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

abrB tests

The part was transformed into the B. subtilis 168 ΔabrB 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 B. subtilis 168 ΔabrB strain with BBa_K802009 should be similar to the wild-type B. subtilis 168 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 medium (dilution ratio of the tested culture compared to the saturated culture : 1/100) supplemented with 2% xylose as abrB gene is under the control of the P_xyl promotor. The negative control was the B. subtilis 168 ΔabrB strain. The positive control was the wild-type B. subtilis 168 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 B. subtilis 168 strain than the B. subtilis 168 ΔabrB strain.

sfp test

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 cuvette. Then, the cuvette was thoroughly vortexed to create an emulsion. After 20 hours the cuvette 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 B. subtilis 168 strain);
• 3 contains the supernatant from the transformed strain B. subtilis 168 ΔarbB with BBa_K802009;
• 4 is a negative control (supernatant from B. subtilis 168 strain transformed with the same plasmid which was used for cloning the part BBa_K802009, but without the part).

Comparison between the cuvettes 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 cuvette 4 confirms that the observed effect in due to the cloned sfp gene and not to the backbone vector.

Confocal microscopy

In order to test the efficiency of the part, the effect of the transformed strain on an E. coli biofilm was observed. The test was performed on glass lamellae in a 12 well plate.

abrB test

Wells containing a B. sbtilis 24 hour biofilm was seeded with a fluorescent adherent E.coli strain and incubated at 30°C for 36 hours before observation. The negative control contains no B. subtilis biofilm.

Positive B. subtilis biofilm (in red). The adherent fluorescent E.coliis shown in green

sfp test

The filtered supernatant from the transformed strain was incubated in wells containing glass lamellae for 24 hours. Then, the supernatant was replaced with LB media diluted 2 times and seeded with a saturated flourescent adherent E. coli strain. In order to compare the surfactin secreted by the strain containing the part and the purified molecule, the positive control wells were incubated with a solution containing surfactin at a concentration of 80 µg/mL. To make sure that the observed effect is not due to the antibiotic or to the backbone, control wells were incubated with the supernatant extracted from a saturated culture of B. subtilis transformed with the empty backbone.

Negative control: fluorescent E. coli biofilm

Positive control: effect of a solution containing purified surfactin on a fluorescent E. coli biofilm

CONCLUSION

BBa_K802009 part was shown to be efficient in controlling 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 760
  Illegal SapI.rc site found at 770