icE. coli Abstract

Escherichia coli's optimal growth temperature is 37°C. Growth rate decreases drastically at lower temperatures, and completely halts below 7°C. Our project aims to increase E. coli’s cold tolerance, by expressing several chaperone proteins normally found in psychrophillic (cold-loving) bacteria. Chaperone proteins regulate the structure of proteins and RNA protecting their function during times of temperature stress. By expressing psychrophillic chaperones we expected E. coli's cold resistance to improve in two ways: increased growth rate at temperatures below 37°C and improved ability to survive freezing. Over the course of our project, our team has successfully expressed two psychrophile chaperones in E. coli and we have demonstrated an enhanced survival rate of bacteria after repeated freeze-thaw cycles.

Applications

Our project is part of the Foundational Advance track. However, some promising applications for our CryoBricks do exist.

Human outreach

Conclusion

Our CryoBrick CspC and Cpn10 both had an enhancing effect on the survivalrate. For Cpn10 we are the first to describe an enhanced survival rate! Both CspC and Cpn10 did not effect the growth at lower temperatures.

E-mail: igemamsterdam@gmail.com

Facebook: iGEM Amsterdam 2011

Twitter: @iGEMAmsterdam

Wiki: 2011.igem.org/Team:Amsterdam

Although the IGEM competition is primarily focused on experimentation, we have dedicated quite some effort to reaching out to the public. Hoping to better inform them and attempting to correct the many existing misconceptions concerning synthetic biology.

Card game Together with Biotexture we are developing a cardgame! A lasting and fun way to inform the public about how synthetic biology is performed in the lab. To see what the cardgame is about, come to ‘the Night of the Nerds’, at the 8th of October in science centre NEMO, or go to www.biotecture.nl.

We have collaborated with the iGEM Wageningen team, who developed a software tool for designing super parts. We have tested this tool. They also sent us some of their constructs to test, since they did not have the right equipment.

We have organized several activities in the fields of education, collaboration and media attention. Below, we list the highlights of our human outreach campaign.

Cpn60/Cpn10 We have taken the chaperone genes Cpn60 and Cpn10 from Oleispira antartica. These work together to form a heteromultimer that can refold enzymes at very low temperatures, thus safeguarding their functionality. Cpn60 is the larger part of the chaperone and Cpn10 is the smaller part. These chaperones have homologues in E. coli, but Cpn60/Cpon10 function at lower temperatures. Figure 1 shows the homologous version found in E.coli.

Chaperone proteins refold misfolded molecules, in our case proteins and RNA’s. This is achieved by creating BioBricks. We like to call these bricks CryoBricks.

Figure 1: GroEL/GroES system that is homologous to Cpn10/Cpn60. The yellow part is GroEL, and the blue and red part GroES. Adappted from Nelson & Cox, Lehninger

Principles of Biochemistry, 3rd ed.

SheDnak In E.coli, Dnak, is involved in many different processes. The precise extent of its functions is still unknown, but its cold-induced unfolding and inactivation has been reported as one of the causes of systems failure at low temperatures. SheDnaK is a DnaK homologue from a shewanella species normally found in the Antarctic.

We speculate, based on literature, expression of SheDnaK in E. coli will recover some of the functions lost by cold-induced inactivation of native DnaK, delaying or even preventing general systems failure.

CspA/CspC Finally, we've investigated two highly similar RNA chaperones from Polaribacter irgensii: CspA and CspC. these are homologous to several of the 9 cold shock proteins found in E. coli. Cold shock proteins bind RNA with two motifs, located on a β-barrel known as the cold shock domain. Through this binding, CspA and CspC can melt stable structures in mRNA. Among others, this can allow mRNA to regain translational activity at low temperatures. Expression of both CspA and CspC have been reported to increase cold resistance in E. coli, which was characterized as an increase in freeze/thaw cycle survival rate.

Project

iGEM team Amsterdam Jantine Broek Paul van Dieken Isoude Kuijper Sandra van der Nat Jorick Mul Bas Stringer Yuri van Warmerdam

Supervisors

Swammerdam Institute for Life Sciences Dr. Pernette Verschure Dr. Frédéric Cremazy Ing. Diewertje Piebes Lisette Anink MSc. Molecular Cellphysiology Prof. dr. Bas Teusink Dr. Domenico Bellomo Dr. Douwe Molenaar

Characterization

Figure 2: Dnak structure Adapted from http://www.chaperone.sote.hu/Examples.html

Figure 3: CspC Adapted from http://wwwuser.gwdg.de/~mstumpe1/gallery/ColdShock2.jpg

Team

We have successfully combined two of our chaperones – Cpn10 and CspC – into protein generator constructs. These CryoBricks are designed to facilitate cold resistance in E. coli. We characterize this cold resistance in two different ways: By measuring the freeze/thaw survival rate and through growth rate measurements at different temperatures.

Selection Replacing an antibiotic resistance gene by a CryoBrick in the plasmid. Selection for bacteria with the right plasmid can be done in the fridge!

Biofuels Organisms that make biofuels in open ponds suffer from cold temperatures in the off-season. Our CryoBricks will allow for higher production rates under inhospitable conditions.

Environmental Projects Naturally environments are most of the time below 37°C . For example, this year’s Brown-Stanford team want to use bacteria on Mars. Our CryoBricks can aid in survival on Mars.

The goal of our project is to create more cold resistant bacteria. Cold resistance can be enhanced in many different ways. We have studied this by looking at the growth rate and the freeze thaw survival. To reach this goal we have used chaperone proteins normally found in the Antarctic.

Before this debate, different meetings were organized to help the PYO’s to get their facts straight. The Dutch iGEM teams were invited to attend these meetings and support the PYO’s.

The Rathenau instituut promotes the formation of political and public opinion on science and technology. They have organized the “Meeting of Young Minds”, where the Dutch political youth organizations (PYO’s) get a platform to debate synthetic biology.

Contact

Rathenau instituut

Figure 4: Freeze/Thaw cycles. This figure indicates that the CryoBricks CspC and Cpn10 have a possitive effect on the survival rate of E.coli.

Wageningen

Mars

Figure 5: Growth curve. This figure shows that the characterized CryoBricks do not enhance growth rate.

Note: this is an overdramatization

Note: this is an overdramatization