Part:BBa_K3505036

pFliC:RBS-LacI-Terminator- pAndersonJ23115:LacO:RBS-ECFP-terminator

Fig.1: Activation of eCFP in absence of SCFAs,Based on NOT GATE

Fig.2: The NOT GATE

Usage and Biology

2 Trancription Units

• LacI BBa_K3505003regulated by inducible promoter FliC BBa_K2924016which is activated from SFCAs and more specifically from Butyrate
• eCFP BBa_K3505019under control of lac regulated AndersonBBa_K3505013.

This way we take signal in the Absence of SCFAs

Design Notes

The coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is present in BBa_K3505010 and has overhangs compatible for GoldenBraid cloning.

Verification of cloning

Fig.3:U2 C2 pFliC:RBS-LacI-Terminator-pAndersonJ23115:LacO:RBS-ECFP-terminator EcoRV, HindIII. Expected bands 3734, 1699.

Experimental Use and Experience

Measurements are the average of 9 total replicates (3 biological replicates and 3 technical replicates per biological replicate). Error bars represent standard deviation of biological replicates

Fig.4:NOT-GATE-regulated eCFP fluorescence after in the presence or absence of 2mM acetate.

Fig.5:Cell growth in the presence or absence of 2mM acetate.

Fig.6:NOT-GATE-regulated eCFP fluorescence after in the presence or absence of 2mM propionate.

Fig.7:. Cell growth in the presence or absence of 2mM propionate.

Conculusion

There is a slight increase in fluorescence in absence of SCFAs - acetate and propionate - as expected. We hypothesize that the gap will be bigger when adding butyrate, which is the main inducer for pFliC, and which we didn’t have in the lab as per Wiki Freeze date. As far as the cell growth goes, we expected a reduction of the cells, since we add acids that are toxic to the cells. These results indicate that our NOT-Gate system works, regarding these acids but in an expected range.

Sequnce and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1321
  Illegal NheI site found at 1344
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]