Part:BBa_K2116027

Nitric oxide and Lactate responsive AND gate

This part is a hybrid promoter, which consists of pNorV promoter between two LldO binding sites. pNorV is a nitric oxide responsive promoter, regulated by NorR. LldO binding sites enable binding of LldR repressor. The promoter exhibits an AND logic and is responsive to L-lactate and nitric oxide. It will be active when both two compounds are present.

Characterization of the Promoter

To study the part, the respective promoter was cloned upstream of the sfGFP gene. Cells were transformed with the before-mentioned plasmid together with a plasmid containing LldR repressor (Part:BBa_K1847001). We did a two dimensional dose response with the two inducers of the promoter, L-lactate and nitric oxide (NO). Diethylenetriamine/nitric oxide adduct (DETA/NO) was used as a source of NO. The dose response curve was studied with the plate reader.

The plate reader experiment

• set an overnight culture of three biological replicas in M9 media
• dilute the overnight cultures 1:100 in a fresh M9 media
• induce the samples at the beginning of the exponential phase (OD₆₀₀=0.3)
• measure the green fluorescence every hour. Fluorescence was normalized with OD₆₀₀

The two dimensional dose response for the hybrid promoter with pNorV embedded between LldR binding sites O1 and O2. The plot is an average of two biological replicas four hours after induction. The dose response for the L-lactate = 0 was omitted from the plot due to outlier values for this dose.

The graph shows an AND gate logic behavior of the promoter, where the three cases where at least one of the inducers is present in a low concentration have a lower fluorescence in comparison to the case where both inducers are present in high concentration. The dose response for the zero value of lactate was replaced with the smallest lactate concentration used due to the outlier values for the dose response where there was no lactate in the media

The two dimensional dose response shows that the hybrid promoter exhibits an AND gate logic with the 1.5-fold activation.

The dose response for the outlier dose response, where the concentration of L-lactate is held constant at 0 mM and the dose response is for the DETA/NO concentrations

</p>

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]