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

GROUP SESSION:

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:

prk404 y7o prk415: resistant to tratracicline, maybe 4 - 9 copies.

pbbr1mcs5: resistant to kentamicine up to 10 copies.

puc: up to 20 copies resistant to ampicillin.

pjet: resistant to ampicillin up to 600 copies.

Plbb: clorma resistant, up to 12 copies with LacI in cis (it can be controlled with IPTG)

REMEMBER not to combine the vector with the same replication origin.

Once the most suitable gene has been chosen, primers must be designed:
• Tm = (2 (A + T)) + (4 * (C + G))
• Choose the single cut site.
• Take in account compatibility of restriction sites.
• Read more to start writing.

GROUP SESSION:
Expositions: Choosing the efflux pump:

Nickel and cobalt
• Very specific
• We don't know how to get the Co inside the cell.
• We can keep 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.
• RcnA & RcnR (~200 & ~300 aa) are used during Ni efflux.
• Pending: How does Co enter the cell 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 and it only operates at low concentrations.
• Problem: It is difficult to regulate their extrusion.
• Zn is essential.

Cadmium
• Its entry is mediated by a transportation system of divalent ions, it is cotransported with Manganese, which is essential for the cell, so the entry is not tightly regulated.
• It is toxic to the cell, but it doesn't seem too serious.
• The efflux can be mediated by multiple systems, all found in E. coli (CzcD, CzcCBA, CadA, ...).
• Legatzki et al. (2003) made an experiment in which they use a mutant E coli GG48 ((delta) zntA & (delta) zitB) that accumulates both Zn and Cd, but when they transform it with a plasmid with zntA & cadA of R. metallidurans, it regained resistance. 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, including through siderophores.
• Problem: On entering the cell, it forms a complex with a global regulator (fur) involved in many important functions. Essential.
• The pump Fief (~920kb) is ok, highly specific and the only way to remove the iron. .

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.
• Entry to the cell is based on a ionic potential in membranes.
• It is not necessary, rather 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 1+, because the extrusion systems only recognize Cu1+.
• Cells hold up to 3.5 miniMolar.
• CusCFAB operon is responsible for efflux, regulated transcriptionally by cusRS. There is probably a biopart. Known in E. coli.
• CusRS is ~1000 aa. Cus CFAB ~2000aa.
• Problem: Its size!
• Admission is ATPase dependent... by pumps? Described in yeast and animals, it is known to enter to E. coli, but how can we regulate it?
• It is not essential, it is highly toxic.
• Pending: How is the entry mediated?

Arsenic
• Resistance is in a plasmid in E. coli. Five genes (Ars [RABCD] ~ 1.4Kb), the plasmid is in total ~ 4.4kb. Some genes on the chromosome are also involved; they are not necessary but the resistance is reduced from 10 to 100 times 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 diffusion, and three carriers are known.
• It is highly toxic, to reduce its toxicity it is reduced.
• Ther is no well-known system of entry.
• The pumps are quite specific.
• Also toxic in the cells environment, therefore cells absorb it, for processing.
• Pending: Getting Hg out of the cells?

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

Not useful for our purposes
• Iron
• Lead
• Mercury
• Tellurium

Probably useful
• Zinc (Cons: It is essential.)
• Copper (Cons: It is very big.)

Favourites
• Cobalt & Nickel
• Cadmium
• Arsenic

GROUP SESSION:
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.

Preliminary design:
• The mechanism of entry of Nickel will remain wildtype.
• In the absence of Nickel, RcnR (whose gene will remain in the chromosome with its normal regulation) will repress rcnA (which will be deleted from the chromosome and inserted into a plasmid).
• By adding (*) to the system we will repress transcription of rcnA, even in the presence of Nickel, so this will be will be our signal to retain the metal inside 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 (*)?

Task: Suggest a molecule for (*)!

Modeling

Pending:

Response vs. Concentration (experimental part).

Set thresholds & limitations.

Efficiency of interactions?

Defining variables:

- Metal concentration.
- Repressor concentration.
- (*) concentration.