Part:BBa_K4685031
Csg Operons
This composite part is composed of the csgBAC genes under the control of a P(BBa_J23119)promoter and the csgEFG genes under the control of a P(BBa_J23100) promoter.
The curli fibers are the main protein component of E. coli biofilms. In the genome, two separate operons (csgBAC and csgDEFG) produce seven proteins (CsgA, CsgB, CsgC, CsgD, CsgE, CsgF, and CsgG) to form the curli fibers. CsgA is the major curli subunit forming the extracellular fibers. However, to successfully form the fibers, CsgB, CsgC, CsgE, CsgF, and CsgG are also required. CsgD is the master regulator of biofilm formation.
Design
Previous iGEM teams worked with curli fibers. Most of the time, CsgA is overexpressed from a plasmid, while relying on the expression of CsgB, CsgC, CsgE, CsgF, and CsgG from the genome. However, those genes are not expressed under all conditions. Therefore, typically those experiments require a special nitrogen-limiting medium (M63) in order to induce expression of the csg operons. Moreover, the curli production is limited by the proteins produced from the genome.Instead, we chose to express six csg genes (without csgD) from a plasmid that we call pC3. We put csgBAC under the control of a P(BBa_J23119) promoter and csgEFG under the control of P(BBa_J23100) a promoter.
Characterisation
We tested the overexpression of this two operon inserted in a strain with a deletion of the csg operons. We grew the colonies on plates with Congo Red and Coomassie Blue to see the biofilm production. The results we obtained show us that M037 strain, with the csg operons deletion cannot produce curli fibers, as we can see the colony is pink (Figure 2, A). While M037 pC3 strain shows an overexpression of curli fibers production, as we can see the colony is red (Figure 2, B).
Figure 1: A: A: M037 in LB Congo Red (CR) Coomassie Blue (CB), colony not producing biofilm, colony is pink; B: M037 pC3 in LB CR CB Amp, colony is red, overexpression of curli fibers production. Pictures taken with a light microscope. .
We managed to find a method for quantifying the the biomass produced by the strains. The idea of the test is to use the vacuum system with a filter with a certain pore size that allows us to retain the microplastics and the biofilm that has been produced. The cells that have not produced biofilm will pass through the filter, allowing us to quantify the biofilm mass produced. We compared the biofilm production in our two strains: M037, which does not produce the Curli fiber, due to the deletion of csg operon in the genome; and M037 pC3, which produces Curli fibers, thanks to the plasmid pC3 constitutively expressing the csg genes. The results prove the overexpression of curli fibers produced by the pC3 plasmid.
Figure 2: barplot of the quantification of biomass produced by M037 strain, with a deletion of the operon csg in the genome and M037 pC3 strain, which contains the plasmid pC3 constitutively expressing the csg operons (Welch two samples t-test: p-value=0.026). This significant result shows us the overexpression of curli fibers produced by the pC3 plasmid.
Having a strain that produced biofilm, we wanted to quantify how much microplastic could be captured.
The idea of this second test is almost the same as for the biomass, only in this case we initially want to have the microplastics captured by the biofilm as pellets, while the microplastic remaining free is present in the supernatant. We then lyse the cells so that only the captured microplastics remain attached to the filter. This allows us to quantify the ability of the biofilm to capture microplastics. Figure 3 shows us that the overexpression of curli fiber allows us to successfully capture more polypropylene (PP) and Polyethylene (PE) microplastics.
Figure 3: The first two barplots show the ability of the strain overexpressing curli fibers (M037 pC3) to capture PP microplastics compared to the strain with the csg operon deletion (M037), not producing curli fiber (Welch two samples t-test: p-value=0.003). The next two barplots show the ability of the M037 pC3 strain to catch PE microplastic compared to the M037 strain (Welch two samples t-test: p-value=0.02). We obtained significant results for the two types of microplastics. That shows us that the overexpression of curli fiber allows us to capture more microplastics.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1651
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1651
Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 1257
Illegal NheI site found at 1280 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1651
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1651
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1651
Illegal AgeI site found at 1924 - 1000COMPATIBLE WITH RFC[1000]
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