Part:BBa_K1510904:Design
Truncated lysostaphin coding circuit
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 50
Illegal NheI site found at 73
Illegal NheI site found at 1092 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 202
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
We cultured S. mutans overnight for 12 hours, and move cultured S. mutans to the modified slide glass which was coated by saliva(37°C, 1 hr). Then we used peristaltic Pump to drop BHI and saliva on slide glass to simulate the flowing atmosphere of oral cavity. All the experiments are conducted in our designed device, which is closed atmosphere mimicking the anoxic atmosphere.
To test whether our modified E. coli has the ability in decreasing formed biofilm, we put modified E. coli (circuit 1 and 2) to treat S. mutans for 12 hr, 24hr and 36hr. Finally, we did biofilm grow assay via crystal violet stain. biofilm grow assay:
gentally washing with ddH2O three times. Air dried at room temperature Stain with 500ul 0.1% crystal violet for 15min at room temperature Add ddH2O three times to wash excess crystal violet Add 1000 atic acid to extract crystal violet stain and wait for10 min. Add 20 extract crystal violet stain and 800 ddH2O to do OD measurement at 595nm wavelength.
Source
The promoter, J23100, comes from the 2014 distributed igem kit. The signal sequence, yebF, encoding signal protein exporting recombinant protein extracellular, is from E.coli K-12. By designing primer, we obtain it together with its naturally occurring ribosomal binding site using PCR and extraction.The truncated lysostaphin coding sequence is from 2013 igem distribution. Finally, the terminator B0015 is from 2014 igem distribution