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ABRHS iGEM Team Project E. Coli Luwak Our Team ➢ Chris Kuffner ➢ Ben Stern ➢ Madhuri Jois ➢ Mayank Mali ➢ William Huang Acton Boxborough Regional High School ➢ Akash Kapoor 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 ○ ○ ○ ➢ 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 assembly protocol done twice using competent cells- failed First- disorganized Second- scheduled with collaboration after second failure we were advised to start our project 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/
