Part:BBa_K3463020:Experience

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Characterization of BBa_K3463020

To get a proof of concept for our all genetic circuit, we decided to build a Proof of concept plasmid containing all the parts of the survival system:

- J23100-RhlR-B0015 / Prhlr-LacI / Ptac-eGFP-B0017

This plasmid was built in 2 steps. First the combination of Ptac-eGFP-B0017 and J23100-RhlR-B0015 and then was added Prhlr-LacI. We then transformed a BL21 strain with our Proof of concept construction and monitored the expression of the eGFP by measuring the fluorescence with a gradient of concentration of synthetic BHL : 100µM / 10µM / 1µM / 100nM and without BHL.

Figure 1 Effect of the BHL on the Proof of concept construct. No significant differences were obtained between the tested concentration and the negative control.

In Figure 1, the BHL didn’t have any effects on the fluorescent level (p-value=0.999). Different hypotheses can be made to explain these data. For instance the genetic networks didn’t function as planned. However different explanations can be given before coming to this conclusion. First the fluorescent expression level obtained is much higher than those obtained classically with the Ptac-eGFP construct and much in line with the level obtained with the classic pUCBB-eGFP suggesting that the construct doesn’t contain the tac promoter. However the plasmid was built following the standard assembly protocol and gave the expected results for the digestion gel control (Figure 2 and 3). Despite having the desired digestion profile some screened colonies show an usual profile with missing restriction sites. To be more precise the PstI and SpeI restrictions were missing as shown in Figures 9 and 10. That loss of restriction sites can be due to a potential rearrangement or alternative cut made by the restriction enzyme. Besides, after the second standard assembly the tac promoter couldn’t be amplified by PCR whereas it could be from the first standard assembly step (not shown). This suggested a mutation or a modification of the Ptac during the second standard assembly explaining the fluorescent level and the dysfonctionnement of our genetic network. A DNA sequencing is necessary to fully understand the issue concerning our proof of concept build, although we had no remaining time to perform it.

Figure 2 Digestion of the positive colonies pUCBB Ptac-eGFP-B0017-J23100-RhlR-B0015 in EcoRI and PstI. All colonies were negative with colonies 3 and 5 as classic pUCBB but 1-2 and 5 showed a missing PstI restriction sites after the standard assembly.

Figure 3 Digestion of the positive colonies pUCBB Ptac-eGFP-B0017-J23100-RhlR-B0015-Prhlr-LacI BBa_K3463020 in EcoRI and PstI. The 1-2-3-6-8-9 colonies were negative i.e. classic pUCBB. The 5 was positive and contained the 3 composite parts. The 4-7 showed an unexpected profile with a missing PstI restriction site

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