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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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.

@@ Line 50: / Line 50: @@
 ==Contribution: iGEM22_DUT_China==
-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.
+. 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.
-The Bacillus subtilis sacB gene encodes the secreted enzyme levansucrase (sucrose: 2,6-b-D-fructan 6-b-D-fructosyltrans- ferase; EC 2.4.1.10; PDB DOI: 10.2210/pdb1PT2/pdb). 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 (4, 6, 16). 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.
+. 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.
-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 1activity and often reduces transfructosylation/hydrolysis and HMW levan/FOS partitions.
+===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.
 <html>
          <div class="col-lg" style="margin:auto;text-align:center;">
-                  <img style="margin:20px auto 5px auto;" src="https://static.igem.wiki/teams/4176/wiki/images/contribution-1.png" width="80%">
+                  <img style="margin:20px auto 5px auto;" src="https://static.igem.wiki/teams/4176/wiki/images/model-10.png" width="80%">
-                  <p style="color:Gray; padding:0px 30px 10px;"> Fig. 1. The sucrose binding site of Bs-SacB. Amino acids at hydrogen bond distances (dashed purple lines) from the fructosyl- and glucosyl-moieties constitute the -1 and +1 subsites, respectively. All residues displayed are at distances ≤ 3.5 Å to sucrose hydroxyl-groups. PDB files 1oyg and 1pt2 were employed to prepare the picture. Sucrose from PDB 1pt2 is de- picted in orange/outlined sticks, highlighting either the fructosyl or the glucosyl unit. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).<sup>[1]</sup></p>
+                  <p style="color:Gray; padding:0px 30px 10px;">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]</p>
           </div>
 </html>
-Residues W85, D86, W163, R246, D247 and E342 are entirely conserved in GH68 enzymes and, with exception of E342, constitute the -1 subsite (Fig. 1). Only alanine or serine are found at positions 164 and 412 in members of family GH68. When serine is present, its side chain can establish hydrogen bonds with sucrose. Thus, we included residues 164 and 412 as part of the -1 subsite of Bs-SacB. E342, R246 and semi-conserved residues R360 and E340 are part of the +1 subsite. <sup>[2]</sup> According to the crystallographic structure of Bs-SacB (PDB1pt2), 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).
+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).
-Invariant residue R246 participates in hydrogen bonds via its guanidinium group with the 3-OH groups of the fructosyl-moiety and the carboxylates of residues E340 and E342 (Fig. 3). Thus, 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. Accordingly, substitution of R246 by alanine nearly in- activates Bm-LS.<sup>[3]</sup>
+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]
-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. Mutation at these two positions by residues of different length and polarity has a negative effect on activity and different impact on K<sub>M</sub>.<sup>[3]</sup>
+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.<sup>[4]</sup>
+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]
-In addition to sacb, we also investigated the tertiary structure of the substrate-bound state of other levansucrases.
+===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.
 <html>
          <div class="col-lg" style="margin:auto;text-align:center;">
-                  <img style="margin:20px auto 5px auto;" src="https://static.igem.wiki/teams/4176/wiki/images/contribution-2.png" width="80%">
+                  <img style="margin:20px auto 5px auto;" src="https://static.igem.wiki/teams/4176/wiki/images/poc-4.png" width="80%">
-                  <p style="color:Gray; padding:0px 30px 10px;">Fig. 2. Binding pocket volume (yellow spheres) of levansucrases of known three-dimensional structure. A frontal and a 90° right-side view of Bs-SacB, Bm-LS, Ea-LS and Gd-LS are displayed. The volumes were predicted using CASTp 3.0 <sup>[5]</sup></p>
+                  <p style="color:Gray; padding:0px 30px 10px;">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..</p>
           </div>
 </html>
-The four levansucrases whose structure is available were selected for a detailed comparison of their central binding pockets, namely Bs-SacB (1oyg and 1pt2), Bm-LS (PDB 3om2), Ea-LS (PDB 4d47) and Gd-LS (PDB 1w18). As expected, the architecture of the sucrose binding site is conserved, while the loops that surround it are variable. Such structural variety equips the enzymes with central pockets exposing dissimilar superficial areas and volumes.
-Topology of Bs-SacBand Bm-LS is very similar. Accordingly, the binding pocket volume and area of both enzymes are comparable (Fig. 2). As mentioned previously, Bm-LS produces FOS with an average molecular weight of 1.6 kDa (Ortiz-Soto et al., 2017, 2018), while Bs-SacB is capable of synthesizing LMW and HMW levan of around 8.3 and 2000 kDa, respectively.<sup>[6]</sup>
-The differences between Ea-LS and Bs-SacB are larger (Fig. 2). Both the volume and superficial area of the catalytic pocket of Ea-LS are smaller than those of Bs-SacB (78% and 75% respectively). It is worth mentioning that Ea-LS synthesizes mainly FOS containing 3–6 fructosyl units but it is also capable of synthesizing levan of 29,700 kDa that forms particles with a mean squared radius of 43.8 nm<sup>[7]</sup>
-Gd-LS displays a 2.3-fold larger surface and 4.3-fold larger volume than Bs-SacB. It can be observed, however, that the sucrose/kestose binding site of Gd-LS is quite narrow and is the least exposed regarding to the sucrose binding site of other levansucrases compared in this section (Fig. 2). Larger area and volume result from the longer loops that enclose the ß-propeller core of Gd-LS, producing a deep and pro- longed central cavity. This enzyme is 1.45 times more hydrolytic than Bs-SacB [8] based on kcat values, and produces inulin-type FOS 1-kestose and 1-nystose<sup>[8]</sup> almost exclusively. Unfortunately, kinetic data (k<sub>cat</sub>) for hydrolysis and transfer under similar conditions is not available for all the enzymes compared here.
 ===References===
-[1] Porras-Domínguez J, Seibel J and López-Munguía] A. A close look at the structural features and reaction conditions that modulate the synthesis of low and high molecular weight fructans by levansucrases [J]. Carbohydrate Polymers, 2019.
-[2] Meng G. Structural framework of fructosyl transfer in Bacillus subtilis levansucrase [J]. Nat. Struct. Biol, 2003, 10.
-[3] Homann A, Biedendieck R, Götze S, et al. Insights into polymer versus oligosaccharide synthesis: mutagenesis and mechanistic studies of a novel levansucrase fromBacillus megaterium [J]. Biochemical Journal, 2007.
+[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.
-[4] Chambert R and Petit-Glatron MF. Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis [J]. Biochemical Journal, 1991, 279 ( Pt 1)(1): 35-41.
+[2] Meng GY, Futterer K. Structural framework of fructosyl transfer in Bacillus subtilis levansucrase. Nat Struct Biol. 2003, 10 (11): 935-941.
-[5] Wei T, Chang C and Jie L. CASTp 3.0: Computed Atlas of Surface Topography of Proteins and Beyond [J]. Biophysical Journal, 2018, 114(3): 50a.
+[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.
-[6] Ortiz-Soto ME, Ertl J, Mut J, et al. Product-oriented chemical surface modification of a levansucrase (SacB) via an ene-type reaction [J]. Chemical Science, 2018: 10.1039.C8SC01244J.
+[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.
-[7] The crystal structure of Erwinia amylovora levansucrase provides a snapshot of the products of sucrose hydrolysis trapped into the active site [J]. Journal of Structural Biology, 2015, 191(3): 290-298.
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