Team:BostonU/ProjectTandemPromoters

From 2014.igem.org

Tandem Promoters

Why Tandem Promoters?

In order to build the priority encoder for phases II and III of Project Chimera, our team needed to add tandem promoters to our current MoClo library. Tandem promoters condense higher order logic gates into fewer transcriptional units and also allow two different inputs for one transcriptional unit. In phase I, we aimed to follow a formalized method for constructing tandem promoters based on what is in the literature.

Image above is Figure 1 (The Genetic NOR Gate) from Tamsir, Tabor, and Voigt [1].
The above figure demonstrates that designing and testing tandem promoters using pBAD and pTetR operators is successful in E. coli.

Condensing three transcriptional units into two through the use of a tandem promoter.
By combining two functional operators from two promoters into one promoter (above), we can create more compact genetic devices and cut down on cloning steps required to make logic gates.

Priority encoder with fusion GFP shown linked to repressor proteins (CDS1-5)
We added fusion proteins to the priority encoder (above) and then broke down the tandem promoters into three categories (below): those requiring two external small molecules (purple boxes), those requiring one (blue boxes), and those requiring none (orange box).

Priority encoder with tandem promoters highlighted: 2 external inputs (purple boxes), 1 external input (blue boxes), and 0 external inputs (orange box).

Design and Assembly

Since we did not yet have all of the repressors required for the priority encoder, we decided to prioritize building the tandem promoters that required two small molecules (purple boxes).

To keep the tandem promoters compatible with a four-part MoClo transcriptional unit, we created a new fusion site, K, with the sequence ‘ATGC’. For each promoter pair, the primers are designed such that one promoter is flanked with ‘A’ and ‘K’ fusion sites and the other has ‘K’ and ‘B’ fusion sites. The two promoters were then combined in a MoClo reaction to form a level 0 tandem promoter part in ‘AB’ destination vector.

Design strategy for building the tandem promoters. Two PCR products (top lines) with A and K or K and B fusion sites flanking the 5' and 3' ends are cloned into a destination vector to form a cohesive tandem promoter containing two promoter operators.

Testing

In order to test this method, we used pBad, pTet, and pA1LacO promoters to form all possible combinations of hybrid promoters:

pBad-pTet, pTet-pBad, pA1LacO-pTet, pA1LacO-pBad, pBad-pA1LacO, pTet-pA1LacO

These three promoters were easily accessible within our lab and are well understood. This summer, we completed the pBad-pTet and pTet-pBad promoters. After creating the tandem promoters, we transformed them into Bioline alpah-Gold E. coli competent cells and confirmed by sequencing. The tandem promoters were then made into level one parts with a fluorescent marker (RFP) and tested in pro-strain competent cells using the flow cytometer. The prostrain cells already have araC and tetR, which means the pTet-pBad and pBad-pTet promoters are turned off when there are no small molecules present. We were then able to add various concentrations of arabinose, atc, and both to obtain transfer curves.

Promoter characterization

After testing and confirming the method for creating tandem promoters, we will be able to increase our variety of tandem promoters and design new tandems for the blue and orange boxes above. This will give us greater flexibility when we build more complex logic circuits. New tandem promoters will be created using pLmrA, pSrpR, and pBetI.

References

[1] A. Tamsir, J. Tabor, C. Voigt (2011). “Robust multicellular computing using genetically encoded NOR gates and chemical ‘wires’.” Nature 469: 212-215

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