Part:BBa_K2116015

PnorV_esabox_(15bp)spacer_esabox_spacer_esabox (p30)

We constructed a selection of AND gates responding to nitric oxide (NO) and 3OC6HSL (AHL). They were designed using the previously described NorV promoter Part:BBa_K1153000. This Promoter (from here on refered to as PnorV) is the native promoter controlling the nitric oxide reduction operon (norRVW) in E. coli [1]. Its transcriptional regulator, NorR, can bind to nitric oxide and activate gene expression. Using the distinct properties of esaboxes, PnorV was designed to also be responsive to AHL, giving it an AND gate behaviour. An esabox is an 18bp sequence to which the transcriptional regulator EsaR Part:BBa_K2116001 can bind. Transcription can be initiated by the specific AHL EsaR responds to [N-(3-oxo-hexanoyl)-L-homoserine lactone]. By placing one, two or three esaboxes at different positions in the vicinity of PnorV, different specificities for AHL and NO were reached. We created and characterized a collection of these kind of AND gates:

• Part:BBa_K2116004
• Part:BBa_K2116005
• Part:BBa_K2116006
• Part:BBa_K2116012
• Part:BBa_K2116013
• Part:BBa_K2116014
• Part:BBa_K2116007
• Part:BBa_K2116008
• Part:BBa_K2116068
• Part:BBa_K2116015

Biology and Usage

Biological logic gates are useful for creating higher order genetic circuits. This AND gate has one esabox placed as a roadblock after PnorV transcription start site.

It is regulated by a transcriptional

activator, NorR, and a transcriptional repressor, EsaR. Transcription can be initiated by NO binding to NorR. EsaR sits on the esabox and blocks RNA polymerase from advancing. As soon as 3OC6HSL binds EsaR it is released and transcription can continue. This design makes the AND gate modular. The esabox/EsaR system can be exchanged for another transcriptional repression system to create another AND gate.

Characterization

This part has not been properly characterized yet. Due to the similarity of it to Part:BBa_K2116006 a similar behaviour is expected. The here discussed part contains three esaboxes downstream of PnorV with 15bp spacing in between each binding site. It is expected, that this leads to a more efficient EsaR binding thereby improving the AND gate behaviour. Further optimization by placing an additional esabox upstream of the NorR binding site 3 might further increase this AND gate behaviour.

References:

• [1] Gardner, A. M. "Regulation Of The Nitric Oxide Reduction Operon (Norrvw) In Escherichia Coli. ROLE OF Norr AND Sigma 54 IN THE NITRIC OXIDE STRESS RESPONSE". Journal of Biological Chemistry 278.12 (2003): 10081-10086.
• [2] Shong, Jasmine and Cynthia H. Collins. "Engineering The Esar Promoter For Tunable Quorum Sensing-Dependent Gene Expression". ACS Synth. Biol. 2.10 (2013): 568-575.




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]