IGEM:UNAM-Genomics Mexico/2009/Notebook/Collaborations/2010/08/10

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Red light sensor and luciferases


Dear Mariana and Hector,

I hope you are well.

After two months of failures we are starting to have a little success at last. We got the firefly luciferases working, think we got the blue light sensor to respond to light :D

For the red light sensor, we think we managed to PCR cph8 out of BBa_M30109 but the PCB genes don't to be in there. We tried PCRing them out of BBa_K098010 but didn't get any DNA, however when we then tried to transform cells with this part we got a few kan resistant colonies. We are going to check these for plasmid DNA, and if this still doesn't work will try PCRing the individual parts. We will try and PCR the individual parts out of some other biobricks, maybe BBa_I15008 or BBa_I15009.

Also, if you just need PCB to make your sensor work, you can add it in the solution. Last year, Harvard (http://2009.igem.org/Team:Harvard) tried to make light sensing yeast. They just purified the PCB from Arthrospira platensis (sold as spirulina in some health food stores, there is a hippy headshop that sells some in Edinburgh), then added it to the solution. Maybe this can be done to test your green light sensor if all else fails? The problem is that the chemicals used are quite toxic. Maybe its possible to extract PCB genes from spirulina too.

In any case we will tell you if we get any PCB stuff working.

About the luciferases:

Please tell us if getting rid of the 6 bases between the RBS and the start codon changes anything. We used last year's part from Edinburgh (BBa_K216015) so it has the nitrate responsive promoter PyeaR in front of it. We will change that for a stronger promoter but haven't chosen which one yet. We will probably get rid of the 6 base insert too.

We are also getting a luciferase which should be brighter (Mutations are described in: Increase in bioluminescence intensity of firefly luciferase using genetic modification, Fujii et al 2007). The mutations combined with the codon optimisation of the gene for E coli will hopefully make a difference. Our ultimate goal is still to get a light sensor to respond to a light producer, so maybe we can test this with your CcaS when it arrives.

For the assay we tested it according to the protocol described on the experience page of the Pyear-Firefly luciferase biobrick (http://partsregistry.org/Part:BBa_K216015:Experience). However the second time we added 10 microlitres of 10 mM D-luciferin instead of 5. The cells were a lot brighter than they were the first time. However our luciferin is a little old and was kept in the -20 freezer instead of -80.
Make sure the external medium is at pH 4.8 or around there because otherwise the luciferin doesn't enter the cell and there is very little brightness. Another option is to treat the cells with EDTA (according to Biosensing of Benzene derivatives in the environment by luminescent Escherichia coli, Kobatake et al 1994).

We are going to do testing with different concentrations of luciferin when we do the proper characterisation of the luciferases. We should start next week after we get the sequences confirmed and have access to the machine that measures the spectral output.

Oh and I'm not sure what plasmid our luciferases are in. We thought it was pSB1A3 but the band seems too big. Last year's team didn't write it down so I will investigate : )

didn't mean to write such an essay. Hopefully I haven't forgotten anything but just email if I did.

Btw if you are bored check this out
http://www.bbc.co.uk/news/world-us-canada-10922843.
Cheers,

Will


Salut Will!

On espère tout aille bien là-bas ;-)


We have good news:

  • Our luciferase should be tested by this Friday; thanks for the tip with the pH; we'll inform you of the detailed protocol we used
  • We *might* have functional luxAB.
  • And our synthesis has FINALLY arrived !!!

About your progress (by the way kudos on getting anything from M30109), we find it most disturbing that M30109 appears to be distributed as an incomplete part. However, that would explain why so many teams have had a bad experience with it :S

About the PCB, best of luck with I15008 & I15009; we've tried many times to obtain anything from those parts but we've been unsuccessful. Since the addition of PCB to the culture is reported to colorize the medium, we fear emergent properties might mess up emission efficiency. We would be most reluctant to add it externally... Please let us know of your progress with ho1 & pcyA. We heard of a particular strain of coli that should produce these genes, but with our Customs woes, we wouldn't have the time to procure it. The reference is here:


Mukougawa K, Kanamoto H, Kobayashi T, Yokota A, Kohchi T (2006)

Metabolic engineering to produce phytochromes with phytochromobilin,

phycocyanobilin, or phycoerythrobilin chromophore in Escherichia coli.

FEBS Lett 580:1333–1338.


Correct me if I'm wrong, but of our synthesized parts you were interested in LuxY & CcaS, right?

Tomorrow we'll organize this on our meeting. We also have to re-re-distribute hands to the newly-arrived parts ;-)


Well, that's all, for now.

Best regards!


Hector Medina & UNAM-Genomics_Mexico

LovTAP, blue promoter, luminometer

Hello Claudia,

here is our small 'progress' with LovTap: It looks like we have stable double transformants with read out system 1 and lovTap, version 09- mutated at pos 427 (Ile-> Phe) seems to grow a bit better. We are repeating our experiments with the daylight and the dark settings; so far clones kept in the dark were more reddish than the one kept in the light- we set up liquid cultures today to check the levels of fluorescence in the luminometer, so we have some numbers to see if it really makes any difference.

We are planing our illumination setting; we want to reapeat Lausanne's assay: http://2009.igem.org/wiki/index.php?title=Team:EPF-Lausanne/Protocols/Illumination

We also want to illuminate it with green and red light and with all possible combinations of light- to ensure that only blue light will activate it- and so we are planning to buy some LEDs with different wavelengths- but I don't have much details let, as soon as we have it done I'll let you know. We would also look o illuminate it with blue light of different wavelengths with very 'sharp peak, to fully characterise the optimal response, but we'll see how broad spectrum can we actually apply.

It's strange that you didn't get any response form Lausanne, the spirit of the competition is to provide the parts for other teams. We'll be happy to send you the parts that you need. Unfortunately, the one that we can't distribute further is trp mutant /commercial strain/, so I hope yours will work ok. We are also very positive about helping you to optimise the promoter- could you actually write on the website what exactly have you don with it?

Our luminometer is from 2006, the name of the company is Modulus, we have manual for versions 9200 and 9201- I couldn't find the name of the model on the machine, but it's definitely one of those two. If you think that your device is not working right- I assume it would be very difficult to characterise response from e.g. blue promoter- we are again very happy to help you with characterisation of anything you need.

We'll also think about the parts that we would like to use, blue promoter is I think one of them and LyxY the other.

Will from our team has written this morning to your team about green light sensor, if you have any further questions just let me or him know :)

Thanks a lot for the email and good luck with your lab work!

Take care, Marta



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