Part:BBa_K2213001
Tet_EutMN
Ethanolamine Utilisation (Eut) bacterial micro-compartment (BMC) proteins EutM and EutN from E.coli , placed under the inducible Tetracycline promoter. Also contains RBS, terminators and all tetp components – thus this part alone can be used to synthesise EutM and EutN at varying concentrations, relevant to the experimental task. EutM is tagged with GFP and His6. EutN is tagged with FLAG. See Figure 1.
Tetracycline Promoter
The tetracycline expression system is based on two regulatory elements, the tetracycline repressor protein (TetR) and the tetracycline operator sequence (tetO); both derived form the tetracycline-resistance operon of the E.coli Tn10 transposon. In our part, tetracycline (Tc) can bind rTetR, increasing its affinity for DNA binding. Thus upon Tc addition, rTetR can bind tetO, permitting transcription of any gene under control of the tet promoter. This is illustrated in Figure 2.
Several iGEM teams have previously carried out characterisation and worked with the Tet promoter system. For your interest and research we recommend looking at the HQ submitted part BBa_R0040 (https://parts.igem.org/Part:BBa_R0040) as a starting point.
EutM and EutN
The Ethanolamine Utilisation (Eut) bacterial micro-compartment (BMC) proteins EutM and EutN from E.coli are clustered together here, as they are found in nature. Coming soon...
Usage and Biology
Although it is possible to use this part for EutM and N expression without further assembly, we do not recommend doing this if the ultimate goal is to produce fully functional Eut BMCs. When forced to produce BMCs, E. coli are placed under a large amount of strain and begin to experience slowed and abnormal growth. Therefore, we suggest using a low copy number plasmid eg. pSB4A5 (https://parts.igem.org/Part:pSB4A5), as we have used in our project. By using a low copy number plasmid, cellular stress is minimised, but the experimenter still has the ability to induce BMC formation.
Characterisation
Optimising conditions for EutM synthesis using 'Design of Experiments'
To find the optimal conditions of EutM microcompartment formation we used a tool called 'Design of Experiments' to vary a multitude of factors including:
- concentration of Tetracyclin inducer (induces EutMN synthesis)
- concentration of IPTG inducer (induces EutS synthesis)
- Harvest time (time after induction)
- Temperature
- Growth Medium (LB and TB)
We determined the concentration of EutM by measuring the GFP fluorescence of the solution and dividing it by the OD of the culture at that time. This gave us the average GFP fluorescence per cell which is proportional to the amount of EutM produced per cell. / from this 22 flask investigation we were able to make surface plots to visualise our findings: /
- 2 TEMPERATURE DoE SURFACE PLOTS***
/
Figure1: The interactions between the concentration of tetracycline inducer (x axis), Temperature after induction (y axis) and Average GFP fluorescence per cell (z axis).
From this graph, it can be deduced that a lower temperature after induction and a higher tetracyclin concentration in the inducer increases the amount of EutM protein produced per cell.
Figure2: This graph is like figure1, except IPTG concentration in the inducer is being compared instead of Tet. IPTG induces the LacUV5 promoter which transcribes the EutS gene. By having a high IPTG concentration in the inducer, the amount of EutM expression increases. This implies that the presence of EutS is increasing the stability of the EutM protein. This may be the combination of EutSMN proteins binding to form partially formed microcompartments.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2190
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1516
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