Difference between revisions of "Part:BBa K4491009"

Latest revision as of 02:34, 12 October 2022

MegaT

The part MegaT is composed of RBS B0032, mVenus and L3S2P21 terminator. The purpose of the part is to show the relative amount of a CDS. In our project, we hope to use a reporter protein to visualise the amount of feedback species X we have in the circuit. To avoid tagging the reporter protein directly onto X, we have decided to put in an RBS and a reporter (mVenus) right after X to visualise the amount of X. We are implementing JUMP assembly. However, JUMP assembly requires us to clone in 4 parts (usually the promoter, RBS, CDS and terminator) at the same time and does not allow polycistronic assemblies as is the case we have in our main project. Therefore, we need a way to design an operon to allow polycistronic assembly.

Design

To allow the reporter to be expressed under the same promoter as X, we have designed MegaT such that the RBS, reporter and terminator are attached together as a fragment. The MegaT part we have designed consists of RBS B0032, CDS mVenus and L3S2P21 terminator (Chen et al., 2013)and is placed as an order with IDT.

We have chosen to use B0032 because we want to start with a medium strength of RBS. If the RBS is too strong, the reporter may form aggregates when existing in high amounts. If the RBS is too weak however, the amount of mVenus produced may not be detectable. In terms of the terminator, the L3S2P21 is a synthetic high efficiency terminator. A strong terminator is important to terminate transcription.The MegaT part itself will not be expressing the reporter itself without the promoter part before it. Therefore, we have built a genetic circuit with a single transcription unit and use MegaT to visualise the relative amount of CDS we have. The circuit we have decided to characterise MegaT in is the autoregulatory negative feedback circuit (nFc). The purpose of this circuit is to characterise the behaviour of autoregulatory feedback circuit and compare it with other adaptation strategies.

Figure 1. Design of the operon MegaT and how it fits into JUMP Golden Gate Assembly

To test for the intensity of the reporter protein in the circuit and show that it is reporting the relative amount of VanR, we have cultured the cells with the plasmid containing the plasmid in 0µM and 100µM Vanillic acid overnight.

Characterisation

The MegaT part itself will not be expressing the reporter itself without the promoter part before it. Therefore, we have built a genetic circuit with a single transcription unit and use MegaT to visualise the relative amount of CDS we have. The circuit we have decided to characterise MegaT in is the autoregulatory negative feedback circuit (nFc). The purpose of this circuit is to characterise the behaviour of autoregulatory feedback circuit and compare it with other adaptation strategies.

To test for the intensity of the reporter protein in the circuit and show that it is reporting the relative amount of VanR, we have cultured the cells with the plasmid containing the plasmid in 0µM and 100µM Vanillic acid overnight. The following table shows the strain of cells and culture conditions overnight we have used for the characterisation of MegaT. The strain SB7 is a strain with mCherry integrated into chromosomal DNA.

We are using the Nikon TI2 Eclipse microscope with phase contrast, mCherry, YFP and have taken 9 images of each cell strain at different fields of views separated from each other to prevent imaging the same cell twice.

Figure 2. Histogram plot of mVenus intensity of the test strain with 0μM and 100μM vanillic acid. The mean of the mVenus intensity of 0μM vanillic acid is 4769 with a standard deviation of 1272 while the mean of the mVenus intensity of 100 μM vanillic acid is 7259 with a standard deviation of 1471.

The above analysis were all performed at a single timepoint during the experiment. To further characterise MegaT, we did the same analysis on an overnight time-lapse imaging to see where the fluorescent intensity of the cells changes with time. As the cells dried up overnight and died, we splice the time period of the experiment within the period where the cells are not dried up and plotted a time series data of how the mean of the fluorescent intensity change with time. As we can see from the graph, the mean intensity of the cells with nFc in 100μM of vanillic acid is always higher than that of the cells with nFc in 0 μM of vanillic acid.

Figure 3. Time series data of MegaT characterisation showing the mean of fluorescent intensity with time

As we are interested in comparing the mean intensities of mVenus expression between 0µM and 100µM Vanillic acid, we perform a t-test between the 2 samples and confirm that the 2 means are statistically significantly different from each other.

The result shows that MegaT can respond to the relative changes of VanR, it shows us that it is a desirable candidate to report the relative amount of the species it is in the same transcription unit with. As we are trying out a combinatorial approach to test for the antithetic integral controller with the best performance, we have decided to design MegaT with a different ribosome binding sites, matching that of the ribosome binding site of species X in our project.