Part:BBa_K802009
Sufactin generator and biofilm repressor for B. subtilis
This part can be used to induce surfactin production (an antimicrobial lipopeptide) and to repress the biofilm formation in B. subtilis strains.
Characterization
The part was transformed into the BSΔabrB B. subtilis strain containing the mutated biofilm repressor gene (abrB). As a result, this strain forms biofilms, so if the part is functional, the transformed strain should not form biofilms. In fact, the transformed BSΔabrB strain with BBa_K802009 should be similar to the wild-type B. subtilis BS168 strain as far as the biofilm formation is concerned.
In order to test the biofilm formation of the transformed bacteria, a qualitative test was performed in a 24-well microplate. Saturated liquid cultures were used to inoculate 2 mL of LB media (dilution ratio of the tested culture compared to the saturated culture: 1/100) supplemented with 2% xylose. The negative control was the BS168ΔabrB strain. The positive control was the wild-type B. subtilis BS168 strain.
Biofilm test
Comparison of biofilm formation between the transformed strain and the wild-type strain
As it can be seen in the video, the transformed strain does not form a pellicular biofilm as opposed to the wild-type strain
The results show that the characteristics of transformed mutated strain resemble more to the BS168 strain than to the BS168ΔabrB strain. Moreover, the two controls do not present a significant difference concerning biofilm formation between the xylose and xylose free media. As a result, the repression of biofilm formation is due to the part BBa_K802009.
In order to confirm the presence of the sfp gene, a qualitative test for surfactant production was performed. 2 mL of filtered supernatant coming from saturated cultures of transformed bacteria was mixed with 2 mL of cooking oil in a spectrometer cuve. Afterwards, the cuve was thoroughly vortexed and the resulted emulsion was observed after 20 hours:
observed spectrophotometer cuves
Sample number details:
- 1 is a positive control (the supernatant was replaced with a 10% SDS solution);
- 2 is a negative control (supernatant from the BS168 strain);
- 3 contains the supernatant from the transformed strain BS168ΔabrB with BBa_K802009;
- 4 is a negative control (supernatant from BS168 B. subtilis strain transformed with the same plasmid which was used for cloning the part BBa_K802009, but without the part).
Comparing the cuves 4 and 2, it is safe to say that the observed effect of the supernatant is due to the part and not to the plasmid in which the part was cloned. Moreover, there is a significant difference between the tested clones and the negative control, so the part BBa_K802009 affects the emulsification capacity of the supernatant produced by the transformed bacteria.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 760
Illegal SapI.rc site found at 770
//collections/antimicrobial
//collections/biofilm
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