While writing DNA, there are a few conventions to be followed. The first one includes the use of Biobricks, standard small sequences of nucleotides that have a specific function. To build a circuit, synthetic biologists would need: promoter, ribosome binding site (RBS), coding sequence (CDS), terminator and a vector (backbone).
In this page, we explain what are the basic and composite parts our team used to make bacteria produce our minicells and how we created a modular plasmid for proteins of interest - exclusively expressed in the minicell.
We chose mainly well-characterized basic parts to work with, extracted from the Registry database of standard biological parts. Table 1 shows all the parts used. Cell-division related coding sequences were amplified directly from the genome of wild type MG1655 E. coli strain.
Composite parts were created to ensure tight control over protein expression, being it either for creation of “minicells producing state” in a range of strains or to express our two main proteins of interest: TMM (trimethylamine monooxygenase) and Taq polymerase.
Throughout the project, the team used TB43 MG1655 E.coli min mutant strain as a continuous source of minicell samples. Lack of sufficient and updated data on minicells encouraged us to start characterizing minicells behaviour.
We noticed that min mutant strains struggle to survive and therefore would not produce an optimum amount of minicells. Whereas by observing their limited lifespan, we could see that by not having a tight regulation of their expression we wouldn’t have clear knowledge of their age and therefore could not know how long they would still produce our proteins.
This led to the design of a tightly regulated minicell production loop. Our hypothesis was that by having a plasmid switching the bacteria on to produce minicells under specific conditions would increase minicells yield and ensure they would be still metabolically active to express protein of interest after purification assays.
We developed a circuit to control minicell expression in any wild type strain by overexpression of the FtsZ ring and deletion of the min operon. Expression of FtsZ is controlled by inactivation of pLac with LacI (BBa_K292002). LacI is under a constitutive promoter from Anderson’s library (BBa_J23106 or BBa_J23100).
While FtsZ overexpression allows minicell production in non-mutant bacterial strains, min operon regulation relies on having a min mutant strain with the min operon depleted. This second approach uses the regulated promoter pLac (BBa_K292002) and pTet (BBa_R0040) and creates a negative feedback loop.
For the application of our project, we decided to focus on the production of the textile dye Indigo through the recombinant production of tmm (Trimethylamine moonoxygenase) gene. What led us to focus on this particular application is the fact that the textile industry is extremely harmful to the environment. Every year, an enormous amount of chemically produced dyes are dumped into lakes which has devastating impacts.
To keep our design as biosafe as possible, we wanted to have the expression of our recombinant protein to be activated only in the minicells. This means that there will only be production of Indigo once the minicells would have been separated from the mother cells (and the mother cells would have lysed). To ensure this controlled production of Indigo, we
designed a device where the production tmm is controlled by the Arabinose operon (pBAD, pC and AraC gene). For the hardware system, this would require the addition of Arabinose into the minicell purified tank to have the production of indigo.
For characterization purposes, we first constructed our device with GFP.
The arabinose operon was amplified from the pBAD-pR plasmid.
GFP was taken from the CIDAR MoClo Toolkit (E0040_CD)
The ftsZ (filamenting temperature-sensitive mutant Z) gene is encoding an essential cell division protein : FtsZ. FtsZ is a tubulin-like protein that has a crucial role during bacterial division as this is the first cell-division protein to arrive on the division site (the septum of dividing cells). Moreover, its GTPase activity enables the contractile movement to promote cell division. FtsZ expression regulates the formation of a contractile ring structure also called ftsZ ring or Z ring, it consists of the assembly of protofilaments at the future cell division site.
This composite part contains the arabinose operon, pET ribosome binding site, the GFP coding sequence and a terminator. The Arabinose operon is very well characterized and leads to tightly controlled gene expression. For characterization purposes, we cloned GFP inside this plasmid. GFP is extremely well characterized and allows for easy and reproducible fluorescence studies, allowing us to determine the tightness and robustness of our device.
The min operon in E.coli is composed of three genes: minC, minD and minE respectively encoding for the proteins MinC, MinD and MinE. More precisely, MinC is one of the main negative regulators of FtsZ by preserving FtsZ polymerization. And overall, this Min system helps to center the position of the Z-ring by restricting FtsZ assembly to the middle of the cell thanks to a high concentration of FtsZ polymerization inhibitor at the pole of the cell .
This part contains the pBAD promoter, B0030 ribosome binding site, TMM gene sequence and B0010 terminator. When transformed into E. Coli, this part allows for the production of TMM (Trimethylamine moonoxygenaseonly) when there is presence of Arabinose and low glucose levels in the medium. The protein of interest needs to be located downstream of the operon to be induced by arabinose.