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

June's summary:--- ---- - --- ---

Session with our advisor Miguel A.

• changes in pH due to colorimetry or electrodes, choose the electrodes wisely.
• Nickel transporter in E.coli. * if they send the mutated strain: Transcriptional merger with the trp operon; induce the repressor off.
• Introducing the gene into a multicopy plasmid and select controls (-) and (+)
• Coli introduces at least 40% less zinc when it has the gene. While the flow is still measurable there is no problem.
• An electrode is not really necessary, this can be done with conventional methods.

Research: Efflux pumps of metals.

(It must be ionized, maybe a simple salt, the anion doesn't matter).

Team work:

• Cobalt - Mariana
• Zinc - Jimena
• Cadmium - Chicken
• Nickel - Libertad
• Iron - Atahualpa
• TeO3 (+2) - Isaac
• Cu (2 +) - Minerva
• CrO4 (2 -) - Daniela
• AsO4 (3 -) - Enrique
• Hg - Carlos
• Pb - Martin

* Primer design

-- Documents / E.coli pumps

-- Vectors we could use:

prk404 y7o prk415 resistant to tratracicline, maybe 4 - 9 copies.
pbbr1mcs5 resistant kentamicine up to 10 copies.
puc up to 20 copies resistant to ampicillin.
pjet, resistant to ampicillin up to 600 copies.
REMEMBER not to combine the vector with the same replication origin.
Plbb: clorma resistant, up to 12 copies with LacI in cis (it can be controlled with IPTG)

• Choosing the most suitable gene and designing primers.

Tm = (2 (A + T)) + (4 * (C + G))

• Choose the single cut site!
• Compatibility of restriction sites.
• Read more to start writing.

Expositions: Choosing the bomb! (efflux pump)

Nickel and cobalt
• Very specific
• We don't know how to get the Co inside the cell.
• We can leave the natural entrance, regulating the output.
• All this in E. coli.
• Co is very toxic, it can hurt many things ... We better use Nickel only.
• We have two pumps we can test.
• It can hold up to 2 minimolar of Ni.
• It has more than one system to get Ni in.
• Help getting out Ni, RcnA & RcnR (~200 & 300 aa).
• Pending: How Co enters and the mechanism to get Ni in.

Zinc
• Getting in ZnuABC, Zupt, ZntB. Getting out ZntA and ZitB.
• The ones that get them in are not specific for Zn (they also work for other metals), except ZnuABC.
• Regulated by Zurt joining Zn.
• To draw: ZntA only works at high concentrations, but is not specific to Zn; also, ZitB is not specific, it only operates at low concentrations.
• Problem: It is very small... it is difficult to regulate their extrusion.
• It is essential.

Cadmium
• Its entry is a transportation system of divalent ions, it is cotransported with Manganese, which is essential for the cell, so the entry would not be regulated.
• It is toxic to the cell, but it seems that nothing too serious.
• The output can be mediated by multiple systems, all present in E. coli (CzcD, CzcCBA, CadA, ...).
• Legatzki et al. (2003) make an experiment in which they use a mutant of E coli GG48 ((delta) zntA & (delta) zitB) that accumulates both Zn as Cd, but when they transform it with a plasmid with zntA & cadA of R. metallidurans, it recover resistance quite well. It is true that we will not regulate all systems involved, but according to their experiment, change is quite significant. It could be useful.
• Genes are large, up to ~800aa.
• Pending: What concentration can the cell hold?

Iron
• There are many ways to get iron. Through siderophores!
• Problem: On entering the cell, it forms a complex with an overall regulator (fur) involved in many important functions. Essential.
• The pump is ok, unique and the only way to remove the iron. About ~ 920kb, Fief.

Tellurium
• Not so much an extrusion pump, because there is a transformation by means of an enzyme, which is not well known.
• All resistance genes are in two plasmids.
• Admission is a potential difference of ions in membranes.
• It is not necessary, toxic.
• We can not regulate the entry and when the Tellurium enters, unless there is resistance, the cell dies immediately.
• The role of the genes involved in resistance is not well understood.

Copper
• When it enters, it is reduced from 2+ to +, because the extrusion systems only recognize this.
• Hold up to 3.5 miniMolar inside the cell.
• CusCFAB operon is responsible for extrusion regulated transcriptionally by cusRS. There is probably a biopart. Known in E. coli.
• CusRS ~1000 aa. Cus CFAB ~2000aa.
• Problem: It's size! --> Bioparts
• Admission is ATPase dependent... by bombs? Described in yeast and animals, it is known that it enters to E. coli, but how can we regulate it in E. coli?
• It is not essential, it is highly toxic.
• Pending: The entry?

Arsenic
• It is in a plasmid in E. coli. Five genes (Ars [RABCD] ~ 1.4Kb), the plasmid is in total ~ 4.4kb. Some genes on chromosome are also involved; they are not necessary, but reduced from 10 to 100 times the resistance if they are absent.
• Do they have a translational control?
• The pump works with ATP.
• The entry is not specific, active transport.
• Toxic.
• This is the most studied bomb.
• This operon is cloned into a vector.
• Pending: What concentration can it hold?

Mercury
• Free admission... and three carrier assets are known.
• It is highly toxic, but it is not drawn as such, because it is reduced... So there is no nice way to remove it.
• It is not a well-known system of entry.
• The pumps are quite specific.
• Also toxic to the cells environment, that's why the cell eats it, for processing...
• Pending: Getting it out?

Lead
• It enters together with manganese, Zn or Co.
• It is highly toxic to E. coli because it affects membranes.
• Calcium pumps that can help it get in were found, but they are animals...
• To remove it, it uses the Cd systems, there are no specific system.
• Pending: Finding a target, Concentration that endures?

Not useful
• Iron
• Lead
• Mercury
• Tellurium

More or less
• Zinc -> Against: It is essential.
• Copper -> Against: It is very big.

Favourites
• Cobalt & Nickel
• Cadmium
• arsenic

Project Design

Experimental

Pump we will use: Nickel.

Articles:

-- Complex Transcriptional Control Links NikABCDE-HYDROGEN with Dependent Nickel Transport Expression in E. coli (2005).
-- Nickel homeostasis in Escherichia coli - the rcnR-rcnA efflux pathway and its linkage to NikR function (2006).
-- Identification of rcnA (yohM), Nickel and Cobalt Resistance Gene in Escherichia coli (2005).

Pending:

-- Check bioparts.
-- Design vectors.
-- Design primers.
-- Strain with deletion of rcnA.

Tentative design:

• The mechanism of entry of Nickel will remain wildtype.
• In the absence of Nickel, RcnR (whose gene will remain in the plasmid with its normal regulation) will repress rcnA (which will be deleted from chromosome and put into a plasmid).
• By putting (*) we will repress transcription of rcnA, even in the presence of Nickel, so this will be will be our signal to retain the metal in the cell and modify the concentration of the medium (if it is not enough to turn off the pump, it will be necessary to find a new level of regulation).
• How will we turn off the signal (*)?

* we don't know what can the (*) be.

Task: Find (*)!

Modeling

Pending:

-- Responding vs. Concentration (experimental part).
-- Set thresholds & limitations.
-- Efficiency of interactions?
-- Defining variables:
> Metal concentration.
> Repressor concentration.
> (*) concentration.

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