Part:BBa_K3889101

P22 expression cassette Bacillus subtilis

BBa_K3889150 contains SRTF1 binding site (BBa_K3889030) allowing expression of P22 c2 repressor (BBa_K3889020) only in the presence of progesterone if SRTF1 (BBa_K3889021) is being produced constitutively.

Azurite was linked with the help of P2A linker (BBa_K3889069)in order to coexpress it with P22 c2 repressor and check protein expression

Usage and Biology

This part along with BBa_K3889100 and BBa_K3889102 was used in order to produce Ovastacin (BBa_K3889022) when progesterone in absent in the fallopian tube. The working of the system is shown below:

Results

In this graph, SRTF1 cassette (BBa_K3889100) and P22 Cassette(BBa_K3889101) were co-expressed in Bacillus subtilis 168 consisting of SRTF1 with mCherry reporter and P22 Cassette with Azurite reporter under various concentrations of progesterone.

Unexpectedly, the mCherry expression decreased at higher concentrations of progesterone. Further experimental evidence is needed to determine the cause. Additionally, the Azurite expression increased with progesterone levels signifying that p22 protein production increased. Hence, there is evidence to suggest that SRTF1 and SRTF1 DNA binding sites were interacting as expected and progesterone sensing is taking place in Bacillus subtilis.

Results Table:

Progesterone Conc. (in nM)	Azurite	mCherry
0	11374	297821
16.5	17101	281770
33	17275	287161
82.5	29714	249459
165	45536	153234
330	78069	117241

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
  COMPATIBLE WITH RFC[1000]