Difference between revisions of "Part:BBa K2213002"

Latest revision as of 02:14, 2 November 2017

araB_eutLK

The ethanolamine utilisation bacterial microcompartment (BMC) proteins, EutLK (from E. coli) under control of the arabinose inducible promoter. Contains RBS, terminators and all tetp components. EutL is tagged with His6. EutK is tagged with FLAG (see figure 1).

Fig 1: Schematic of the araBAD_EutLK part (BBa_K2213002)

This part is used in the composite part: https://parts.igem.org/Part:BBa_K2213013

araBad Promoter

The ara expression system is advantageous as in addition to being induced by arabinose, gene expression can be rapidly downregulated as repression can occur due to catabolite repression in the presence of glucose or by competitive binding of the anti-inducer fucose. Plasmids containing the ara expression system have minimal levels of background expression. This is not the case with the lac expression system, which is leaky (Siegele and Hu, 1997).

In this construct, the expression system consists of an araBad promoter, an operator region, the araBad repressor (araC), and a bidirectional terminator. Addition of arabinose reduces the affinity of araC to DNA, causing it to dissociate from the operator region. As a result, genes under the control of the araBad promoter can be transcribed.


EutLK

The Ethanolamine Utilisation (Eut) bacterial micro-compartment (BMC) proteins EutL and EutK from E.coli are clustered together here, as they are found in nature.

EutL is the longest of the Eut microcompartment proteins, at 216 amino acids in length (Takenoya et.al, 2010). It is similar in structure to EutS and forms a pseudohexamer with two other EutL proteins (Tanaka et.al, 2010). The protein has a centralized pore that is negatively charged, in contrast to that of EutM. EutL and M have been shown to form a 2D proteinaceous membrane in solution (Takenoya et.al, 2010).

EutK is distinct to the other Eut shell proteins, in that whereas the rest of the Eut BMC proteins form hexamers (or pseudohexamers such as the case of EutL), EutK exists as a monomer in solution. The apparent inability of EutK to assemble into a hexamer by itself suggests that different BMC paralogs might form mixed hexamers during assembly of the shell (Tanaka et.al, 2010).

Usage and Biology

Although it is possible to use this part for EutL and K 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 EutLK protein expression
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 2). 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 2. 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.

Sequence and Features

References
Siegele, D.A. and Hu, J.C., 1997. Gene expression from plasmids containing the araBAD promoter at subsaturating inducer concentrations represents mixed populations. Proceedings of the National Academy of Sciences, 94(15), pp.8168-8172.

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.