Team:Ionis Paris/Measurement

CelloCAD Plasmid Optimization

CelloCAD is a newly created software, allowing plasmid optimization through powerful bioinformatics tools. It was described in 2016 in “Genetic Circuit Design Automation” (Nielsen et al.), and is freely available here

CelloCad takes a genetic construction as an input, and gives as an output the best possible plasmid for this genetic construction.
Our team wanted to work with the Cello software in order to try to optimize our plasmid. Though we may not have the most complex genetic circuit, we were interested in the possible outcomes of such an experiment.

We heard about Cello through one of our advisors, and were immediately appealed by the characterization possibilities of such a software. We wanted to design an enhanced version of our biosensor plasmid, and then compare its properties (detection range, response rapidity…) to the one we designed ourselves.

However, we encountered an issue during the realization of this side-projects: in order for the software to work, we needed to know the RPU (Relative Promoter Unit) of our promoters. This RPU method is fairly recent, and no RPU database currently exists. We therefore wanted to determine by ourselves the RPU for the two promoters used in our biosensor, (Pr and Pu), and maybe add our results as the beginning of a crowdsourced RPU database. In order to perform this experiment, we designed a series of BioBricks (see Parts) where our promoters coupled to GFP. As the Pr promoter is constitutive, its GFP BioBrick was pretty simple. However, as Pu requires the XylR/Toluene complex to be activated, we modified our biosensor so it produces GFP instead of luciferase. The cells were then induced with toluene prior manipulation, in order to trigger GFP expression.

To determine the RPU of a promoter, the GFP expression has to be calculate cells per cells. Therefore, we collaborate with Paris Bettencourt iGEM team that kindly give us access to a flow cytometer. Experiments were realized according to the Cello protocol described in article "Genetic circuit design automation" published in Science in 2016.[1] The test device 1 from this year interlab study was used as the reference as this device contains the reference promoter BBa_J23101, a RBS (BBa_B0034), the GFP (BBa_E0040) and the terminator BBa_B0015.
We used the following parameters to set up the flow cytometer : FSC voltage of 437 V, SSC voltage of 289 V, and a green laser (488 nm) voltage of 425 V. An SSC and FSC threshold of >200 was used to limit collection to cell-sized particles. We analyzed 50,000 events for each sample. GFP intensity and OD600 values were determine four times.
The fluorescence of several bacterial culture was determine :

  • DH5a transformed with the test device 1

  • DH5a transformed with Pr-GFP

  • DH5a transformed with XylR coding device-Pu-GFP in presence of toluene 1 mg/L

  • DH5a transformed with XylR coding device-Pu-GFP in presence of toluene 10 mg/L

  • Several negative controls were realized :

  • DH5a transformed a plasmid made of GFP-Term

  • DH5a with BBa_K2023015 without toluene addition

  • At a OD600= 0.2, the several cells culture were pelleted and resuspended into PBS. Samples were then transfered to flow cytometer tubes and the fluorescence of each cells was assessed.
    We obtain the following results:

    As shown on the Figure above, we did not get fluorescence for the test device 1 which contains the standard promoter. Although we observed fluorescence for both our BioBricks, we couldn't calculate RPUs due to that lack of standard data. The fluorescence measurement will however serve as characterization data.
    RPU could have been calculate using the formula:
    𝑅𝑃𝑈=〈G𝐹𝑃〉−〈G𝐹𝑃〉0〈G𝐹𝑃〉𝑅𝑃𝑈−〈G𝐹𝑃〉0 where is the median fluorescence of the cells containing the sensor, RPU is the median fluorescence of the cells containing the standard plasmid, and 0 is the median autofluorescence.[1]
    [1] Nielsen, A. A. K. et al. Genetic circuit design automation. Science 352, aac7341–aac7341 (2016).