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 8°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.)

CryoBrick Candidates

The different proteins we intend to use for making cold resistant E. coli strains widely fall into two different categories: antifreeze proteins, and chaperones. For more detailed information on any of the parts listed below, refer to our Basic Parts page in the BioBricks section.

Antifreeze Proteins

Antifreeze proteins, or AFPs, facilitate survival at subzero temperatures by binding small ice crystals and inhibitting their growth, preventing fatal ice crystallization. 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 are being considered for use 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)

Chaperones

Chaperone proteins are closely involved with the folding of other proteins. As such, certain chaperones have been observed to bestow cold resistance to the organisms that express them, by refolding proteins denatured by the cold. When (re)folded correctly, these proteins can function properly and the cell can carry on as usual. To the best of our knowledge, we are the first and thusfar only iGEM team to try and achieve cold resistance through the expression of chaperone proteins. The following chaperones are being considered for use in our CryoBricks:

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

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 AFPs and chaperones 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

@@ Line 1: / Line 1: @@
 {{:Team:Amsterdam/Header}}
+__NOTOC__
 ==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 8°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 at very low temperatures, possibly even '''at or below freezing point'''.
+''Escherichia coli'''s optimal growth temperature is 37°C. Its growth rate decreases sharply at temperatures deviating from this optimum, and growth beneath 8°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 the expression of heterologous proteins in the Top10 ''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.
+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 selecting factor in plasmid backbones. Our CryoBricks may also be useful in various projects of other iGEM teams. (See also our [[Team:Amsterdam/Project/Applications|application]] and [[Team:Amsterdam/Project/Collaborations|collaboration]] pages.)
+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 [[Team:Amsterdam/Project/Applications|application]] and [[Team:Amsterdam/Project/Collaborations|collaboration]] pages.)
 ==CryoBrick Candidates==
-The proteins and parts listed below are currently being considered as viable CryoBrick candidates. That is to say, they will be synthesized or ordered from the registry, and combined with promoters, ribosome binding sites or terminators as required. We'll transform Top10 cells with the resulting constructs and characterize how they modulate the effect of temperature on growth speed. Note that the parts submitted by previous teams aren't exclusively enzyme coding regions, as they also include, for example, a promoter that's active at very low temperatures.
+The different proteins we intend to use for making cold resistant ''E. coli'' strains widely fall into two different categories: antifreeze proteins, and chaperones. 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.
-<br><br>
-'''Synthesized genes'''
+===Antifreeze Proteins===
-*''Cpn10'' - Part of ''Oleispira antarctica'''s ''Cpn10/Cpn60'' chaperonin system
+Antifreeze proteins, or AFPs, facilitate survival at subzero temperatures by binding small ice crystals and inhibitting their growth, preventing fatal ice crystallization. Other iGEM teams have already worked with AFPs, such as team [https://2010.igem.org/Team:Mexico-UNAM-CINVESTAV/Home Mexico 2010] and team [https://2009.igem.org/Team:Tokyo_Tech Tokyo 2009] before them. This year, the teams of [[Team:KULeuven|Leuven]] and [[Team:Yale|Yale]] also incorporate AFPs in their respective projects. The following AFPs are being considered for use in our CryoBricks:
-*''Cpn60'' - See above
+*RiAFP (From the holarctic longhorn beetle ''Rhagium inquisitor'')
-*''SheDnaK'' - A homolog of ''E. coli'''s ''DnaK'' found in ''Shewanella'' species
+*TmAFP (From the mealworm beetle ''Tenebrio molitor'')
-<br>
+*ZeAFP (From the demersal eelpout ''Zoarces elongatus'')
-'''Parts submitted by previous teams'''
-*(To be updated soon.)
+===Chaperones===
+Chaperone proteins are closely involved with the folding of other proteins. As such, certain chaperones have been observed to bestow cold resistance to the organisms that express them, by refolding proteins denatured by the cold. When (re)folded correctly, these proteins can function properly and the cell can carry on as usual. To the best of our knowledge, we are the first and thusfar only iGEM team to try and achieve cold resistance through the expression of chaperone proteins. The following chaperones are being considered for use in our CryoBricks:
+*OaCpn10 (From ''Oleispira antarctica'')
+*OaCpn60 (From ''Oleispira antarctica'')
+*PiCspA (From ''Polaribacter irgensii'')
+*PiCspC (From ''Polaribacter irgensii'')
+*SheDnaK (From ''Shewanella'' sp. AC10)
+==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 AFPs and chaperones 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
 {{:Team:Amsterdam/Footer}}