Synthetic Biology: Biopharmaceuticals & Insulin-

generating Enteric Bacteria

Vi Nguyen

Biopharmaceuticals & MDR-TB/XDR-TB

What Are Biopharmaceuticals?

• Medical drugs created using biotechnology

• Include:– interferons– hormones– clotting factors– vaccines– antibodies

Tuberculosis

• caused by Mycobacterium tuberculosis

• 8.7 million contracted TB in 2011

• 630,000 cases of MDR-TB– resistant to most powerful tuberculosis drugs

Synthetic Gene Circuit for Drug Screening

Going Further

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Weber, Wilfried, Ronald Schoenmakers, Bettina Keller, Marc Gitzinger, Thomas Grau, Marie Daoud-El Baba, Peter Sander, and Martin Fussenegger. "A Synthetic Mammalian Gene Circuit Reveals Antituberculosis Compounds." Proceedings of the National Academy of Sciences of the United States of America 105.29 (2008): 9994-998. Web. 6 July 2013. <http://www.pnas.org/content/105/29/9994>.

Weber, Wilfried. "Synthetic Biology in Drug Discovery and Combating Drug Resistance." Lecture. Synthetic Biology Workshop - From Science to Governance. Sofitel Hotel, Brussels. 18 Mar. 2010. Public Health. European Commision. Web. 6 July 2013. <http://ec.europa.eu/health/dialogue_collaboration/docs/ev_20100318_co10.pdf>.

World Health Organization. "Tuberculosis (TB)." WHO. United Nations, 2013. Web. 7 July 2013. <http://www.who.int/topics/tuberculosis/en/>.

Insulin-generating Enteric Bacteria (IGEBs)

Purpose

• Provide a more convenient means of insulin therapy for diabetics

• Modify native gut flora to produce insulin (E. coli)

• Bacteria that produce insulin at ideal times (during glucose intake)

Competing Technologies

• Insulin injections– Pros:

• Relatively inexpensive• Relatively simple to

administer

– Cons:• Requires daily injections• Blood glucose spikes• Insulin resistance may occur in repeated needle

stick areas

Competing Technologies

• Insulin pumps– Pros:

• More accurate doses• Fewer blood glucose spikes• More flexible lifestyle• Fewer needlesticks

– Cons:• Expensive• Bulky system constantly attached to body• Requires extensive training to use

Design

• Determining when to produce large amounts of insulin

• No glucose no insulin– Glyoxylate cycle in absence of glucose

• Modified quorum sensing– Produce large amounts of insulin at certain

times

SuccinateSignaling molecule

Repressor Insulinproduction

LuxS

lsr promoterLuxS TAT peptide export signal

INS

succinate

AI-2 signaling molecules

ABC transporter

ATP ADPATP

ADP

lsr transport cassette

LsrR

P

LsrRP

TAT export

prepoinsulin

insulin molecules

AI-2

insulin

ATP

ADP

Expected Results

• During times of carbohydrate intake insulin production by IGEBs

Glucose Insulin production

0 0

1 1

Glucose Insulin production

absent 150 units

present 2000 units

Advantages

• Fewer required treatments

• Completely internal system

• Self-adjusting system

• Very flexible lifestyle

Potential Problems

• Surviving gastrointestinal tract

• Adhering to villi in small intestine

• Ensuring adequate absorption of insulin

• Horizontal gene transfer?

Testing

• Insulin production in absence/presence of glucose in environment– Cells exposed to various cycles of glucose

absence and presence– Insulin production measured and tracked over

time

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Human Small Intestine." Diabetes 17 (1968): 625-27. Print."Human Insulin Gene, Complete Cds." National Center for Biotechnology Information. U.S. National Library of

Medicine, 12 Feb. 2001. Web. 10 July 2013. <http://www.ncbi.nlm.nih.gov/nuccore/J00265.1>."MetaCyc Pathway: Glyxoylate Cycle." MetaCyc. BioCyc Database, 04 Dec. 2007. Web. 10 July 2013.

<http://www.biocyc.org/META/NEW-IMAGE?type=PATHWAY&object=GLYOXYLATE-BYPASS>.Miller, MB, and BL Bassler. "Quorum Sensing in Bacteria." Annual Review of Microbiology 55 (2001): 165-

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Taqa, ME, JL Semmelhack, and BL Bassler. "The LuxS-dependent Autoinducer AI-2 Controls the Expression of an ABC Transporter That Functions in AI-2 Uptake in Salmonella Typhimurium." Molecular Microbiology 42.3 (2001): 777-93. PubMed.gov. Web. 9 July 2013. <http://www.ncbi.nlm.nih.gov/pubmed/11722742>.