Part:BBa_K4769420
spoIIE Knockout Construct (5' & 3' HR)
The below figure shows the number of vegetative cells and spores for wild type B. subtilis 3610 and ΔspoIIE B. subtilis 3610 – these results show almost a total loss of spore formation in the ΔspoIIE strain: 35% of the wild type culture are spores, whereas only 0.005% of ΔspoIIE culture are spores!
From this CFU count data, ΔspoIIE appears to have a reduced number of viable cells; to test this, we grew wild type and ΔspoIIE B. subtilis 3610 in 96-well plates and recorded their growth rate over 24 hours by measuring absorbance at 600nm (OD600). These growth curves suggest that ΔspoIIE does not have a negative effect on cell growth rate. As the goal of knocking out sporulation was to increase the long-term stability of the biofilm, we also wanted to check how the biofilm biomass of ΔspoIIE compared to wild type <i>B. subtilis over time. For this, we optimised a biofilm biomass assay by staining with safranin – as seen below, there does not appear to be significant amounts of biofilm dispersal/breakdown in wild type by 2 weeks, so from this data we cannot conclude if ΔspoIIE is less affected by biofilm dispersal; however, ΔspoIIE appears to form a mature biofilm significantly faster than wild type B. subtilis and appears to have slightly greater total biofilm biomass overall. Increased rate of biofilm formation and increased total biofilm biomass were also aims of our project, hence the spoIIE knockout construct was a great success!
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1557
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1557
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1557
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1557
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1224
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