Difference between revisions of "Part:BBa K4719027"
 
(2 intermediate revisions by one other user not shown)
Line 3: Line 3:
 
==Sequence and Features==
 
==Sequence and Features==
 
<partinfo>BBa_K4719027 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4719027 SequenceAndFeatures</partinfo>
−
 
 
<html>
 
<html>
 
<body>
 
<body>
Line 15: Line 14:
 
<a href="https://parts.igem.org/Part:BBa_K4719013">BBa_K4719013</a>. <br>
 
<a href="https://parts.igem.org/Part:BBa_K4719013">BBa_K4719013</a>. <br>
 
<b>2.</b>The second one was the production of N-acetylglucosamine by <i>K. xylinus</i> from common sugars such as glucose, fructose, and sucrose in the growth medium <a href="https://parts.igem.org/Part:BBa_K4719014">BBa_K4719014</a>. <br>
 
<b>2.</b>The second one was the production of N-acetylglucosamine by <i>K. xylinus</i> from common sugars such as glucose, fructose, and sucrose in the growth medium <a href="https://parts.igem.org/Part:BBa_K4719014">BBa_K4719014</a>. <br>
−
After achieving bacterial cellulose-chitin copolymer, we had to deacetylase this material to produce bacterial cellulose-chitosan copolymer <a href="https://parts.igem.org/Part:BBa_K4719019">BBa_K4719019</a>, <a href="https://parts.igem.org/Part:BBa_K4719020">BBa_K4719020</a>, <a href="https://parts.igem.org/Part:BBa_K4719024">BBa_K4719024</a>. Our final objective was to combine the two necessary production steps for bacterial cellulose-chitosan copolymer into a single process.
+
After achieving bacterial cellulose-chitin copolymer, we had to deacetylase this material to produce bacterial cellulose-chitosan copolymer <a href="https://parts.igem.org/Part:BBa_K4719019">BBa_K4719019</a>, <a href="https://parts.igem.org/Part:BBa_K4719020">BBa_K4719020</a>, <a href="https://parts.igem.org/Part:BBa_K4719024">BBa_K4719024</a>. <b> This part was specifically used in our final objective to combine the two necessary production steps for bacterial cellulose-chitosan copolymer into a single process.</b>
  
 
<h2>Usage and Biology</h2>
 
<h2>Usage and Biology</h2>
Line 35: Line 34:
 
<center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/lyderine-koloniju-pgr.png" style = "width:400px;"></center>
 
<center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/lyderine-koloniju-pgr.png" style = "width:400px;"></center>
 
</div>
 
</div>
−
<figcaption><center><b>Figure 1:</b> Results of colony PCR of <i>K. xylinus</i> transformed with pSEVA331-Bb-AGM1-GFA1-GNA1-UAP1-AnCDA. <b>L</b> -  Invitrogen™ 1 Kb Plus DNA Ladder. 1 - positive control. 2-8 - selected colonies. <b>The positive clones (2,3 and 4) had a PCR product of 767bp as expected</b>.  </center></figcaption>
+
<figcaption><center><b>Figure 1.</b> Results of colony PCR of <i>K. xylinus</i> transformed with pSEVA331-Bb-AGM1-GFA1-GNA1-UAP1-AnCDA. <b>L</b> -  Invitrogen™ 1 Kb Plus DNA Ladder. 1 - positive control. 2-8 - selected colonies. <b>The positive clones (2,3 and 4) had a PCR product of 767bp as expected</b>.  </center></figcaption>
 
</figure>
 
</figure>
 
</p>
 
</p>

Latest revision as of 14:41, 12 October 2023

AGM1-GFA1-GNA1-UAP1-AnCDA operon for one-step bacterial cellulose-chitosan synthesis

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2076
    Illegal EcoRI site found at 2574
    Illegal EcoRI site found at 4000
    Illegal EcoRI site found at 5091
    Illegal XbaI site found at 3174
    Illegal SpeI site found at 37
    Illegal SpeI site found at 4867
    Illegal SpeI site found at 5755
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2076
    Illegal EcoRI site found at 2574
    Illegal EcoRI site found at 4000
    Illegal EcoRI site found at 5091
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 5908
    Illegal SpeI site found at 37
    Illegal SpeI site found at 4867
    Illegal SpeI site found at 5755
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2076
    Illegal EcoRI site found at 2574
    Illegal EcoRI site found at 4000
    Illegal EcoRI site found at 5091
    Illegal BglII site found at 1484
    Illegal BglII site found at 1583
    Illegal BglII site found at 3033
    Illegal BamHI site found at 4339
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2076
    Illegal EcoRI site found at 2574
    Illegal EcoRI site found at 4000
    Illegal EcoRI site found at 5091
    Illegal XbaI site found at 3174
    Illegal SpeI site found at 37
    Illegal SpeI site found at 4867
    Illegal SpeI site found at 5755
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2076
    Illegal EcoRI site found at 2574
    Illegal EcoRI site found at 4000
    Illegal EcoRI site found at 5091
    Illegal XbaI site found at 3174
    Illegal SpeI site found at 37
    Illegal SpeI site found at 4867
    Illegal SpeI site found at 5755
    Illegal NgoMIV site found at 5916
    Illegal NgoMIV site found at 5970
    Illegal NgoMIV site found at 6499
    Illegal AgeI site found at 5955
  • 1000
    COMPATIBLE WITH RFC[1000]

Introduction

Vilnius-Lithuania iGEM 2023 team's goal was to create synthetic biology tools for in vivo alterations of Komagataeibacter xylinus bacterial cellulose polymer composition. Firstly, we chose to produce a cellulose-chitin copolymer that would later be deacetylated, creating bacterial cellulose-chitosan. This polymer is an easily modifiable platform when compared to bacterial cellulose. The enhanced chemical reactivity of the bacterial cellulose-chitosan polymer allows for specific functionalizations in the biomedicine field, such as scaffold design. As a second approach, we designed indigo-dyed cellulose that could be used as a green chemistry way to apply cellulose in the textile industry. Lastly, we have achieved a composite of bacterial cellulose and polyhydroxybutyrate (PHB), which is synthesized by K. xylinus.

Bacterial cellulose-chitin polymer was achieved by increasing the production of UDP-N-acetylglucosamine, which can be recognized as a viable substrate for cellulose synthase and incorporated in the bacterial cellulose polymer.
We employed two strategies to produce this material:
1.The first approach was to add N-acetylglucosamine into the growth medium BBa_K4719013.
2.The second one was the production of N-acetylglucosamine by K. xylinus from common sugars such as glucose, fructose, and sucrose in the growth medium BBa_K4719014.
After achieving bacterial cellulose-chitin copolymer, we had to deacetylase this material to produce bacterial cellulose-chitosan copolymer BBa_K4719019, BBa_K4719020, BBa_K4719024. This part was specifically used in our final objective to combine the two necessary production steps for bacterial cellulose-chitosan copolymer into a single process.

Usage and Biology

This construct is a combination of BBa_K4719014, BBa_K4719025 and BBa_K4719011. Expression of this composite in K. xylinus results in a single-tube synthesis of bacterial cellulose-chitosan copolymer. The composite consists of an operon for synthesis and incorporation of N-acetylglucosamine, producing a bacterial cellulose-chitin copolymer, followed by signal peptide sequence fused to deacetylase for direct deacetylation reaction extracellularly. Incorporation of N-acetylglucosamine has been previously demonstrated by M. H. Tan (2019) and V. Yadav (2010), since the cellulose synthase can recognize both UDP-glucose and UDP-N-acetylglucosamine as substrates. K. xylinus requires genetic modification to incorporate GlCNAc into the bacterial cellulose as the natural conversion to UDP-GlcNAc is inefficient. The first four genes in this composite code for proteins responsible for converting various carbon sources such as glucose, fructose, and sucrose to UDP-GlcNAc. Another part of this composite is a signal peptide sequence (BBa_K4719025) characteristic to K. xylinus fused to chitin deacetylase AnCDA (BBa_K4719011).

Bacterial cellulose-chitosan copolymer has applications in the biomedicine field due to in vivo biodegradability by the lysosome. Also, the bacterial cellulose-chitosan copolymer is a convenient platform for further modifications that would aid solving the need for the promotion of tissue development. The uncovered amino groups are susceptible to enzymes catalyzing an addition of targeted organic groups. For instance, after modifying reaction conditions, deacetylase ClCDA (BBa_K4719024) can propylate chitosan, which can later be used for click chemistry reactions [3]. In the future, specific targets like drugs or amino acids could be linked to the polymer, promoting the healing properties of the material [4].

Since polymer production occurs in K. xylinus it requires a specific plasmid backbone (pSEVA331-Bb) for successful replication. We choose to use BBa_K1321313 as it was characterized by iGEM14_Imperial team as the most suitable plasmid backbone for Komagateibacter species. We performed PCR of the plasmid eliminating mRFP in order to preserve Anderson promoter J23104 (BBa_J23104), ribosome binding site (BBa_B0034) and terminator (BBa_B0015). The construct was cloned by utilizing BBa_K4719014 as a plasmid backbone containing bacterial cellulose-chitin synthesis operon where signal peptide sequence and deacetylase AnCDA (BBa_K4719011) were assembled into the backbone by Gibson assembly.

Experimental characterization

Verification and transformation of the single-tube synthesis of bacterial cellulose-chitosan copolymer composite

Whole plasmid sequencing revealed pSEVA331-Bb-AGM1-GFA1-GNA1-UAP1-AnCDA did not contain any deleterious mutations and was successfully transformed into electrocompetent K. xylinus cells as seen in Figure 1.

Figure 1. Results of colony PCR of K. xylinus transformed with pSEVA331-Bb-AGM1-GFA1-GNA1-UAP1-AnCDA. L - Invitrogen™ 1 Kb Plus DNA Ladder. 1 - positive control. 2-8 - selected colonies. The positive clones (2,3 and 4) had a PCR product of 767bp as expected.

References

1.Teh, M.Y. et al. (2019) ‘An expanded synthetic biology toolkit for gene expression control in acetobacteraceae’, ACS Synthetic Biology, 8(4), pp. 708–723. doi:10.1021/acssynbio.8b00168.
2.Yadav, V. et al. (2010) ‘Novel in vivo degradable cellulose-chitin copolymer from metabolically engineered gluconacetobacter xylinus’, Applied and Environmental Microbiology, 76(18), pp. 6257–6265. doi:10.1128/aem.00698-10.
3.Lima, R., Fernandes, C. and Pinto, M.M. (2022) ‘Molecular modifications, biological activities, and applications of chitosan and derivatives: A recent update’, Chirality, 34(9), pp. 1166–1190. doi:10.1002/chir.23477.
4.Torkaman, S. et al. (2021) ‘Modification of chitosan using amino acids for wound healing purposes: A Review’, Carbohydrate Polymers, 258, p. 117675. doi:10.1016/j.carbpol.2021.117675.