably little RFP production even in dark, something expected according with the advices of Dr. Devin.

These results have to be improved because the optical densities of the cultures are not  the same.

On the other hand, I tried a quantitative measurement  using  the fluorimeter (with a 96 well plate) and considering de OD (our spectrophotometer collect crazy data, so the OD of my samples was still variable). Besides, my initial data were lost in the machine so I couldn’t compare the differences before and after the light and dark exposition (during 12 hours) of the samples in the plate. I started the protocol before loading the plate under blue light to test the dark condition, expecting that the RFP levels in dark increase in comparison with those samples that were always under blue light. I did this in order to face better the issue of the long half life of the RFP due to it doesn’t have a degradation tag. Analyzing the only data that I recovered (normalizing with the controls, the fluorescence and OD backgrounds), the results are crazy, there is no a clear difference between light and dark samples .As well there is a lot of variance in the samples that are replicates of the same culture and condition (dark/light). But it seems that there is lower levels of RFP in co-transformants vs  the cells harboring only the RFP reporter system. I hope to repeat the experiment during the week and have better luck. Having the initial measuring (that unfortunately lost) I expect to see a difference.

Yesterday I was seeing your information about LovTAP and I noticed that your team ligated LovTAP without promoter with the RFP reporter system. If so, maybe you are not getting good results because LovTAP is not being expressed. In the shipment I sent you LovTAP ligated to four promoters.

Those are our results, in my experience characterisation by itself is a huge challenge.

We will be repeating some experiments, besides we will be working with the presentation and poster.

Well, iGEM is finishing... something exciting but also a little bit sad.

I and my team are looking forward to meet you, and hope everything works out with our projects.

Let's continue working during the end of this great experience.

Best,

Claudia and UNAM-Genomics-Mexico Team.


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