Difference between revisions of "Part:BBa K5246035"
 
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===Introduction===
 
===Introduction===
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Vilnius-Lithuania iGEM 2024 project <HTML><b><a href="https://2024.igem.wiki/vilnius-lithuania" target="_blank">Synhesion</a></b></html> aspires to create biodegradable and environmentally friendly adhesives. We were inspired by bacteria, which naturally produce adhesives made from polysaccharides. Two bacteria from aquatic environments - <I> Caulobacter crescentus </I> and <I> Hirschia baltica </I> - harness 12 protein synthesis pathways to produce sugars, anchoring them to the surfaces. We aimed to transfer the polysaccharide synthesis pathway to industrially used <I>Escherichia coli</I> bacteria to produce adhesives. Our team concomitantly focused on creating a novel <I>E. coli</I> strain for more efficient production of adhesives.
 +
 +
This is a <b>part of the complete holdfast polymerization and export apparatus </b> <HTML><b><a href="https://parts.igem.org/Part:BBa_K5246046" target="_blank">BBa_K5246046</a></b></html> used in Vilnius-Lithuania iGEM 2024 project <HTML><b><a href="https://2024.igem.wiki/vilnius-lithuania" target="_blank">Synhesion</a></b></html>. This part can also be used separately for polysaccharide export, but this feature needs more characterization.
  
 
===Strain description===
 
===Strain description===
 +
 +
<i>Escherichia coli</i> BL21(DE3) protein expression strain derived from  <i>Escherichia coli</i>  B strain. It produces recombinant proteins under the control of T7 RNA polymerase [1]. This strain lacks the proteases Lon and OmpT, which reduces the likelihood of recombinant protein degradation [2]. This strain is resistant to phage T1 (fhuA2), which improves survival and growth of the cells [3].
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  <meta charset="UTF-8">
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  <title>BL21(DE3) Genotype Table</title>
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    table {
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<center><b>Table 1.</b> Genotype of BL21(DE3) <i>E. coli</i> strain</center>
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<table>
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  <tr>
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    <th>Genotype</th>
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    <td>fhuA2 [lon] ompT gal (λ DE3) [dcm] ∆hsdS λ DE3 = λ sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5</td>
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  </tr>
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</table>
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</body>
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</html>
  
 
===Usage and Biology===
 
===Usage and Biology===
−
WecA is Undecaprenyl-phosphate &#945;-N-acetylglucosaminyl transferase initiating the biosynthesis of enterobacterial common antigen (ECA) and O-antigen PS by catalyzing the transfer of N-acetylglucosamine (GlcNAc)-1-phosphate onto undecaprenyl phosphate to form Und-P-P-GlcNAc. WecA is the first glycosyltransferase, and deleting the wecA gene impairs the ECA synthesis pathway in E.coli. Therefore, lipopolysaccharides containing it are not produced. We wanted to disable the ECA pathway to reduce the metabolic burden on the cell. We aimed to optimize polysaccharide production by creating a more efficient strain. BL21(DE3) is an E.coli expression strain suitable for expressing non-toxic heterologous genes. The BL21(DE3) strain has a lambda DE3 prophage that allows for the expression of T7 RNA polymerase with IPTG. This strain is an E. coli B derivative that lacks the lon and OmpT proteases, which help preserve foreign proteins from degradation.
+
 
 +
===Biology===
 +
 
 +
The enterobacterial common antigen (ECA) is an outer membrane glycolipid shared by all members of the Enterobacteriaceae and is restricted to this family. It is a glycophospholipid located in the outer leaflet of the outer membrane, composed of an L-glycerophosphatidyl residue linked to an aminosugar heteropolymer [4]. The carbohydrate consists of trisaccharide repeat units of N-acetyl-α-D-glucosamine, N-acetyl-β-D-mannosaminuronate, and N-acetylthomosamine [5]. The function of this molecule has remained largely unknown, partly because the biosynthesis pathways for ECA, O-antigen, and peptidoglycan overlap and partly because there are three forms of ECA that cannot currently be genetically separated [6]. Current research suggests that its function is to maintain outer membrane stability and immunogenicity, and it is involved in biofilm formation [7],[8],[9].
 +
 
 +
WecA is Undecaprenyl-phosphate α-N-acetylglucosaminyl transferase, which initiates the biosynthesis of enterobacterial common antigen (ECA) and O-antigen by catalyzing the transfer of N-acetylglucosamine (GlcNAc)-1-phosphate onto undecaprenyl phosphate to form Und-P-P-GlcNAc[10]. The O antigen is located on the cell surface and could be recognized by the host immune system and bacteriophages [11].
 +
 
 +
Eliminating the wecA gene results in ECA and lipopolysaccharide 08-side chain inactivation without causing high-stress levels [12].  
 +
 
 +
===Usage===
 +
 
 +
<i>Escherichia coli</I> BL21(DE3) protein expression strain is used to produce recombinant proteins under the control of T7 RNA polymerase [1]. BL21(DE3) is widely used in both research and industrial settings. It is particularly popular due to its high efficiency in producing recombinant proteins. More specifically, it is widely used as medical diagnostic reagents in human healthcare, including vaccines, drugs, or antibodies, and in biochemical analysis[13].  
 +
 
 +
Knocking out the <i>wecA</I> gene and inactivating the ECA pathway loads off the metabolic burden of synthesizing polysaccharides. It prevents ECA biosynthesis and increases precursor availability for peptidoglycan biosynthesis [14]. BL21(DE3)∆wecA strain could be used to investigate the ECA pathway further and the cell interactions with other cells or bacteriophages. This strain could be used in biotechnology as cells with altered surface properties. Biofilms are increasingly recognized as a critical global issue in many industries and come out with economic costs. Biofilms contaminate manufacturing equipment and pharmaceuticals, compromising product quality and safety. Biofilms can foul heat exchangers, pipelines, and other industrial equipment, leading to decreased performance and increased energy consumption. A strain with reduced biofilm formation could save power consumption and finances [15]. On top of that, exposed glycan structures often serve as initial receptors for host - N4 bacteriophage - recognition. Subsequent binding to a terminal or secondary receptor directly on the cell surface triggers irreversible adsorption and injection of the phage genome. [16] N4 bacteriophage infects industrially grown bacteria. Eliminating recognition antigens on these bacteria could provide them additional resistance to N4 phage infection. [17].
 +
 
 +
In our project, Synhesion, we aimed to produce an efficient polysaccharide in <i>E. coli</I> by introducing a new synthesis pathway. This pathway originated from the bacteria <i>C. crescentus</> and <i>H. baltica</I>, which inhabit aquatic environments. Although polysaccharide synthesis pathways in <i>E. coli</i> and marine bacteria are analogous, we aimed to engineer an <i>E. coli</I> strain that could produce desired polysaccharides more efficiently by reducing its metabolic burden. For this reason, we chose to eliminate the polysaccharide-producing ECA pathway from <i>E. coli</I> by knocking out the <i>wecA</I> gene.
  
 
===Sequence and Features===
 
===Sequence and Features===
Line 17: Line 76:
  
 
===Experimental characterization===
 
===Experimental characterization===
 +
To create an ECA pathway deficient E. coli BL21(DE3)∆wecA strain, we used a homology recombineering <b>Red/ET recombination system</B>. Our edited colonies had kanamycin-resistance cassettes as markers in the edited region, and we tested them by colony PCR (Fig 1.). We could distinguish edited and non-edited colonies by size: PCR’ed non-edited region 1544 bp and with edited region 1840 bp.
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<html>
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</p>
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<figure>
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<div class = "center" >
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<center>
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<img src = "https://static.igem.wiki/teams/5246/registry/bl21-weca-ko-alone.webp" style = "width:300px;"></center>
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</div>
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<figcaption><center>
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<b>Fig. 1.</b> Knocked out wecA gene in E.coli strain BL21(DE3). C- is the wecA gene and its region from an unedited strain (~1.5 kb), and C+ is the kanamycin cassette, indicating knockout (~1.8 kb). 1-10 is the cPCR of knocked-out colonies. Colonies the same size as C+ indicate a knockout.  M - molecular weight ladder, GeneRuler Mix DNA Ladder (Thermo Scientific). 1% agarose gel in TAE buffer with EtBr.</center></figcaption>
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</figure>
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<p/>
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</html>
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===References===
 +
1. Langenscheid, J., Killmann, H., & Braun, V. (2004). A FhuA mutant of Escherichia coli is infected by phage T1 independent of TonB. FEMS Microbiology Letters, 234(1), 133–137. https://doi.org/10.1016/j.femsle.2004.03.019
 +
<br>
 +
2. Reduction of protein degradation by use of protease-deficient mutants in cell-free protein synthesis system of Escherichia coli. (2002). Journal of Bioscience and Bioengineering, 93(1), 19–25. https://doi.org/10.1016/S1389-1723(02)80007-X
 +
<br>
 +
3. Davis, B. M., & Waldor, M. K. (2009). Genome sequences of Escherichia coli B strains REL606 and BL21(DE3). Journal of Molecular Biology, 394(4), 1026–1036. https://doi.org/10.1016/j.jmb.2009.09.052
 +
<br>
 +
4. Rick, P. D., Hubbard, G. L., & Barr, K. (1994). Role of the rfe gene in the synthesis of the O8 antigen in Escherichia coli K-12. Journal of Bacteriology, 176(9), 2877–2884. https://doi.org/10.1128/jb.176.9.2877-2884.1994
 +
<br>
 +
5. Klena, J. D., & Schnaitman, C. A. (1993). Function of the rfb gene cluster and the rfe gene in the synthesis of O antigen by Shigella dysenteriae 1. Molecular Microbiology, 9(2), 393–402. https://doi.org/10.1111/j.1365-2958.1993.tb01705.x
 +
<br>
 +
6. Alexander, D. C., & Valvano, M. A. (1994). Role of the rfe gene in the biosynthesis of the Escherichia coli O7-specific lipopolysaccharide and other O-specific polysaccharides containing N-acetylglucosamine. Journal of Bacteriology, 176(22), 7079–7084. https://doi.org/10.1128/jb.176.22.7079-7084.1994
 +
<br>
 +
7. Sala, R. F., Morgan, P. M., & Tanner, M. E. (1996). Journal of the American Chemical Society, 118(12), 3033–3034. https://doi.org/10.1021/ja960266z
 +
<br>
 +
8. Jorgenson, M. A., Kannan, S., Laubacher, M. E., & Young, K. D. (2016). Dead-end intermediates in the enterobacterial common antigen pathway induce morphological defects in Escherichia coli by competing for undecaprenyl phosphate. Molecular Microbiology, 100(1), 1–14. https://doi.org/10.1111/mmi.13304
 +
<br>
 +
9. Zeidan, A. A., Poulsen, V. K., Janzen, T., Buldo, P., Derkx, P. M. F., Øregaard, G., & Neves, A. R. (2017). Polysaccharide production by lactic acid bacteria: From genes to industrial applications. FEMS Microbiology Reviews, 41(Suppl_1), S168–S200. https://doi.org/10.1093/femsre/fux017
 +
<br>
 +
10. Rai, A. K., Carr, J. F., Bautista, D. E., Wang, W., & Mitchell, A. M. (2021). ElyC and cyclic enterobacterial common antigen regulate synthesis of phosphoglyceride-linked enterobacterial common antigen. mBio, 12(5), e02846-21. https://doi.org/10.1128/mBio.02846-21
 +
<br>
 +
11. Highmore, C. J., Melaugh, G., Morris, R. J., & Rice, S. A. (2022). Translational challenges and opportunities in biofilm science: A BRIEF for the future. npj Biofilms and Microbiomes, 8(1), 68. https://doi.org/10.1038/s41522-022-00327-7
 +
<br>
 +
12. Nobrega, F. L., Vlot, M., de Jonge, P. A., Drost, M., & Brouns, S. J. J. (2018). Targeting mechanisms of tailed bacteriophages. Nature Reviews Microbiology, 16(12), 760–773. https://doi.org/10.1038/s41579-018-0070-8
 +
<br>
 +
13. McPartland, J., & Rothman-Denes, L. B. (2009). The tail sheath of bacteriophage N4 interacts with the Escherichia coli receptor. Journal of Bacteriology, 191(2), 525–532. https://doi.org/10.1128/JB.01423-08
 +
<br>
 +
14. Rick, P. D., Hubbard, G. L., & Barr, K. (1994). Role of the rfe gene in the synthesis of the O8 antigen in Escherichia coli K-12. Journal of Bacteriology, 176(9), 2877–2884. https://doi.org/10.1128/jb.176.9.2877-2884.1994
 +
<br>
 +
15. Klena, J. D., & Schnaitman, C. A. (1993). Function of the rfb gene cluster and the rfe gene in the synthesis of O antigen by Shigella dysenteriae 1. Molecular Microbiology, 9(2), 393–402. https://doi.org/10.1111/j.1365-2958.1993.tb01705.x
 +
<br>
 +
16. Alexander, D. C., & Valvano, M. A. (1994). Role of the rfe gene in the biosynthesis of the Escherichia coli O7-specific lipopolysaccharide and other O-specific polysaccharides containing N-acetylglucosamine. Journal of Bacteriology, 176(22), 7079–7084. https://doi.org/10.1128/jb.176.22.7079-7084.1994
 +
<br>
 +
17. Sala, R. F., Morgan, P. M., & Tanner, M. E. (1996). Journal of the American Chemical Society, 118(12), 3033–3034. https://doi.org/10.1021/ja960266z

Latest revision as of 19:30, 1 October 2024


BL21(DE3) ΔWecA

Introduction

Vilnius-Lithuania iGEM 2024 project Synhesion aspires to create biodegradable and environmentally friendly adhesives. We were inspired by bacteria, which naturally produce adhesives made from polysaccharides. Two bacteria from aquatic environments - Caulobacter crescentus and Hirschia baltica - harness 12 protein synthesis pathways to produce sugars, anchoring them to the surfaces. We aimed to transfer the polysaccharide synthesis pathway to industrially used Escherichia coli bacteria to produce adhesives. Our team concomitantly focused on creating a novel E. coli strain for more efficient production of adhesives.

This is a part of the complete holdfast polymerization and export apparatus BBa_K5246046 used in Vilnius-Lithuania iGEM 2024 project Synhesion. This part can also be used separately for polysaccharide export, but this feature needs more characterization.

Strain description

Escherichia coli BL21(DE3) protein expression strain derived from Escherichia coli B strain. It produces recombinant proteins under the control of T7 RNA polymerase [1]. This strain lacks the proteases Lon and OmpT, which reduces the likelihood of recombinant protein degradation [2]. This strain is resistant to phage T1 (fhuA2), which improves survival and growth of the cells [3].

BL21(DE3) Genotype Table

Table 1. Genotype of BL21(DE3) E. coli strain
Genotype fhuA2 [lon] ompT gal (λ DE3) [dcm] ∆hsdS λ DE3 = λ sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5

Usage and Biology

Biology

The enterobacterial common antigen (ECA) is an outer membrane glycolipid shared by all members of the Enterobacteriaceae and is restricted to this family. It is a glycophospholipid located in the outer leaflet of the outer membrane, composed of an L-glycerophosphatidyl residue linked to an aminosugar heteropolymer [4]. The carbohydrate consists of trisaccharide repeat units of N-acetyl-α-D-glucosamine, N-acetyl-β-D-mannosaminuronate, and N-acetylthomosamine [5]. The function of this molecule has remained largely unknown, partly because the biosynthesis pathways for ECA, O-antigen, and peptidoglycan overlap and partly because there are three forms of ECA that cannot currently be genetically separated [6]. Current research suggests that its function is to maintain outer membrane stability and immunogenicity, and it is involved in biofilm formation [7],[8],[9].

WecA is Undecaprenyl-phosphate α-N-acetylglucosaminyl transferase, which initiates the biosynthesis of enterobacterial common antigen (ECA) and O-antigen by catalyzing the transfer of N-acetylglucosamine (GlcNAc)-1-phosphate onto undecaprenyl phosphate to form Und-P-P-GlcNAc[10]. The O antigen is located on the cell surface and could be recognized by the host immune system and bacteriophages [11].

Eliminating the wecA gene results in ECA and lipopolysaccharide 08-side chain inactivation without causing high-stress levels [12].

Usage

Escherichia coli BL21(DE3) protein expression strain is used to produce recombinant proteins under the control of T7 RNA polymerase [1]. BL21(DE3) is widely used in both research and industrial settings. It is particularly popular due to its high efficiency in producing recombinant proteins. More specifically, it is widely used as medical diagnostic reagents in human healthcare, including vaccines, drugs, or antibodies, and in biochemical analysis[13].

Knocking out the wecA gene and inactivating the ECA pathway loads off the metabolic burden of synthesizing polysaccharides. It prevents ECA biosynthesis and increases precursor availability for peptidoglycan biosynthesis [14]. BL21(DE3)∆wecA strain could be used to investigate the ECA pathway further and the cell interactions with other cells or bacteriophages. This strain could be used in biotechnology as cells with altered surface properties. Biofilms are increasingly recognized as a critical global issue in many industries and come out with economic costs. Biofilms contaminate manufacturing equipment and pharmaceuticals, compromising product quality and safety. Biofilms can foul heat exchangers, pipelines, and other industrial equipment, leading to decreased performance and increased energy consumption. A strain with reduced biofilm formation could save power consumption and finances [15]. On top of that, exposed glycan structures often serve as initial receptors for host - N4 bacteriophage - recognition. Subsequent binding to a terminal or secondary receptor directly on the cell surface triggers irreversible adsorption and injection of the phage genome. [16] N4 bacteriophage infects industrially grown bacteria. Eliminating recognition antigens on these bacteria could provide them additional resistance to N4 phage infection. [17].

In our project, Synhesion, we aimed to produce an efficient polysaccharide in E. coli by introducing a new synthesis pathway. This pathway originated from the bacteria C. crescentus</> and <i>H. baltica, which inhabit aquatic environments. Although polysaccharide synthesis pathways in E. coli and marine bacteria are analogous, we aimed to engineer an E. coli strain that could produce desired polysaccharides more efficiently by reducing its metabolic burden. For this reason, we chose to eliminate the polysaccharide-producing ECA pathway from E. coli by knocking out the wecA gene.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 185
    Illegal SpeI site found at 458
    Illegal PstI site found at 715
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 458
    Illegal PstI site found at 715
    Illegal NotI site found at 164
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 185
    Illegal SpeI site found at 458
    Illegal PstI site found at 715
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 185
    Illegal SpeI site found at 458
    Illegal PstI site found at 715
    Illegal AgeI site found at 323
  • 1000
    COMPATIBLE WITH RFC[1000]

Functional Parameters

genotypeF- ompT hsdSB (rB-, mB-) gal dcm (DE3)(KanR) ΔwecA

Experimental characterization

To create an ECA pathway deficient E. coli BL21(DE3)∆wecA strain, we used a homology recombineering Red/ET recombination system. Our edited colonies had kanamycin-resistance cassettes as markers in the edited region, and we tested them by colony PCR (Fig 1.). We could distinguish edited and non-edited colonies by size: PCR’ed non-edited region 1544 bp and with edited region 1840 bp.

Fig. 1. Knocked out wecA gene in E.coli strain BL21(DE3). C- is the wecA gene and its region from an unedited strain (~1.5 kb), and C+ is the kanamycin cassette, indicating knockout (~1.8 kb). 1-10 is the cPCR of knocked-out colonies. Colonies the same size as C+ indicate a knockout. M - molecular weight ladder, GeneRuler Mix DNA Ladder (Thermo Scientific). 1% agarose gel in TAE buffer with EtBr.

References

1. Langenscheid, J., Killmann, H., & Braun, V. (2004). A FhuA mutant of Escherichia coli is infected by phage T1 independent of TonB. FEMS Microbiology Letters, 234(1), 133–137. https://doi.org/10.1016/j.femsle.2004.03.019
2. Reduction of protein degradation by use of protease-deficient mutants in cell-free protein synthesis system of Escherichia coli. (2002). Journal of Bioscience and Bioengineering, 93(1), 19–25. https://doi.org/10.1016/S1389-1723(02)80007-X
3. Davis, B. M., & Waldor, M. K. (2009). Genome sequences of Escherichia coli B strains REL606 and BL21(DE3). Journal of Molecular Biology, 394(4), 1026–1036. https://doi.org/10.1016/j.jmb.2009.09.052
4. Rick, P. D., Hubbard, G. L., & Barr, K. (1994). Role of the rfe gene in the synthesis of the O8 antigen in Escherichia coli K-12. Journal of Bacteriology, 176(9), 2877–2884. https://doi.org/10.1128/jb.176.9.2877-2884.1994
5. Klena, J. D., & Schnaitman, C. A. (1993). Function of the rfb gene cluster and the rfe gene in the synthesis of O antigen by Shigella dysenteriae 1. Molecular Microbiology, 9(2), 393–402. https://doi.org/10.1111/j.1365-2958.1993.tb01705.x
6. Alexander, D. C., & Valvano, M. A. (1994). Role of the rfe gene in the biosynthesis of the Escherichia coli O7-specific lipopolysaccharide and other O-specific polysaccharides containing N-acetylglucosamine. Journal of Bacteriology, 176(22), 7079–7084. https://doi.org/10.1128/jb.176.22.7079-7084.1994
7. Sala, R. F., Morgan, P. M., & Tanner, M. E. (1996). Journal of the American Chemical Society, 118(12), 3033–3034. https://doi.org/10.1021/ja960266z
8. Jorgenson, M. A., Kannan, S., Laubacher, M. E., & Young, K. D. (2016). Dead-end intermediates in the enterobacterial common antigen pathway induce morphological defects in Escherichia coli by competing for undecaprenyl phosphate. Molecular Microbiology, 100(1), 1–14. https://doi.org/10.1111/mmi.13304
9. Zeidan, A. A., Poulsen, V. K., Janzen, T., Buldo, P., Derkx, P. M. F., Øregaard, G., & Neves, A. R. (2017). Polysaccharide production by lactic acid bacteria: From genes to industrial applications. FEMS Microbiology Reviews, 41(Suppl_1), S168–S200. https://doi.org/10.1093/femsre/fux017
10. Rai, A. K., Carr, J. F., Bautista, D. E., Wang, W., & Mitchell, A. M. (2021). ElyC and cyclic enterobacterial common antigen regulate synthesis of phosphoglyceride-linked enterobacterial common antigen. mBio, 12(5), e02846-21. https://doi.org/10.1128/mBio.02846-21
11. Highmore, C. J., Melaugh, G., Morris, R. J., & Rice, S. A. (2022). Translational challenges and opportunities in biofilm science: A BRIEF for the future. npj Biofilms and Microbiomes, 8(1), 68. https://doi.org/10.1038/s41522-022-00327-7
12. Nobrega, F. L., Vlot, M., de Jonge, P. A., Drost, M., & Brouns, S. J. J. (2018). Targeting mechanisms of tailed bacteriophages. Nature Reviews Microbiology, 16(12), 760–773. https://doi.org/10.1038/s41579-018-0070-8
13. McPartland, J., & Rothman-Denes, L. B. (2009). The tail sheath of bacteriophage N4 interacts with the Escherichia coli receptor. Journal of Bacteriology, 191(2), 525–532. https://doi.org/10.1128/JB.01423-08
14. Rick, P. D., Hubbard, G. L., & Barr, K. (1994). Role of the rfe gene in the synthesis of the O8 antigen in Escherichia coli K-12. Journal of Bacteriology, 176(9), 2877–2884. https://doi.org/10.1128/jb.176.9.2877-2884.1994
15. Klena, J. D., & Schnaitman, C. A. (1993). Function of the rfb gene cluster and the rfe gene in the synthesis of O antigen by Shigella dysenteriae 1. Molecular Microbiology, 9(2), 393–402. https://doi.org/10.1111/j.1365-2958.1993.tb01705.x
16. Alexander, D. C., & Valvano, M. A. (1994). Role of the rfe gene in the biosynthesis of the Escherichia coli O7-specific lipopolysaccharide and other O-specific polysaccharides containing N-acetylglucosamine. Journal of Bacteriology, 176(22), 7079–7084. https://doi.org/10.1128/jb.176.22.7079-7084.1994
17. Sala, R. F., Morgan, P. M., & Tanner, M. E. (1996). Journal of the American Chemical Society, 118(12), 3033–3034. https://doi.org/10.1021/ja960266z