Project Modules

In order to successfully create a cold resistant strain of E. coli, we intend to express and combine the chaperones and AFPs listed in the Description page.

Since all action in a cell grinds to a halt at low temperatures due to reduced protein function it is essential for us to use a promoter that can effectively operate at low temperatures. Even though some of our targets are documented to convey increased cold growth we will still have to confirm this. Moreover, our other genes of interest may have an influence on cold growth as well and, as such, will have to be characterized in the same way. Similarly, the effect of our targets conveying resistance to repeated freezing and thawing will have to be validated as well. For this experiment we will once again study all of our targets to see if any undocumented effects emerge.

The Project was divided up into several project modules. Note that the first module has to be executed independently, allowing for the second and third to be executed in tandem, modules four and five can be started as soon as the first three modules are done. The final item can be performed based on the information gained from the fourth point.

1. Characterizing and modeling promoters

When growing bacteria at lower temperatures certain problems can occur other than the misfolding of proteins and RNA. Some promoters are known to be affected by lowering the temperatures; making them less sensitive or lowering their activity. We wanted to select a promoter that wasn’t negatively affected by lower temperatures. Therefore a plan was made to test different promoters.

First the ODE’s were written down, describing the route from DNA to protein.

mRNA'[t]=V_{transcription}-V_{mRNAdegradation}
Protein'[t]=V_translation-V_{proteindegradation}

The concentration of mRNA over time depends on the transcription rate and the rate of mRNA degradation. The protein concentration over time depends on the translation rate, and the rate of protein degradation, as shown above. The rates in these ODE’s depend on different factors. These are shown below.

V_{transcription} ->signal*k_{transcription}
V_{mRNAdegradation} -> k_{mRNAdegradation}*mRNA[t]
V_translation -> k_translation*mRNA[t]
V_{proteindegradation} -> k_{proteindegradation}*Protein[t]

The transcription constant is influenced by multiple factors, for example the promoter activity and RBS strength. We want to calculate if there is a difference in the transcription constant if you change the promoter ([http://partsregistry.org/Part:BBa_R0010 pLacI], and [http://partsregistry.org/Part:BBa_K206000 pBAD]) or temperature. Some promoters are always active; others are influenced by environmental signals.

First the degradation factors should be measured. This will be done by using antibiotics that stop the translation or transcription. To calculate the protein degradation the protein concentration should be measured before and after stopping the translation. From the decline in protein concentration we can than calculate the protein degradation constant. For mRNA it is a bit harder, because of the time limit mRNA concentration can’t be measured. This means that the only information that can be gathered is about the protein level. The solution is to stop the transcription, and see when the protein levels decline. This gives an indication for the mRNA degradation. The parameters that are left are signal strength, transcription and translation constants. With over expression of the input signal, the signal strength parameter can be neglected. The transcription and translation constants can not be separated easily. By testing both promoters with the same detection protein the translation constant is the same. This means we can give an estimation of the transcription constant on an arbitrary scale.

2. Characterizing growth rate of individual Growth rate enhancing, antifreeze & freeze resistance proteins

Information on this subject can be found on the Characterisation page.

3. Characterizing freeze resistance proteins on cell viability

Information on this subject can be found on the Characterisation page.

4. Combining BioBricks based on their characterization in order to maximize growth rate

An additional four final constructs will be designed as 'operons' comprising Cpn10 and Cpn60 together. These will be tested at 37°C and below, measuring OD600 at set intervals.

5. Combining Growth rate enhancing with antifreeze for subzero characterization

Theoretically the growth rate enhancer should stimulate the growth at lower temperatures than 0°C. With an addition of anti-freeze proteins E. coli has an protection against crystallization and thus can grow below 0°C.

6. Make and test a cold resistance backbone

Information on this subject can be found on the Application page.

Due lack of time module 1 was not completed, still both promoters were used for creating the assemblies. Modules 4, 5 and 6 were not completed due lack of time and the shipment time of the parts we received from Yale.

Team:Amsterdam/Project/Modules

From 2011.igem.org