Team:Amsterdam/Project/Description

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icE. coli

Escherichia coli's optimal growth temperature is 37°C. Its growth rate decreases sharply at temperatures deviating from this optimum, and growth beneath 7°C is impossible for wildtype E. coli. The aim of the project is to increase the cold tolerance of E. coli, allowing it to grow faster at temperatures below 37°C. We expect to be able to culture the resulting strain(s) at very low temperatures, possibly even at or below freezing point.

Our approach involves heterologous expression of proteins in the DH5α E. coli strain. Following iGEM's standardized protocols as much as possible, we will create cold resistance BioBricks - or CryoBricks - out of different (combinations of) synthesized genes and parts submitted to the registry by previous iGEM teams.

Succesfully increasing E. coli's cold tolerance is valuable to both fundamental and applied science. One of many possible applications is replacing antibiotic resistance with cold resistance as a selection property in plasmid backbones. Our CryoBricks can also be useful in various projects of other iGEM teams, and may open new avenues for heterologous expression of heat-sensitive proteins. (See also our application and collaboration pages.)

CryoBricks

The different proteins we intend to use for making cold resistant E. coli strains widely fall into two different categories: chaperones and antifreeze proteins. We expect expression of chaperones from psychrotrophic organisms will allow our E. coli to grow more easily at temperatures below 37°C, possibly even shifting their minimum growth temperature from 7°C (the wildtype minimum) to 0°C or lower. If we can achieve this, additional expression of antifreeze proteins might push the minimum temperature even further down. For more detailed information on any of the parts listed below, refer to our Basic Parts page in the BioBricks section.

Chaperones

Chaperone proteins are closely involved with the folding of other molecules. They bind substances like RNA or other proteins, and can then inhibit or stimulate conformational changes. Such interactions may facilitate protection against degradation or unfolding, or even refolding of denatured proteins. This, in turn, can safeguard the functionality of a chaperone's target. As such, certain chaperones have been observed to bestow cold resistance to the organisms that express them, by protecting molecules that are sensitive to cold. To the best of our knowledge, we are the first and thus far only iGEM team to try and achieve cold resistance through the expression of chaperone proteins. The following chaperones will be used in our CryoBricks:

OaCpn10 (From Oleispira antarctica)
OaCpn60 (From Oleispira antarctica)
PiCspA (From Polaribacter irgensii)
PiCspC (From Polaribacter irgensii)
SheDnaK (From Shewanella sp. AC10)

Antifreeze Proteins

Antifreeze proteins, or AFPs, facilitate survival at subzero temperatures by binding small ice crystals and inhibiting their growth, preventing fatal ice crystallization. Some of them are also suspected to interact with cell membranes to protect them from cold damage. Note that they functionally differ from commercial antifreeze products - used in cars for example - in that they do not lower the freezing point in a manner linearly proportional to their concentration. Other iGEM teams have already worked with AFPs, such as team Mexico 2010 and team Tokyo 2009 before them. This year, the teams of Leuven and Yale also incorporate AFPs in their respective projects. The following AFPs will be used in our CryoBricks:

RiAFP (From the holarctic longhorn beetle Rhagium inquisitor)
TmAFP (From the mealworm beetle Tenebrio molitor)
ZeAFP (From the demersal eelpout Zoarces elongatus)

Project Modules

[Under construction]

In order to succesfully create a cold resistant strain of E. coli, we intend to express an ideal combination of the chaperones and AFPs listed above. Unfortunately, this is not as straightforward as it sounds.

Characterizing and modelling promoters and ribosome binding sites

Assembling individual CryoBrick candidates into PRCT assemblies

Characterizing and modelling strains transformed with above assemblies

Assembling combinations of PRCT bricks based on their characterization

Characterizing strains transformed with combinations of CryoBricks

In parallel

Try combining parts with previous iGEM teams' parts, if time allows?

Send parts to current iGEM teams if desired -- might help them out.

Make and test a cold resistance backbone

Try to heterologously express EFE and ButB

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 ==CryoBricks==
-The different proteins we intend to use for making cold resistant ''E. coli'' strains widely fall into two different categories: chaperones and antifreeze proteins. We expect expression of chaperones from psychrotrophic organisms will allow our ''E. coli'' to grow more easily at temperatures below 37°C, possibly even shifting their minimum growth temperature from 7°C (the wildtype minimum) to 0°C or lower. If we can achieve this, additional expression of antifreeze proteins might push the minimum temperature even further down. For more detailed information on any of the parts listed below, refer to our [[Team:Amsterdam/BioBricks/Basic Parts|Basic Parts]] page in the [[Team:Amsterdam/Biobricks/Overview|BioBricks]] section.
+The different proteins we intend to use for making cold resistant ''E. coli'' strains widely fall into two different categories: chaperones and antifreeze proteins. We expect expression of chaperones from psychrotrophic organisms will allow our ''E. coli'' to grow more easily at temperatures below 37°C, possibly even shifting their minimum growth temperature from 7°C (the wildtype minimum) to 0°C or lower. If we can achieve this, additional expression of antifreeze proteins might push the minimum temperature even further down. For more detailed information on any of the parts listed below, refer to our [[Team:Amsterdam/BioBricks/Basic_Parts|Basic Parts]] page in the [[Team:Amsterdam/Biobricks/Overview|BioBricks]] section.
 ===Chaperones===