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Engineering

During our project, we fully experienced the concept of the engineering cycle.

In order to make our design and the entire ARBO-BLOCK project evolve, we tested each step of our thinking and analysing our results was necessary to rethink our first idea.

Here, we will describe an example of how we worked with this concept. Indeed, it was especially applied during the construction of our bacterial timer lysis device (permitting Cry11Aa et Cyt1Aa toxins production, toxins release through cell lysis using Cal).

Design 1

The pRL1 plasmid used to design our lysis device already contained the lexA promoter and coding sequences for ColA toxin, Cai immunity protein and Cal lysis protein (Figure 1).

First, to assess the correct construction of the plasmid, the coding sequence for a GFP protein was inserted in the caa sequence (encoding for ColA). This allowed us to measure GFP fluorescence to quantify the toxin expression, as gfp gene is under the control of caa promoter. If the toxin is released in the extracellular medium after lysis of the bacteria by Cal, it will be possible to detect it and to validate the asynchronous lysis/toxin system. Theoretically, the lysis protein is supposed to be produced exponentially 7 hours after induction, permitting cell membrane breakage and release of the cytosolic compounds into the extracellular medium.

Three constructions were tested in this experiment: pRL1 wild-type (WT) (positive control), a mutant version of pRL1 containing the gfp sequence (pRL1-GFP) and a mutant version of pRL1 containing the rfp coding sequence fused to the cal sequence (negative control, pRL1-RFP) (Figure 2). With the latter construction no cell lysis should occur, and the GFP fluorescence should be present in the bacterium. For each experiment, increasing doses of mitomycin C were used.

Figure 1: Operonic structure and regulation of the colicin A system. Caa: colicinA gene; Cai: immunity protein caI gene; Cal: lysis protein.

Figure 2: Scheme of the first design of the pRL1 plasmid containing the gfp coding sequence and the design of the rfp-cal negative control.

Testing 1

This first designed pRL1 plasmid was tested in E. coli cells. As the lexA promoter is commonly activated by stress stimuli in nature, mitomycin C induction was used to mimic this phenomenon. Indeed, this chemical is known to induce DNA mutations, which triggered the lexA promoter. Due to bacterial lysis by Cal, the toxin is detectable in the extracellular medium by Optic Density measurement using TECAN methods. The three previously described construction were tested.

Analysis 1

GFP is used in our design as a reporter gene, the production of ColA is therefore followed using fluorescence intensity measurements. The production of lysis proteins induce the lysis of the bacterium, which leads to release GFP in the extracellular medium.

After induction with mitomycin C, WT cultures show a decrease in OD600 (Optical Density at 600nm) signal when high concentrations of the inducer are added to the culture. Indeed, when lexA is induced in increasing levels of mitomycin C, cell lysis is observed. When the induction of cultures containing pRL1-RFP is done, the GFP signal does not decrease, which means that cell lysis does not occur, as we expected. For the engineered plasmid with the gfp sequence, the same trend is observed as the negative control. The result is not the one expected. Indeed, a decrease in the OD600 signal, reflecting an effective lysis of the bacterial cell, should have been detected. In all 3 cases, as GFP is detected, the toxin is produced.

Hypothesis: While inserting gfp coding sequence in the system, the regulation of cal expression could have been altered.

Design 2

Given the previous results, another plasmid was constructed according to our hypothesis (Figure 3).

For this second design, a longer caa sequence was cloned in the pRL1 plasmid. An alternative lexA box was also added in this sequence. This box is naturally present within the caa sequence. However, when the gfp sequence was cloned in the caa gene, it seemed to alter the expression of the alternative lexA box, potentially leading to no transcription of the lysis gene, cal. In this new construct, caa sequence is still implemented with the gfp sequence as shown on the figure below, but the alternative lexA box is kept in the sequence encoding for the toxin.

With this second engineered plasmid, we expect to induce Cal production as well as ColA. The latter should be released into the extracellular medium.

Figure 3: Scheme of the second design of the pRL1 plasmid containing the gfp coding sequence.

Future experiments

With this experiment, we have been able to execute the engineering cycle at least one time.

In future experiments, our 2nd design should be tested and analyzed. If the latter is successful, it would be more than interesting to replace the colA sequence by the sequences encoding for Cry11Aa, Cyt1Aa and P20. In order to observe and quantify if toxins are well-produced, fluorescent coding sequences will be fused to each toxin coding sequence. This will allow us to compare toxins production.

If this 3rd design is successful, reporter genes could be deleted and plasmids could be tested in vivo in E. coli cells or even in Asaia sp cells.