Team:VIT Vellore/Project

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In Vivo Drug Factory

Our project for iGEM 2011 consists of the design, characterisation and proof of concept experiments for the development of an In-Vivo Drug Factory. Our proposal also covers the development of a generalised synthetic gene circuit for in-vivo drug production that can be used for the treatment of other disorders requiring frequent or continuous medication with an optional special provision for increase/decrease in production depending on the diurnal meal rhythm.

General Project

Development of purpose-built recombinant strains of pre-existing intestinal flora or E.coli, using synthetic biology with parts in the BioBricks™ standard, to treat inherited deficiencies of key metabolites. Production of the key metabolites will be activated during mealtimes in the duodenum, and controlled by a feedback regulation loop. Also, we propose the inclusion of a safety switch, which can be externally triggered in case of any unexpected/awry behaviour.

BioBrick Circuit

Our strategy relies on:
Use of enteric bacteria residing in the duodenum as hosts:
Makes for a much safer host with less risk of unfavourable side effects.
Use of glucose repressible promoters to link the production of the desired drug to glucose levels in the duodenum. (related to mealtimes)
The use of a PoPS (polymerase per second) inverter to relate the drug production either positively or negatively to glucose concentration.
Continuous production of drug throughout the day at low concentrations as a basal dose, with an increased concentration during lactose intake.
Use of R1 plasmid hok/sok system, in order to ensure stable plasmid replication. This provides a double level of control over protein production.
Use of already characterised bacteriophage holin BioBricks or the development of newer holing BioBricks under the control of a specific environmental trigger as an emergency safety switch.
Holins are intracellular acting proteins and hence unlike antibiotics pose a much lower chance of affecting other intestinal flora.

Proof of Concept

We have selected lactose intolerance as the disease of interest for our project and to conduct proof of concept experiments. This is because this disease is characterised by the deficiency of a fairly simple and commonplace enzyme, lactase. In prokaryotic systems, β-galactosidase is the functional equivalent of lactase and acts in much the same manner. The production of a simpler enzyme, rather than more complicated compounds such as insulin or cyanocobalamin leaves us with the freedom to focus more on the multiple genetic controls and the characterisation of new BioBricks which form the basis of our project.
Lactose metabolism and how it affects patients with Lactose Intolerance: Lactic acid bacteria have the enzymes β- galactosidase, glycolases and lactic dehydrogenase (LDH) which produce lactic acid from lactose. Lactic acid is reported to have some physiological benefits such as:
a) Enhancing the digestibility of milk proteins by precipitating them in fine curd particles.
b) Improving the utilization of calcium, phosphorus and iron.
c) Stimulating the secretion of gastric juices
d) Accelerating the onward movement of stomach contents
e) Serving as a source of energy in the process of respiration.
In humans, both isomers are absorbed from the intestinal tract. Whereas L(+) lactic acid is completely and rapidly metabolized in glycogen synthesis, D(-) lactic acid is metabolized at a lesser rate, and the un-metabolized acid is excreted in the urine. The presence of un-metabolized lactic acid results in metabolic acidosis in infants. L. acidophilus produces the D (-) form and is therefore of disputable clinical benefit, although it has earlier been the probiotic of choice in various therapeutic formulations. L. sporogenes* on the other hand produces only L(+)- lactic acid and hence is preferred.
The ability of lactobacilli to convert lactose to lactic acid is used in the successful treatment of lactose intolerance. People suffering from this condition cannot metabolize lactose due to lack or dysfunction of the essential enzyme systems. Lactic acid, by lowering the pH of the intestinal environment to 4 to 5, inhibits the growth of putrefactive organisms and E. coli, which require a higher optimum pH of 6 to 7. Some of the volatile acids produced during fermentation also possess some antimicrobial activity under conditions of low oxidation-reduction potential.
Since lactose intolerance can be treated to some extent by current formulations/ probiotics we also propose a comparison study between a standard LAB and our Synthetic Factory to highlight the advantages of such a synthetic biological system with multiple safety locks and controls. Also, our project emphasises on the controls present and the development of a generalised system with lactose intolerance only as a proof of concept.