Difference between revisions of "Part:BBa K322921"

Latest revision as of 15:48, 12 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.

In our project of directed evolution , we added SacB in our constructed vector as a counter-selection element , and we collected some related information from the past papers to verify our experiment.

Fig.1 SacB addition in our constructed vector , which serves as a counter-selection element.

Fig.2 In the process of counter-selection,we add sucrose on the surface of solid culture medium . (A): No colonies are found on the medium supplemented with sucrose;(B):There are many colonies on the medium without added sucrose.

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.

Contribution: iGEM22_DUT_China

1. We have compiled the information about sacB in the literature, such as the structure and function of the protein, to facilitate the follow-up investigation of this protein by other teams.

2. We developed a CRISPR-based purification system "strainer" in E. coli, which can remove the unsuccessfully edited cells, and then improve the overall editing efficiency. This system can be used on most scenes.

1 New information learned from literature

The description of BBa_K322921 has briefly introduced the lethal effect of sacB, and the DUT_China team will add information on its structure and function.

The sacB gene encodes the secreted enzyme levansucrase. The enzyme catalyzes hydrolysis of sucrose and synthesis of levans, which are high-molecular-weight fructose polymers. In the gram-negative bacteria E. coli, E. chrysanthemi, and L. pneumophila, expression of sacB in the presence of sucrose is lethal. In E. coli, levansucrase activity is mostly located in the periplasm. The molecular basis of the toxicity is still unclear, but the toxicity could be due to an accumulation of levans which might encumber the periplasm because of their high molecular weight or a transfer of fructose residues to inappropriate acceptor molecules, which could thereafter have toxic effects on the bacterial cells.[1]

We learned that, modification of the tuned interplay among the ten residues that originate the sucrose-binding site (first layer comprising subsites -1 and +1) has generally a drastic effect on the enzyme’s activity and often reduces transfructosylation/hydrolysis and HMW levan/FOS partitions.

Fig. 1 The first layer means that the amino acid shown in the figure is the closest layer to the substrate (sucrose), and the distance between all amino acids and the substrate is less than 3.5 Å. W85, D86, W163, R246, D247, E342 are completely conservative in GH68 family.[1]

W85, D86, W163, R246, D247 and E342 are entirely conserved in such enzymes. When serine is present, its side chain can establish hydrogen bonds with sucrose. E342, R246 and semi-conserved residues R360 and E340 are part of the +1 subsite. [2]. According to the crystallographic structure of SacB, the side chains of residues from -1/+1 subsites (excluding W163 and positions 164 and 412 when occupied by alanine) that are able to act as a donor/acceptor of the hydrogen bond are at distances ≤3.5 Å from sucrose hydroxyl-groups (Fig. 1).

R246 is an invariant residue, which participates in hydrogen bonds via its guanidinium group with the 3-OH groups of the fructosyl-moiety. So, researchers thought that the function of R246 may involve both the stabilization of sucrose binding and the support of an optimal orientation of E340 and E342 with respect to the substrate.[3]

Scientists found that S164 forms a hydrogen bond with the nucleophile D86 and the 4-OH of the fructosyl-moiety, while S412 modulates the position of the nucleophile regarding to the fructosyl-donor also via a hydrogen bond. S412 coordinates the 1-OH group of the fructosyl unit and the invariant residue R343 of the 339DxxER343 motif (Fig. 1) as well. [3]

Arginine or histidine can be found at corresponding positions to 360 in the +1 subsite of fructansucrases, respectively. These residues form hydrogen bonds with the 2- and 3-OH groups of the glucosyl-moiety (Fig. 1, right panel). Mutations at this position reduce the rate of formation of the covalent intermediate fructosyl-enzyme leaving the hydrolyzing activity unaffected and favoring the synthesis of kestose.[4]

2 New data collected from laboratory experiments

Improve The CFU/μg of "strainer" Method with SacB Mutant

The results showed that the editing efficiency of SacB_S164T mutant (in EC85) is 25% higher than the control without “strainer” system. The CFU/μg of SacB_S164T mutant (in EC85) increased 3-fold compared to the original “strainer” system while still keep high editing efficiency (Fig. 2A).

We also used the same condition to test SacB_S164T mutant in EC88. The results showed that the editing efficiency using “strainer” system with SacB_S164T mutant is 4-fold higher than the control, although the CFU/μg of SacB_S164T mutant (in EC88) is still 71% lower than the control without “strainer” system (Fig. 2B). We also used the same condition to test SacB_S164T mutant in EC88. However, the results showed that the toxicity of the modified SacB protein was low, and the screening effect served by the lower concentration of sucrose chosen at this point was no longer obvious, so we further optimized the sucrose concentration and found that testing the mutants at 0.1% concentration conditions, the CFU/μg using the "strainer" system with the SacB_S164T mutant was 2-fold higher than the control, although the SacB_ S164T mutant (in EC88) still had a lower CFU/μg than the control without the "strainer" system.

These results showed that our strainer system works as a good purification system to remove the unedited cells, and SacB_S164T mutant is less toxicity than the original SacB.

Fig. 2. Improve the CFU/μg of “strainer” method with SacB mutant. (A) The CFU/μg and editing efficiency of the control group, SacB and SacB_S164T under 0.01% sucrose condition in EC85. (B) The CFU/μg and editing efficiency of the control group, SacB and SacB_S164T under 0.1% sucrose and 0.2% sucrose condition in EC88..

References

[1] Ortiz-Soto ME, Porras-Dominguez JR, Seibel J, Lopez-Munguia A. A close look at the structural features and reaction conditions that modulate the synthesis of low and high molecular weight fructans by levansucrases. Carbohydr Polym. 2019, 219: 130-142.

[2] Meng GY, Futterer K. Structural framework of fructosyl transfer in Bacillus subtilis levansucrase. Nat Struct Biol. 2003, 10 (11): 935-941.

[3] Homann A, Biedendieck R, Gotze S, Jahn D, Seibel J. Insights into polymer versus oligosaccharide synthesis: mutagenesis and mechanistic studies of a novel levansucrase from Bacillus megaterium. Biochem J. 2007, 407: 189-198.

[4] Chambert R,Petitglatron MF. POLYMERASE AND HYDROLASE ACTIVITIES OF BACILLUS-SUBTILIS LEVANSUCRASE CAN BE SEPARATELY MODULATED BY SITE-DIRECTED MUTAGENESIS. Biochem J. 1991, 279: 35-41.