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Contribution

Overview

This year, our team submitted some new experimental data to existing bio bricks, which will be useful tools for other iGEM teams in the future.

Contribution

1.Capsular Polysaccharide (CPS)

1.1Construction 

We have constructed a composite part (BBa_K5248053) utilizing KpsE (BBa_K5248007) and KpsT (BBa_K5248008) (see Figures 1 and 2).

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Figure 1：pACYCDuet-1-KpsE-KpsT plasmid map
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Figure 2：pACYCDuet-1-KpsE-KpsT Plasmid electrophoresis image

1.2 Validation

We have successfully transformed the aforementioned plasmids into our chassis strain, Escherichia coli Nissle 1917 (DE3), and conducted quantitative polymerase chain reaction (qPCR) to verify the expression levels of the target genes. The results indicate a substantial overexpression of mRNA for both KpsE and KpsT genes.

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Figure 3 & 4

Additionally, we performed the extraction and quantification of bacterial capsular polysaccharides from the engineered strain, using the strain transformed with an empty vector as a control. As depicted in Figure 5, the KpsE-KpsT genetically engineered strain showed a significant increase in capsular polysaccharide production compared to the strain with the empty vector. All these findings may be of assistance to other teams. We hope that this work will contribute to the iGEM community.

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Figure 5: The production of capsular polysaccharides when cultures of pACYCDuet-1-1917 (DE3), pACYCDuet-1-KpsE-KpsT, and pETDuet-1-galU-pgmA, grown to an OD600 of 0.6, were induced with 0.5 mM IPTG for 3 hours and diluted to an OD600 of 0.4.

2. Antimicrobial peptide

2.1 Construct

We have constructed a composite part, pETduet-1-mcmI-2(6his)-mcmA(strep) (BBa_K5248058), on the pETduet-1 vector backbone (see Figures 1 and 3).


Furthermore, we have also constructed another composite part, pACYCDuet-1-mCherry-mchI(6his)-EGFP-mchB(strep) (BBa_K5248055), on the pACYCDuet-1 vector backbone (see Figures 2 and 4).

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Figure 1：pETduet-1-mcmI-2（6his）-mcmA(strep) plasmid map
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Figure 2：pACYCDuet-1-mCherry-mchI（6his)-EGFP-mchB (strep) plasmid map
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Figure 3:pETduet-1-mcmI-2（6his）-mcmA(strep) Plasmid electrophoresis image
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Figure 4：pACYCDuet-1-mCherry-mchI（6his)-EGFP-mchB (strep) Plasmid electrophoresis image

2.2 Verify

Regarding antimicrobial peptides, we have completed the experimental characterization of two parts (BBa_K5248058 and BBa_K5248055) and have added the new data to their respective BioBrick entries.

BioBricks Plasmid name Characterization Method
BBa_K5248058 pETduet-1-mcmI-2（6his）-mcmA(strep) Antibacterial zone assay
BBa_K5248055 pACYCDuet-1-mCherry-mchI（6his)-EGFP-mchB(strep) Antibacterial zone assay

Antibacterial zone assay：

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1. Escherichia coli Nissle 1917 DE3 containing the expression plasmid with the mcmA gene.
2. LB blank validation.
3. Acinetobacter baumannii ADP1 without the expression plasmid containing the mcmA gene.
4. Escherichia coli Nissle 1917 without the expression plasmid containing the mcmA gene.
5. Escherichia coli Nissle 1917 DE3 without the expression plasmid containing the mcmA gene.
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1. Escherichia coli BL21 DE3 containing the expression plasmid with the mchB gene.
2. LB blank validation.
3. Acinetobacter baumannii ADP1 without the expression plasmid containing the mchB gene.
4. Escherichia coli BL21 without the expression plasmid containing the mchB gene.
5. Escherichia coli BL21 DE3 without the expression plasmid containing the mchB gene.

The results indicate that the strains harboring the designed plasmids for mcmA or mchB expression exhibit visible zones of inhibition, capable of protecting the host cells from the antimicrobial peptides they produce. Further use and characterization of these two antimicrobial peptides in our project can be found on our Engineering Success and Results pag.