Difference between revisions of "Part:BBa K322921"

Revision as of 13:51, 8 October 2022

B. subtilis levansucrase. Lethal to E. coli in presence of sucrose.

sacB encodes the Bacillus subtilis levansucrase, which catalyses hydrolysis of sucrose and synthesis of levans (high molecular weight fructose polymers). It is lethal to gram-negative bacteria E-coli.

It works with cat as an alternative method for inserting BioBricks into the genome by using homologous recombination rather than restriction digestion. SacB is used as a negative selection marker, which allows to insert genes onto the chromosomes without leaving a selection marker. The method can thus be reused indefinitely.

The protocol for BRIDGE can be found on the Edinburgh 2010 igem wiki.

http://2010.igem.org/Team:Edinburgh/Project/Protocol

Measurement of SacB promoter:

SacB promoter is the starting sequence of part BBa K322921. It is separately documented as BBa_K2224001 [1] by SMS_Shenzhen team in 2017.

We, SMS_Shenzhen Team, tested the strength of this promoter by comparing it with J23100. According to our measurement,SacB promoter is a functional promoter in E.coli expression system.

For detailed information about SacB promoter, please see the ‘measurement’subtitle on page [2].

Sequence and Features

Contribution: iGEM22_WHU-China

This is a sucrose lethal gene that encodes the enzyme levosucrase, which catalyzes the production of high molecular weight fructose polymers called Levans. Given that heterologous expression of SacB is lethal in the presence of sucrose in many Gram-negative bacteria, SacB is widely used as a genetic engineering tool. The introduction of this simple and reliable counter-selection method greatly reduced the investment in the practice of bacterial genetic engineering. Counter-selection markers are great candidates for seamless genome modification.

Bacillus subtilis sacB gene with its 463 bp upstream region including its native promoter has been used for marker-free gene deletion in Corynebacterium glutamicum, but the role of this upstream region is not clear. In this study, it was demonstrated that the upstream region of sacB failed to efficiently promote its expression in C. glutamicum, and the native promoter of sacB is weak in C. glutamicum. The expression level of sacB under its native promoter in C. glutamicum is not high enough for cells to confer sucrose sensitivity. Therefore, a new promoter PlacM and a novel vector pDXW-3 were constructed. PlacM is 18 times stronger than the native promoter of sacB in C. glutamicum. The pDXW-3 contains B. subtilis sacBwith the PlacM fused at the 50-end, a general Escherichia coli replicon oriE for easy cloning, a kanamycin resistance marker for selection, and a multiple unique restriction sites for XhoI, NotI, EagI, SalI, SacI, BamHI, and NheI, respectively. By using pDXW-3, the aceE gene in the chromosome of C. glutamicum was deleted. This sacB-based system should facilitate gene disruption and allelic exchange by homologous recombination in many bacteria.

Fig.1 (A) Comparison of maps of vectors pDXW-1, pDXW-2, pDXW-3 and pDXW-3-LRaceE. (B)Construction of the C. glutamicum aceE mutant strain YTW-1. (C and D) Growth difference ofC. glutamicum ATCC13032 and YTW-1 on CGXII media in the presence (C) or absence (D) of sodium acetate.

Reference:
[1] Ambrosis N, Fernández J, Sisti F. Counter-Selection Method for Markerless Allelic Exchange in Bordetella bronchiseptica Based on sacB Gene From Bacillus subtilis. Curr Protoc Microbiol. 2020 Dec;59(1):e125.
[2] Chen W, Li Y, Wu G, Zhao L, Lu L, Wang P, Zhou J, Cao C, Li S. Simple and efficient genome recombineering using kil counter-selection in Escherichia coli. J Biotechnol. 2019 Mar 20;294:58-66.
[3] Logue CA, Peak IR, Beacham IR. Facile construction of unmarked deletion mutants in Burkholderia pseudomallei using sacB counter-selection in sucrose-resistant and sucrose-sensitive isolates. J Microbiol Methods. 2009 Mar;76(3):320-3.
[4] Tan Y, Xu D, Li Y, Wang X. Construction of a novel sacB-based system for marker-free gene deletion in Corynebacterium glutamicum. Plasmid. 2012 Jan;67(1):44-52.