Team:LCG-UNAM-Mexico/Notebook/2008-May

GROUP SESSION:
Preliminary Projects Group Presentation (2nd Round)

Discussion of Papers & Ideas:
Sprinzak & Elowitz, 2005.

Combinatorial experiment.

Three TF and 5 binding sites.

Combinatorial and phenotypes analysis.

With a few well characterized elements much can be done.

Things to consider:

Next steps, future models:

• Protein level phosphorylation (example: Phytocrome found in cyanobacteria that get phosphorylated when activated with a specific wavelength and eventually trigger the activation of TF.
• miRNA (only in eukaryotes, not found in yeast)

Sequence-specific Ribosomes. (few well characterized)

Repressilator: Three genes that repress each other.

Signaling cascades

• Not exploited yet.
• Response time delay.
• It is mentioned as the simplest system.
• The noise is accumulated in these systems.
• Design a signaling cascade and see how noise affects and spreads throughout the system.
• Compare the noise of a two element cascade with the one of a three-element cascade.
• There is evidence that to reduce noise in a noisy system, noise is introduced into the noise, and as a result the system becomes ordered.
• Increase copies of the elements and see how does the noise is affected. Theory says that noise should decrease, see if this system converges to a deterministic system.
• Compare strains with different protein production ratios. *EXPLOIT MORE THIS POINT*.
• See if it is easy to do. However in this system it is very unlikely to find multi-stationary states.
• Measuring noise is a potential problem. Do we have access to technology to measure it?

Experimental Technique: Promoters recombination.

Suggestion: Use PCR to avoid direct recombination.

Investigate the difficulty to study the competition between the two enzymes for same metabolite in a metabolic pathway.

In positive circuits it is possible to see multiple states.

It is not enough to have the network topology, the context also influences the outcome.
We need to check whether the module works in the desired conditions. Two modules working separately doesn't mean that they will work when joined together.

GROUP SESSION:
Ideas:

• Halobacterium salinarum NRC1 and R1 have the two genes:hop (825bp) and bop (789pb)
• Modified it replicates in about an hour, the WT in about 12hrs(NRC1)
• Small genes (>3Kb)
• We could use Halorodopsines and Bacteriorodopsines, and as an alternative efflux pumps contained in plasmids and that work on the antibiotics, so we could transform light into sound or only measure the concentrations.
• We love the idea :P
• The electrode part is not difficult
• We would make a model of the system to regulate both pumps
• Program a script for the “singing sound”
• Rhodopsines react to different light frequencies(colors) generating different concentrations in the medium and depending on those concentration, they could “say” the color of the light that it is receiving.
• There is a paper that has much to do with our problem: "Cl Concentration Dependence of Photovoltage Generation by Halorhodopsin from Halobacterium salinarum"
• It is possible to move this system into E. coli(Luis found a paper that proves it)
• There is a specific medium for Halobacterium and its cost is $12 USD
• Paper: “Genetic Transfer in Halobacterium volcanii" Moshe Mevarech & Ruth Werczberger. Journal of bacteriology 1985. Vol 162 No 1.. In the Methods section the author speak about some culture media. They point out some auxotroph mutants. It took 6-10 days to grow
• Paper: "Bacteriorhodopsin production by cell recycle culture of Halobacterium halobium" Sang Yup Lee et. al. Biotechnology Letters 1998. Vol 20 No 8. Talk about some cultures of Halobacterium salinarum R1.
• Different culture media
• General information about Halobacterium salinarum
• Halobacterium salinarum divides itself each 4-6h (it could last even a day).
• About the general genome, with an image about the membrane proteins. Watch out the two CI
• Halobacterium was sequenced
• Halobacterium salinarum growing conditions: 50ºC, pH 7.2, [NaCl]=3.5-4.3 M

GROUP SESSION:
Preliminary Projects Groups Presentation(3rd Round)
Points to review…
Singing Bacteria:

• How to control the salinity.
• What happens if you knock out this channels? Does a compensatory mechanism exist in the cell?
• Wave length at which the cell reacts. Do cells react to colors?
• If we use tetracycline, use a naturally resistant mutant
• We need to know if the cell is transporting tetracycline in an efficient way.
• Why don’t we use efflux pumps but with heavy metals? They seem to be auto-regulated and the insertion is specific

• Usin metal pumps
• Magnetic proteins?

GROUP SESSION:
Project Analysis and Tasks Assignation
Two models:

Study the bioparts:

Take into account the external signal
Regulation
Mainly a cascade, ‘cause it is easier
People in charge: Martin, Libertad & Minerva

Study the pumps in E. coli:

People in charge: Mariana, Mariana-GS, Jimena & Isaac

Investigate about the electrodes:

People in charge: Carlos, Enrique & Luis

Writing:

• People in charge: Sur, Daniela & Atahualpa

GROUP SESSION:
Points to check:

• Define a model
• Manage the medium to make fluctuations and measure it.
  Machines to measure fluctuations of even one ion. (Mariana Reyes will contact the Chemestry Faculty. There’s no machine in the IBT, at least no with such sensitivity. Electrodes aren’t expensive, neither hard to obtain, the hard part will be passing the info into the computer. We can get one adapter in 800 UK pounds

• Mariana
• Martin
• Libertad
• Jimena
• Luis
• Atahualpa
• Minerva
• Isaac
• Enrique

• Mariana GS
• Daniela
• Carlos
• Sur