Difference between revisions of "Part:BBa K2213000"

Revision as of 00:55, 1 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 Salmonella spp 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 it.

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 this part. By using a low copy number plasmid, cellular stress is minimised, but the experimenter still has the ability to induce BMC formation.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 1260
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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.

@@ Line 20: / Line 20: @@
 EutS is one of the shell proteins that make up the Ethanolamine utilisation bacterial microcompartment (Eut BMC) in <i>Salmonella</i> spp  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 we 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 it.
+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 it.
 ===Usage and Biology===
-Despite not being able to demonstrate this in our project, literature has shown that it is possible to make functional BMCs using only EutS. However, when forced to produce BMCs, <i> E. coli</i>  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.
+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, <i> E. coli</i>  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 this part. By using a low copy number plasmid, cellular stress is minimised, but the experimenter still has the ability to induce BMC formation.
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