Revision as of 14:42, 28 October 2016

bxb1 integrase under the control of nitric oxide and AHL responsive AND gate

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. Our favourite AND gate Part:BBa_K2116011 is here combined with our codon optimized Bxb1 recombinase Part:BBa_K2116025. Bxb1 is a phage integrase that mediates unidirectional site-specific recombination between two DNA recognition sequences, the phage attachement site, 'attP', and the bacterial attachment site, 'attB'. This recombination event is hereon referred to as a flip. Bxb1 belongs to the family of serine integrases and uses a catalytic serine for strand cleavage, recognizes shorter attP sequences, and does not require host cofactors. It mediates efficient site-specific recombination between two different sequences that are relatively short yet long enough to be specific on a genomic scale. These properties make Bxb1 an efficient tool, especially in combination with the here presented AND gate, for creating genetic logic gates, as demonstrated in various papers.

Kinetic Characterisation

We used a reporter construct Part:BBa_K2116024 with a promoter flanked between attB and attP sites Part:BBa_K2116021. Thus, we could investigate the kinetics of Bxb1 using flow cytometry (BD LSR Fortessa SORP) on time course samples.

Genetic Design

• Bxb1 was expressed under an AND gate promoter Part:BBa_K2116011, without any degradation tag Part:BBa_K2116026.
• We applied a dual fluorescence reporter system Part:BBa_K2116024 that expresses sfGFP Part:BBa_K2116017 when Bxb1 flips a directional promoter. In the non-flipped state the reporter expresses the red fluorescent protein mNectarine Part:BBa_K2116016.

Results

The behaviour of the composite part is close to what is expected from the characterisation of its individual parts. Some improvement could be made by changing the EsaR content of the cell. We expect the recombinase expression to therevy decrease when only AHL is present. For more information, see the discussion on Part:BBa_K2116026.

Figure 1: AND gate expressing Bxb1. Bxb1 flips a promoter between two att sites, expressing either mNectarine (non-flipped) or sfGFP (flipped).

Figure 2:Simulation of the full system. Once NO and AHL are detected at the same time, our memory element start switching.

Visit our team wiki http://2016.igem.org/Team:ETH_Zurich/Switch_Module for a full discussion of these parts.

Sequence and Features