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. If the part is functional, the transformed strain should not form biofilms as AbrB will repress genes involved in biofilm formation. 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-wells 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 as the abrB gene is under the control of the Pxyl promotor. 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 sticky pellicular biofilm as opposed to the wild-type strain
The results show that the characteristics of the transformed mutated strain resemble more the BS168 strain than the BS168ΔabrB strain.
In order to confirm the presence of the sfp gene, encoding a regulator of surfactin synthesis, a qualitative test for surfactant production was performed. 2 mL of filtered supernatant extracted from saturated cultures of transformed bacteria were mixed with 2 mL of sunflower oil in a spectrometer cuve. Then, the cuve was thoroughly vortexed to create an emulsion. After 20 hours the cuve was observed, in case of surfactant production the emulsion should still be visible:
Surfactin production test
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).
Comparision between the cuves 3 and 4 shows that the emulsion is due to a surfactant present in the supernatant of the transformed bacteria with BBa_K802009. The absence of an emulsion in cuve 4 confirms that the observed effect in due to the cloned sfp gene and not to the backbone vector.
CONCLUSION
BBa_K802009 part was shown to be efficient in controling the ON and OFF biofilm states in B. subtilis. Moreover the production of surfactin was efficient, so this part can be a source of surfactin that can be easily extracted from the supernatant of the cultures.
Usage and Biology
This part can be used in B. subtilis for the control of biofilm formation and for the production of a surfactant that will prevent the growth of other 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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