ABRHS iGEM Team Project E. Coli Luwak Our Team Chris Kuffner - - PowerPoint PPT Presentation

ABRHS iGEM Team

Project E. Coli Luwak

Our Team

➢ Chris Kuffner ➢ Ben Stern ➢ Madhuri Jois ➢ Mayank Mali ➢ William Huang ➢ Akash Kapoor

Acton Boxborough Regional High School Together we promote respect for self, others and learning.

Team Attributions

• 1. Research
• 2. Lab Work
• a. 3A
• b. High Efficiency
• c. CaCl2
• 3. Wiki

Background on Kopi Luwak

➢ One of the most expensive and rarest types of coffee in the world ○ $600 per pound ➢ Asian Palm Civet or Luwak (Paradoxurus hermaphroditus), a cat-like omnivore that fills the niche of a racoon in Asia ➢ Eats raw coffee beans ➢ During digestion, enzymes break down the parts of the bean that cause it to be bitter

Background on Kopi Luwak (cont.)

➢ The civet only digests the fleshy outer layer, not the beans themselves ➢ The feces includes clumps of the coffee beans that have been processed by enzymes ➢ Due to high demand, businesses resort to animal cruelty ➢ Thousands of civets are kept in battery cages, and are force fed the berries ➢ Population is diminishing

Proposition on Kopi Luwak

➢ Protect the civet and other mammals from use as enzyme factories. ➢ Engineer E. coli with genes for various digestive enzymes to replicate the digestive process of the civets on coffee berries ➢ Make the process more humane, efficient, and sanitary by using bacteria to produce the coffee

Research

Yeast vs. E.coli ➢ E.coli can live in a higher range of pH, are easier to engineer ➢ Yeast are more productive because they have a secretion mechanism, ➢ Chose E. Coli ○ k12 strain of E. coli from Carolina Labs ○ highly-competent strain of E. coli from NEB. DNA sequences ➢ wanted 3-5 ORFs: pepsin, salivary amylase, trypsin, chymotripsin, and pancreatic amylase. ➢ found pepsin and alpha amylase on the registry- went to work on assembling the part ○ Backbone- BBa_K318502- resists acid for pepsin production ○ Promoter- BBa_J23100- strong promoter ○ RBS- BBa_B0030- strong RBS ○ Terminator- BBa_B1006, a reliable option

ORFs- Amylase and Pepsin

Pepsin ➢ breaks up large proteins into smaller peptides ➢ It is produced in the stomachs of many animals ➢ breaks down bitter proteins in coffee ➢ it’s precursor, pepsinogen, is converted to pepsin in an acidic environment. Amylase ➢ breaks down complex carbohydrates into simple sugars ➢ In Kopi Luwak, amylase breaks down starch into sweeter, simple sugars

Safety

➢ Followed NSTA’s “Tips for the Safer Handling of Microorganisms in the School Science Laboratory” guidelines ➢ Our E.coli fall under risk group 1 of WHO’s microorganism classification: they do not pose a threat to the environment ➢ Used 10% Bleach and 70% Ethanol for sterilization

Project Labs

➢ 3A assembly protocol done twice using competent cells- failed ○ First- disorganized ○ Second- scheduled with collaboration ○ after second failure we were advised to start our project ➢ Attempted transformation with the protocol that came with the competent cells-failed ○ cells no longer competent ➢ Attempted transformation using CaCl2 and K12 cells-failed ○ more technique practice and equipment familiarity ○ not going to use competent cells without a -80

Proposed Parts

Pepsin Existing planned part in registry has human signal peptide Attempted to find a pepsin gene more similar to Paradoxurus hermaphroditus, to more closely mimic the civet’s digestive system

Pepsin Problem

• Part included signal peptide from production in human cells, wouldn’t work in e Coli, despite

claiming to be optimized for production in e Coli

• analyzed sequence through National Center for Biotechnical Information(ncbi)and ExPASy Translate

Tool

• shows which part of sequence codes for what
• contained codons optimized for production in e Coli
• essentially, human pepsinogen sequence with codons more frequently used by e Coli, but that code

for the same amino acid

• needs an e Coli signal peptide, without this peptide, the cell will have no method of secretion
• our proposed part contains the e Coli signal peptide
• part BBa_K208004 contains the e Coli signal sequence

In the Future

➢ Parts ○ Synthesize Pepsin ○ Trypsin Research ○ Periplasmic enzyme secretion ➢ Lab Protocols ○ Improve familiarity with materials ○ Different E.coli Chassis

Acknowledgements

References (Websites and Pictures)

http://en.wikipedia.org/wiki/Asian_palm_civet http://ab.mec.edu/abrhs/ http://en.wikipedia.org/wiki/Kopi_Luwak http://animalsadda.com/asian-palm-civet/ http://en.wikipedia.org/wiki/Kopi_Luwak#mediaviewer/File:Luwak_(civet_cat)_in_cage.jpg http://catpoopcoffeeinc.com/