Module 1: Protein Production

GENERAL COMMENTS: ADD IN SOME STUFF ON MOTIVATION. I.E. WHY WE ARE TACKLING THESE DISEASES... ETC. PKU... ATM, YOU HAD SOME INFO BUT I COMMENTED IT OUT. I WOULD RETRIEVE SOME PICS FROM YOUR OTHER PGS.

Module 2: Encapsulation

The E.ncapsulator has been designed to produce and deliver polypeptides (amino acid polymers) to the intestine. Here in our system, a single gene is responsible for producing them. In order to perform this function successfuly, the polypeptide must be synthesised at a rate that will be sufficient to facilitate its accumulation inside the cell. Once the culture has grown to an optimal level, polypeptide production is chemically induced using IPTG [ref IPTG induction]. With our generic design, it is possible to synthesise any polypeptide. In our project, we have focused on:

PAH (phenylalanine hydroxilase): Include an explanation
Cellulase: Include an explanation
Opiorphin: Include an explanation

Genetic circuit

ADD MORE DETAILS ABOUT GENETIC CIRCUIT AND LITERATURE REVIEW OF GENES. OTHER STUFF HAS MOVED TO DETAILS OF GENETIC CIRCUIT PG. GET CHARLES TO UPLOAD THE GEN.CIRCUIT VIDEO. This is the genetic circuit responsible protein drug fabrication (Module 1).

LacI is produced constitutively by the E. coli bacterium, and it represses the pLambda promoter. This inhibits production of the proteins of interest (PAH and cellulase).

To start the drug manufacturing process, IPTG is pipetted into the system. IPTG will repress LacI. As a result, protein production is de-repressed. Then, the enzymes PAH or cellulase will be produced by the E.ncapsulator.

About the genetic circuit!

Polypeptide Showcase

To demonstrate The E.ncapsulator's versatility, we have chosen to showcase it with both enzymes and peptides. These two classes of polypeptide have very different properties that we have considered and catered for in The E.ncapsulator's design.

About the difference between enzymes and peptides.

AFTER THIS: I HAVE ADDED ANOTHER SECTION FOR RESULTS AND DRYLAB, TO STANDARDIZE.

Our results

Wetlab

Different concentrations of IPTG were added to a 96-well plate [link to assay] of e-coli Top 10 cells containing LacI-RFP genetic constructs. Here we measured the optical density of the cells over-time, for different concentrations of IPTG to monitor the effects in the culture. The IPTG growth curve results are plotted in the figure (ADD A FIGURE CAPTION TO IT: FIG1, FIG2 ETC). By visual assessment,the growth rate of the culture prior the saturation phase stays within a narrow range and does not change significantly with concentrations of [IPTG]. Therefore, we can conclude that [IPTG] in the concentration ranges we are using does not affect cellular growth and is non toxic. The effects of IPTG have been widely studied [SAY THAT WE ARE CONFIRMING SOMETHING WELL KNOWN ANYWAY BY OTHERS]

Drylab

Taking into account the indications from the wetlab, we can then draw a relationship between different input IPTG concentrations and the amount of polypeptide produced.

When we introduce IPTG into the system, it temporarily removes LacI from the system. Hence, during this period of time, we produce the drug of interest.
When the effects of IPTG wear off, the system returns to equilibrium.
The more IPTG we add in, the higher the amount of output protein.

DAVE: FROM THE BOTTOM ONWARDS WE MAY NEED TO SHORTEN OR ADD SOME STUFF TO LEARN MORE TABS.IVE COMMENTED IT OUT, NOT DELETED IT. ALSO ADD LEARN MORE TABS FOR WETLAB AND DRY LAB LIKE IN M3 OVERVIEW!