Part:BBa_K2116005

AND gate regulated by norR and esaR (two esaboxes)

Summary: AND gate constructed by placing 2 esaboxes after the transcription start site of PnorV.

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

Figure 1: Design of AND gate. The spacing between the esaboxes is 8bp long. NRB;NorR binding site, Sigma54B; RNA polymerase binding site

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 of this part

When characterizing our parts collection we initially confirmed functionality. Below you can see a graph depicting AND gate behaviour of this biobrick.

Figure 2: AND gate behaviour. This part does not resemble an optimal AND gate behaviour.

The AND gate behaviour shown in Figure 2 can be explained due to the placing of only two esabox after PnorV. This seems not to be enough to reach optimal balance between repression and derepression. An improvement could be achieved by either increasing the number of esaboxes, their placement or by decreasing the amount of EsaR production. We followed all these steps, and recommend you have a look at our favourite AND gate Part:BBa_K2116011.

Secondly we tested the system with and without EsaR present, in order to show that the AND gate behaviour is due to repression by EsaR.

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Figure 3: AND gate behaviour compared to cells containing an EsaR expressing plasmid vs non expressing. Lighter colors depict cells lacking EsaR. This difference in repression suggests that EsaR is the major protein acting, producing the desired AND gate function

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.</i>
• [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]