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

Session with Dr. Miguel

We did some brainstorming in regards to the project. How to measure the efflux, and how to build the system.
• Monitor changes in pH using colorimetry or electrodes, choose the electrodes wisely.
• If we use a nickel transporter in E. coli we can easily obtain the mutated strain (deleted trasporter).
• Introducing the gene into a multicopy plasmid and establish positive and negative controls
• An electrode is not really necessary, measurements can be done with conventional methods.

Researching Metal Efflux Pumps:
(The metal must be ionizable, maybe using a simple salt, the anion doesn't matter).

We decided to research how are metals transported by E. coli , each member of the team chose a specific metal:

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

Vectors we could use:

Expositions: Choosing the efflux pump:

Nickel and cobalt
• Very specific
• We don't know how to get the Co inside the cell.
• We can leave the wild type entry, and regulate the efflux.
• All this in E. coli.
• Co is very toxic, it can damage the cell, we better only use Nickel.
• We have two pumps we can test.
• The cell hold up to 2 millimolar of Ni.
• It has more than one system for Ni entry.
• Used during Ni efflux, RcnA & RcnR (~200 & ~300 aa).
• Pending: How does Co enter and the mechanism of Ni entry.

Zinc
• Uses ZnuABC, Zupt and ZntB to enter the cell. To leave the cell it uses ZntA and ZitB.
• The only specific entry for Zn is ZnuABC, the others also transport other metals.
• Regulated by Zurt, which binds Zn.
• To withdraw Zn: ZntA only works at high concentrations, but is not specific for Zn; ZitB is not specific either, but it only operates at low concentrations.
• Problem: It is difficult to regulate their extrusion.
• Zn 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.