Part:BBa_K2213001

Tet_EutMN

UZurich 2019 added more detailed characterization data for this part. Scroll to the bottom of the page to find it.

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.

This part is used in the composite part: https://parts.igem.org/Part:BBa_K2213012

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.

EutM is one of the shell proteins that constitute the Eut BMC. Six EutM subunits self-assemble to make a flat cyclic hexamer with a bowl-shaped depression on one side, punctuated by a narrow central pore (Tanaka et.al, 2010). This central pore is positively charged, in contrast to EutL, indicating that they may facilitate movement of different molecules into the BMC (see the Manchester team's characterization of Eut LK at https://parts.igem.org/Part:BBa_K2213002). EutM is also able to form tightly packed two-dimensional arrays similar to that of EutL (Takenoya et.al, 2010).

EutN is a non-BMC domain protein that may be a part of the Eut BMC (Held et.al, 2013), though is more likely than not as EutN was shown to be important for growth in high ethanolamine conditions (Tanaka et.al, 2010). EutN appears to form pentameric assemblies and displays an oligonucleic/ oligosaccharide-binding fold (OB) fold, a domain which is usually associated with nucleic acid binding (Held et.al, 2013).

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 (see characterisation data below). 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

Understanding growth defects upon Eut protein expression
Following the succesful transformation of Eut constructs into E. coli Manchester iGEM 2017 noticed that cultures grew at a slower rate after Eut subunit protein expression had been induced. This lead to the investigation of how each of Eut construct https://parts.igem.org/Part:BBa_K2213000 , https://parts.igem.org/Part:BBa_K2213001 and https://parts.igem.org/Part:BBa_K2213002 affected growth rate after it had been induced.

Manchester iGEM 2017 recorded optical density measurements at 600nM for EutS, EutMN, EutSMN and EutLK. OD measurements were taken at 0 hours, 4 hours and at 20 hours (see figure 3). It was observed that between 4 and 20 hours, the OD of cultures containing the constructs EutMN, EutSMN and EutLK were reduced by 75.53%, 81.77% and 67.93% respectively. In contrast to this, the OD of the EutS culture continued to rise and had increased by 45.28% when the final reading was taken at 20 hours. This suggests that the production of microcompartment subunits EutM, EutN, EutL and EutK are toxic to the cell, however, the production of EutS may be less toxic. This may be due to less strain being put on the cell due to the expression of a single microcompartment subunit, rather than multiple subunits being expressed simultaneously. Overall this data indicates that the expression of complete microcompartments is likely to be toxic to the cell and should be highly regulated.


Figure 3. Average optical density at 600 nM of EutS, EutMN, EutSMN constructs induced and non-induced. Measurements were taken at 0 hours, 4 hours and 20 hours.


Measuring BMC subunit expression
Manchester iGEM 2017 measured GFP fluorescence to determine if the expression of EutM had been successful. They found a significant increase in fluorescence at both a 4 and 20-hour time point (p = 0.0016 and p = 0.0054 respectively), produced by cells containing the EutMN construct under inducing conditions. Similarly, there was also found to be s significant increase in fluorescence produced by cells containing the EutSMN construct at both the 4 and 20-hour time points (p = 0.002 and p = 0.0007 respectively). This confirmed that the TetR promoter was working as expected, controlling the induction of the EutMN construct (see figures 4 and 5.


Figure 4. Average OD corrected fluorescence (Ex. λ 470-15 / Em. 515 – 20 nM) measurements of EutS, EutSM, EutSMN and EutLK constructs, non-induced and induced taken after 4 hours. Error bars show the SEM.



Figure 5. Average OD corrected fluorescence (Ex. λ 470-15 / Em. 515 – 20 nM) measurements of EutS, EutSM, EutSMN and EutLK constructs, non-induced and induced taken after 20 hours. Error bars show the SEM.

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' (DoE) 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. With the data we collected from this round of DoE we were able to design surface plots to visualise our findings:

Figure 6: A surface plot of 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.

Figure 7: 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 concentration of EutM per cell increases. This implies that the presence of EutS is increasing the stability of the EutM protein. This may be due to the binding of EutSMN proteins to form partially formed microcompartments, potentially improving the stability of EutM protein.

Figure 8: The interactions between the concentration of tetracycline inducer (x axis), Harvest time (y axis) and Average GFP fluorescence per cell (z axis). From this graph, it can be seen that a low harvest time and a high harvest time yields the highest EutM synthesis per cell.

Figure 9: This graph is like figure3, except IPTG concentration in the inducer is being compared instead of Tetracycline concentration. IPTG induces the LacUV5 promoter which transcribes the EutS gene. At a lower harvest time, IPTG concentration in the inducer has little effect on the EutM concentration (z axis). However, at higher harvest times, IPTG concentration has a significant effect on EutM concentration. We believe that this relationship is caused by the binding of EutS, M and N proteins forming partially formed microcompartments and thus increasing the half-life of the EutM protein.
With this data collected, a further round of DoE could be designed to optimise EutM expression further. To find out how this would be done, visit the Manchester 2017 iGEM page at http://2017.igem.org/Team:Manchester/Model/DoE

Approach

In order to better understand the apparent toxicity of the EutM and EutN proteins we carried out a more thorough OD measurement while also trying out a different inducer.

Procedure

Three 100 mL LB flasks were inoculated with an overnight transformation of pSEVA441 plasmids carrying the EutMN part. Bacteria used: E. coli strain DH5alpha (chemically competent).
Cultures were grown for ~4 hours and then induced with 45 ng/mL final concentration of Anhydrotetracycline(ATC) and Tetracycline(Tetc), one was not induced (control). Measurements were taken every hour for 8 hours and then a final measurement was taken 20 hours past after induction. Cultures were grown in 37° shaking at 300 rpm.

Results

The production of the EutN and EutM proteins seems to be detrimental to growth of the bacteria, OD does not go higher than ~1.6 and even declines after that. However the decrease in OD after 20 hours was not as strong as in the original Measurement. We were not able to replicate this drastic decrease in OD. Therefore, we propose that the EutM and EutN proteins are detrimental to growth of bacteria but are not generally toxic to the cells such that they would die.

Imaging

We confirmed with imaging that the protein is produced at high enough levels to form compartment, as you can clearly see the localization of gfp which is tagged to EutM:

Conclusion

We advice people who want to use this part to use inducer concentrations 10x or even 100x lower than we used in this experiment to produce BMC's if the cells should be healthy after induction. Another alternative would be to exchange their RBS with a very weak RBS (f.e J61100) as we have done in this part of theirs: https://parts.igem.org/Part:BBa_K2213012

Here you can see how we improved that particular part : https://parts.igem.org/Part:BBa_K3265026

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 2190
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1516
  

References
Tanaka, S., M. R. Sawaya, and T. O. Yeates. 2010. Structure and mechanisms of a protein-based organelle in Escherichia coli. Science, 327, pp.81-84.

Takenoya M, Nikolakakis K, and Sagermann M, 2010: Crystallographic insights into the pore structures and mechanisms of the EutL and EutM shell proteins of the ethanolamine-utilizing microcompartment of Escherichia coli. Journal of Bacteriology, 192, pp.6056-6063.

Held, M., Quin, M. and Schmidt-Dannert, C. (2013). Eut Bacterial Microcompartments: Insights into Their Function, Structure, and Bioengineering Applications. Journal of Molecular Microbiology and Biotechnology, 23(4-5), pp.308-320.