IGEM:IMPERIAL/2008/Projects/Water filter/Pimping

Pimping E.coli

Given updates in other areas and more information on topic, E.coli as a chassis has returned to the decision table, however for use, customisation is required..

What do we need to pimp?

Active temperature range

Local area pH

Increased ability to secrete proteins

Any other suggestions...

Homeostasis v Differentiation

In pimping our E.coli to make it more able to operate in adverse conditions it would be necessary to use circuitry to sense and respond. The circuitry would however differ upon circumstance. In a small volume of still water, the affects of the E.coli's modifications could rapidly alter the local environment and making it hostile to the E.coli once more but for a different reason (eg. first too hot, now too cold). In this instance a homeostatic circuit (where heat production, heat reduction or neither is turned on) would be more beneficial to the cell and waste less resources.

If the E.coli was in a large volume of water however or the water was flowing past the bacterium the local envirnoment will constantly be being replaced and so there will be no accumulation of affects. As such, a semi-permanent or even premanent differentiation step (to say relative thermophhile or relative alkalophile) would be more beneficical as it would allow the E.coli to turn off the other counteracting pathway completely, at least for a short period of time, or depending on where it was used, permanently.

Temperature

To allow E.coli to work for us in a greater temperature range a way of increasing or decreasing the E.coli's temperature and that of the surrounding local environment is required.

This will be best achieve by altering the metabolic or synthetic processes of the E.coli to utilse an exothermic or endothermic reaction pathway to increase or reduce the temperature.

By increasing the working temperature range will allow use of the E.coli in water outside it's normal capacity particularly the cool waters of rivers

Water river temperature varies worldwide. From the list of the 10 most polluted places, the furthest north is Romania and Africa will have the warmest water. This can be used to establishment a suitable temperature range: 11°C < E.coli < 17°C

Sensing

High Temperature (>40°C)

E.coli possesses a heat shock promoter (htpG) which only becomes active at high temperatures. This could be used to switch on a homeostatic circuit or a differnetiation pathway to enable the bacterium to be more able to cope with the temperature. The htpG promoter was found by UCSF 2005 to have a very poor output and so another sensing promoter or system may have to be found. Notably, water temperature is not going to get this high so is a rather moot point.

Low Temperature (<17°C)

E.coli also possessses a cold shock promoter (HybB) which activates at low temperature (<30°C) and could form the basis for a differentiation pathway to a relative psychrophile. For a homeostatic circuit however, this temperature cut off may be too high. There is also the cspA promoter which may be turned on at the same or lower temperature and genes after this promoter also have a 'cold box' an mRNA element which makes them unstable and untranslateable when warm

[cold shock protein system]

Response

Exothermic Response (Heat Gain)

The simplest way to give out heat is to increase metabolic flux by upregulating a bottleneck gene in the E.coli's metabolism, causing the energy production system to give out plenty of heat.

Endothermic Response (Heat Loss)

Increased working pH range

A greater pH range is useful in this context as many soils are fairly acidic or alakline, a characteristic that is reflected in the groundwater. An E.coli able to alter the pH around it to a degree would work more efficiently in these conditions. pH for drinking water will not fluctuate much reasonaly between 6 and 8.5 (6.5 and 9.5 for tap water). For industry however it is not uncommon to get large swings in pH, in this setting, a pH range of 2 to 12 is more useful.

Sensing

Acidic pH (<5)

pCad detects low pHs, however the lower limit to which it does this may be too low and cause a severe reduction in E.coli working activity and so a more sensitive sensor may be required.

Alkaline pH (>11)

[[http://www.igem.upv.es/igem06/index.php?title=The_idea Valencia2006

Response

Acidic pH (<5)

Urease can be produced to breakdown urea into ammonia and Carbon dioxide, raising the pH of the surrounding environment. Coupled to a pH sensitive promoter, this could make a basic homeostatic cicuit or part of a acidophile differentation pathway depending on usage.

Alkaline pH (>11)

Lactate dehydrogenase could be produced to convert pyruvate to lactate a by-product of which is H+ which has an effect of lowering the pH of the environment. If coupled to a pH sensor, this could again be part of a homeostatsis mechanism or part of an alkalophile differentiation pathway depending on usage.

Secretion

E.coli is a gram-neagtive bacterium and as such has two membranes and a periplasm. The inner membrane is highly permeable but the outer membrane is not, making secretion of non-E.coli proteins more diifcult. An increased protein secreation ability would be useful for our E.coli in case it was necessary to bind or chelate the metals outside the cell (to prevent the cell becoming gummied up or allow precipitation of metal for collection).

To increase the ability of E.coli to secrete proteins, it is likely that porins will need to be incorporated into the membrane however this may be a two edged sword. As proteins will be allowed to diffuse out of the E.coli, other molecules which are normally barred entery may be able to enter and damage or potentially kill our E.coli.

Suicide (and clumping)

For safety when removing the bacterium, it may be required for the bacterium to die.

Due to it containing heavy metal ions however, it would be useful if it would not die and lyse as lysis would allow the metal ions to diffuse back into the water.

One known suicide switch in E.coli is the B.subtilis sacB gene which causes the E.coli to become sucrose sensitive, so when exposed to sucrose th E.coli cells would lyse (not ideal, but a sucrose jump trigger AFTER completion is useful) but clean up may be managed using a precipitant or clumping material.

Alternatively, it may be possible to cleave up the host DNA using encoded restriction endonucleases and leave the cell intact (other than the digested DNA).