for polypeptide release in the small intestine Team UW Madison 2010 - - PowerPoint PPT Presentation

A fully automated system for polypeptide release in the small intestine

Team UW–Madison 2010

Growth

iDIET in Brief

Dietary Ingestion Acid Survival Lysis & Product Release Product Absorption

Acid Survival Lysis & Product Release Product Absorption Dietary Ingestion Growth

Genetic Disorders & More: Celiac Disease

• 1 in 133 are diagnosed in the US
• US healthcare cost: $14.5 - $34.8 billion

annually

• No cure
• Extreme dietary measures

Genetic Disorders & More: High Cholesterol

• Cholesterol made from bile acids
• Bile Salt Hydrolase enzyme deconjugates
• Deconjugates bile salts cannot be reused

Genetic Disorders & More: High Cholesterol

Cholesterol Bile Salts Bile Salts Deconjugated Bile Salts

Genetic Disorders & More: High Cholesterol

Cholesterol Bile Salts Bile Salts Deconjugated Bile Salts Serum Cholesterol Excreted

Genetic Disorders & More: Lactose Intolerance

• Unable to digest lactose found in

dairy products

• Model system for experiment

– Deliver beta-galactosidase by timed-lysis to small intestines – Interchangeable system

Growing Micro-Organisms

Growth: Encapsulation & Enzyme Production

RcsA RcsB B-Gal

REPLACABLE ENZYME

constitutive

RcsA RcsB B-Gal

REPLACABLE ENZYME

Ingestion

Encapsulation Testing Constructs

RcsA RcsB B-Gal

REPLACABLE ENZYME

RcsA RcsB RcsB RcsA

IPTG Inducible

Testing Constructs

Constitutive

Colonic Acid

Encapsulation: Proof of Colonic Acid Production

Encapsulation: Proof of Colonic Acid Production

2 4 6 8 10 (ug/ml)/OD600 of L-Fucose

Quantification of L-Fucose

Control RcsB RcsA

Encapsulation: Cell Survivability in Low pH

1E+10 4.1E+11 8.1E+11 1.21E+12

CFU per ml

Cells Survivability at pH4

RcsA RcsB RcsA+RcsB Control

Enzyme Release in Small Intestine

1) Inducible Repressible

• pH promoter
• Activator and Repressor
• A promoter sensitive to

activator and repressor 2) Bile Salt Inducible Expression

• Salmonella enterica strain LT2
• PCR Amplify and clone ramA and

acrRA genes 3) Encryption System

• See our poster

Inducible-Repressible Lysis

Lysis Cassette

pH I/R

cI

LuxR

Lysis Cassette cI + LVA LuxR cI + LVA LuxR Low pH Duodenum of Small Intestine Stomach Before Stomach

Inducible-Repressible Lysis: Testing Constructs

Lysis Cassette

gadAP I/R

cI + LVA LuxR RFP

Testing Construct

Protein expression behind the gadA promoter is dependant on growth phase

5x

• Tight control of expression below OD 3.0

(indistinguishable from baseline fluorescence)

• Limited expression from OD 3.0 to OD 5.5
• Induction as culture approaches a true

stationary state

gadAp + mRFP1, pSB1A2 MG1655 placI + mRFP1, pSB1A2 MG1655 WT MG1655

1 2 3 4 5 6 7 2000 4000 6000 8000 10000 12000 5 10 15 20 25 30 OD600 RFU/OD600 Time (hrs)

gadAp RFP Expression vs Time and OD (pH 5.5)

• 2. Bile Inducible Lysis:

System

RamA

constitutive

AcrRA

RamA

BS BS BS BS

Lysis Cassette Lysis Cassette

Duodenum of Small Intestine Before Duodenum

• 2. Bile Induced Lysis:

Testing Constructs

RamA

constitutive

AcrRA

Lysis Cassette RFP RamA

Testing Constructs

AcrRA

constitutive

Enzyme Action

Large Intestine

Large Intestine

Registry Maintenance: BBF RFC 67

20 40

EXPERIENCE TESTING SEQUENCING GENBANK

C

Achievements

Lysis Systems in Small Intestine Applications

Characterization

Registry Additions

• 1. Inducible-Repressible System
• 2. Induction in the presence of Bile
• 3. Encryption (see wiki/poster)
• K318500 *
• K318501 *
• K318502 *
• K318506
• K318507
• K318511
• K318512 *
• K318513 *

1. gadA promoter

• OD
• pH

2. Expression of RcsA, RcsB, RcsA&RcsB

• Colonic acid production
• Cell survivability

1. More efficient delivery of functional enzyme to the small intestine 2. Decrease in cost of production and purification for pharmaceuticals 3. Accessibility of treatments for genetic disorders

* Characterized

Lysis Systems in Small Intestine Registry Additions

Characterization

Applications

References

IMAGES http://diet.lovetoknow.com/wiki/Lactose_Intolerance_Symptoms http://popsop.com/wp-content/uploads/realgoodness_blue.jpg http://topnews.in/health/files/heart-attacks_0.jpg http://media.photobucket.com/image/atherosclerosis/Kacchan_08/atherosclerosis-2.jpg BACKGROUND

1 Fasano A, Berti I, Gerarduzzi T, et al. Prevalence of celiac disease in at-risk and not-at-risk groups in the United States. Archives of

Internal Medicine. 2003;163(3):268–292. Martini,M., G. Bollweg, M. Levitt, and D. Savaiano. 1987. Lactose Ingestion of yogurt β-galactosidase: influence of pH and microbial cell

• integrity. Am. J. Clin Nutr. 45:432-436.

NICHD, NIH. 2006. Lactose Intolerance: Information for Health Care Providers. <http://www.nichd.nih.gov/publications/pubs/upload/NICHD_MM_Lactose_FS.pdf> NIDDK, NIH. 2009. Lactose Intolerance. <http://digestive.niddk.nih.gov/ddiseases/pubs/lactoseintolerance/> BILE INDUCTION Nikaido E., A. Yamaguchi, and K. Nishino. 2008. AcrAB Multidrug Efflux Pump Regulation in Salmonella enterica serovar Typhimurium by RamA in Response to Environmental Signals. J. Biol. Chem. 283:24245-24253. Liong, M.T. and N.P. Shah. 2005. Bile salt deconjugation ability, bile salt hydrolase activity and cholesterol co-precipitation ability of lactobacilli strains. Int. Dairy J. 15:391-398. INDUCIBLE/REPRESSIBLE Tucker, D., et al. Gene Expression Profiling of the pH Response in Escherichia coli. J Bacteriol. 2002. 184(23): 6551–6558. Waterman, S., et al.Transcriptional Expression of Escherichia coli Glutamate-Dependent Acid Resistance Genes gadA and gadBC in an hns rpoS Mutant. J Bacteriol. 2003. 185(15): 4644-4647. Castanie-Cornet, M., et al. Control of Acid Resistance in Escherichia coli. J Bacteriol. 1990. 181(11): 3525-3535.

Questions?