Results

Objective

We designed four gates, two XOR gates and two AND gates. (here)
Our objective was to characterize these gates with the recombinases as input and RFP as output, Tp901.1, Bxb1 for BBa_K1132001 and BBa_K1132002 and FimE and PhiC31 for BBa_K1132003 and BBa_K1132004. With the RFP as output, it will be easy to see the switch of the recombinases.

Conception

The gates have been implemented into pSB1C3, and the RFP gene inserted after the gate or inside it (depending of the gate). For XOR2 and AND2, the T7 promoter was used and the polymerase T7 was also inserted in the construct, with promoter, rbs and terminator.

Furthermore, for the input, the recombinases Tp901.1, Bxb1, FimE and PhiC31 need to be with at least a weak promoter and a ribosome binding site (it is better to add a terminator but as pSB1C3 contains a terminator then it can be usable). We succeeded in assembling Bxb1 with a ribosome binding site and a promoter and FimE with a ribosome binding site, a promoter and a terminator.

The gates and the recombinases have been submitted, here is the detailed description of the parts.
We did not manage to finish the cloning of PhiC31 and Tp901.1 but (thanks to Suiti’s and Bonnet’s help!!), we produced the recombinases directly on the original plasmids (view other parts), and use the produced recombinases for characterization.

Results

The following table presents our plan for testing the efficiency of the recombinase-based switch and the results we should expect. The color of each cell of the table corresponds to the expected colors of the resulting colonies.

In vitro

The basic idea is to perform the switch with a cell lysis containing the recombinase and the plasmid containing the gate to be switched. After incubation, transformation of the plasmid containing the gate allows the quantification of switched versus non switched plasmids. Experiments have been performed as described in the protocol.

Phenotype study (visual inspection or fluorimetric assays)

XOR1

In the absence of recombinase or in recombination assays with FimE, the resulting colonies are white. Therefore, the XOR1 gate does not present any transcriptional leakage and non-specific recombinases cannot switch the door. When XOR1 was put in the presence of Bxb1, the switch occurred as attested by the presence of numerous red colonies (RFP expression).

To test the time dependency of the reaction, the reaction mixture incubated was analyzed at different time from 15 min to 2.5 hours.

From the graph above, one can see that switching occurs after 15 min. After 2 hours reaction, over 90 % of XOR1 gates were switched and the percentage of XOR1 gates switched from 2 hours to 5 hours reaction is almost constant (not shown).
In the in vitro protocol, DNAses were not eliminated. Therefore, longer times reactions also means that plasmids containing the gates can also be degraded. This could explain why, when incubation lasted longer than 2 hours, the number of colonies dropped as shown in the following graph.
We also tried to switch XOR1 with the two recombinases. The XOR1 gate was tested with the Dual Controller Plasmid provided by Bonnet (KC529324) and containing Tp901.1 and Bxb1. No switch was ever obtained. This negative result might be due to low expression levels of any of the two recombinases that would lead to partial switch in in vitro conditions. Further experiments will have to be performed to understand this phenomenon.

XOR2

In the absence of recombinase or in recombination assays with Bxb1, the resulting colonies are white. Therefore, the XOR2 gate does not present any transcriptional leakage and non-specific recombinases cannot switch the door. When XOR2 was put in the presence of FimE, the presence of red colonies validated the switch.
However, only 5% of the plasmids were switched. Hence, the switch of XOR2 using FimE was not highly efficient. Several explanations can be made: i) low expression of FimE; ii) low activity of FimE in the tested buffer; iii) low activity of FimE on our designed gate. Improvement on FimE protocols or redesign of new sites could have been performed with our Michigan buddies but, unfortunately, time was running out.

We also performed a second switch, using the XOR2 gate that has been switched Phenotype study: white colonies appear as a function of time. We prepared the DNA from the XOR2 gate that has been previously switched by FimE (a red colony). This plasmid DNA was tested in the presence of PhiC31 recombinase. The picture below demonstrates that the second switch occurred: white colonies are now present.

We also tried the switch with PhiC31 only. We used the original Siuti’s plasmid containing PhiC31, but no switch was obtained. The results might be due to low expression levels of PhiC31 that would lead to partial switch in in vitro conditions. A second explanation would be that the site we have designed is less efficient when the FimE recombinase has not yet performed the primary switch. More experiments are needed to verify PhiC31’s efficiency.

AND1

In the absence of recombinase or in the presence of FimE, the colonies are white. As a result, AND1 gate does not have transcriptional leakage and non-specific recombinases cannot switch the door.

We tested the AND1 gate with Bxb1. After incubation with Bxb1, the colonies are still white (this is normal!), therefore visual inspection of the switch was not possible. The only possibility was to sequence independent clones (see genotype section). We analyzed five independent colonies by sequencing, all of them were switched.

AND2

In the absence of recombinase or in the presence of Bxb1, the colonies are white. As a result, AND2 gate does not have transcriptional leakage and non-specific recombinases cannot switch the door.

We tested the AND2 gate with FimE. After incubation with FimE, the colonies are still white (this is normal!), therefore visual inspection of the switch was not possible. The only possibility was to sequence independent clones (see genotype section). We analyzed five independent colonies by sequencing, but none was switched. Several hypotheses can be made. First, the presence of the PhiC31 switching sequences may inhibit.

The AND2 was also tested with the Siuti’s construction parts containing PhiC31, but not switched has been obtained. As with the XOR2 gates, results might be due to low expression levels of PhiC31 that would lead to partial switch in in vitro conditions. A second explanation would be that the site we have designed is less efficient when the FimE recombinase has not yet performed the primary switch. More experiments are needed to verify PhiC31’s efficiency.

Genotype study

We analyzed XOR1 and AND1 switched plasmids by sequencing. The sequences of both switched plasmids correspond to the expected switches.

In vivo

The principle of this characterization is the co-transformation of the gate and the recombinase. The gate construct (BBa_K1132032) is on chloramphenicol and the Bxb1 construct (BBa_K1132027) is on kanamycine, we used 20 ng of each plasmid to transform E. coli and let it growth on chloramphenicol and kanamycine. Some colonies were red after transformation. However, in these conditions, the genotype of the gate (presence of the switch) is hard to verify due to the presence of two plasmids in the cotransformed strain. We therefore resorted to deeper characterization with fluorescence measurements. The positive control is a plasmid expressing constitutively RFP and the negative control is the co-transformation of XOR1 with a plasmid not containing any recombinases but on the same antibiotic resistance.

On the above graph, there is a clear difference when XOR1 is cotransformed with Bxb1 contrarily to the negative control .A deeper analysis of the fluorescence of the strain demonstrated that the level of fluorescence of the negative control is actually just diffusion of light. This result confirms the switch. The low fluorescence of our strains compared to the positive control could be due to the presence of a weak promoter in our gates and/or the long distance between the promoter and the coding sequence.

Summary
The following table summarizes our results with the different logic gates and the recombinases. Green cells are successful experiments while gray ones are.

Discussions

The gates are amazing, so cool, ultra bright!!!

Titre 4

Titre 5