Difference between revisions of "Part:BBa K2213000"

Revision as of 00:27, 2 November 2017

LacUV5_EutS

The ethanolamine utilisation bacterial microcompartment (BMC) protein, EutS, under control of the LacUV5 inducible promoter. Also contains a bidirectional terminator, RBS and all inducible components of the Lac operon. Thus, this part can be used to synthesize EutS at varying concentrations, depending on the task at hand. EutS is tagged with His6 (see Figure 1).

Fig 1: Schematic of the LacUV5_EutS part (BBa_K2213000)

Lac UV5 Promoter

The Lac expression system is one of the most commonly used systems for expressing recombinant proteins. The Lac UV5 promoter is very similar to the standard E.coli Lac promoter, with only two base mutations in the -10 hexamer region, compared to the lac promoter. The expression system is primarily composed of the Lac UV5 promoter, the Lac repressor (LacI) and an operator region. In our part, lactose (lac) can bind LacI, reducing its affinity for DNA. Thus upon lac addition, LacI dissociates from the operator , permitting transcription of any gene under control of the LacUV5 promoter.

EutS

EutS is one of the shell proteins that make up the Ethanolamine utilisation bacterial microcompartment (Eut BMC) in E. coli and other enterobacteria species. It is a hexameric protein, and seem to function as the outer edges of the BMC shell (Held et.al, 2013).

A study conducted by Held et.al (2016) and Choudhary et.al (2012) has shown that Eut S is necessary and sufficient for the successful formation of the Eut BMC. This property was also observed by the CU-Boulder iGEM team in 2016 (http://2016.igem.org/Team:CU-Boulder). While the Manchester team did not observe the sufficiency of EutS to form microcompartment, our data suggests that EutMN becomes more stable when co-expressed with EutS (see below). This seems to be in line with previous findings on the necessity of EutS for proper BMC formation and further substantiates them.

Usage and Biology

The CU-boulder team has shown that it is possible to make functional BMCs using only EutS in 2016. However, 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, the Manchester team recommends the use of a low copy number plasmid eg. pSB4A5 (https://parts.igem.org/Part:pSB4A5) for the expression of EutS. 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.

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:

Figure 4: 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 5: 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 6: 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 7: This graph is like figure 3, 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.

Sequence and Features

References
-Held, M., Kolb, A., Perdue, S., Hsu, S., Bloch, S., Quin, M. and Schmidt-Dannert, C. (2016). Engineering formation of multiple recombinant Eut protein nanocompartments in E. coli. Scientific Reports, 6(1).
-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.
-Choudhary, S., Quin, M., Sanders, M., Johnson, E. and Schmidt-Dannert, C. (2012). Engineered Protein Nano-Compartments for Targeted Enzyme Localization. PLoS ONE, 7(3), p.e33342.