Part:BBa_K4491009
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.
Contents
| MegaT | |
|---|---|
| Function | Reporter |
| Use in | Bacteria |
| Chassis Tested | E. coli |
| Abstraction Hierarchy | Part |
| Related Device | |
| RFC standard | RFC10 & RFC12 & RFC21 & RFC23 & RFC25 & RFC1000 compatible |
| Backbone | pJUMP18-Uac |
| Submitted by | Cambridge iGEM 2022 |
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.
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<img src="
" alt="Design of MegaT" style="width:10%">
<figcaption>Figure 1. Design of the operon MegaT and how it fits into JUMP Golden Gate Assembly</figcaption>
</figure>
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.
| None |