LA Biohackers presents: A Strategy to Create a Chassis to Boot an Artificial Genome
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Authors and their Affiliated Institution(s) Tony Manzo Dan Wright Cory Tobin, Caltech Keoni D o u g F o s t e r David Sophia Hewlitt C S U N Gandall, UCI, McDuffee Edison High Wolfy Hutton Not shown : Joseph Ayar and Cambell Yore, of Santa Clara School Milken Community University School of Law Middle School
Abstract The goal of this project was to integrate the entire Streptococcus thermophilus genome into Bacillus subtilis , demonstrating the robustness of the system. Obtaining a suitable strain of S. thermophilus was more challenging than we expected, but we did conduct an initial proof of concept using yeast ( Saccharomyces cerevisiae ) gDNA. We successfully integrated a short 20 kBp DNA yeast fragment into the locus specified by our design. We showed that the terminus is non- essential, and also showed that the pos/neg selection cassettes are viable for potential recombination. This is promising, and we will continue working on this project after the 2014 iGEM competition is over.
Project goals ● Characterize B. subtilis as a chassis for future genome engineering techniques ● Demonstrate the robustness of the system using the S. thermophilus ● Demonstrate B. subtilis’ use to other iGem teams
Genomic engineering: Which organism? Casting call: pool of known life forms... We are HERE E. coli B. subtilis & S. thermophilus are HERE
Bacteria of course! Closer look S. thermophilus & E. coli is B. subtilis HERE are HERE
Strains Comparison of bacterial species Trait Bacteria Species E. coli B. subtilis S. thermophilus rod/spheres/spirals rod rod sphere gram +/- - + + base pairs in 5 mb 4.2 mb 1.8 mb chromosome plasmids naturally yes yes yes present
Strains cont. E. coli ( K-12 strain ) ● Used for cloning ● Well studied, robust, large integrations used in past ● GRAS Bacillus subtilis ● Model organism ● Sporulates ● Gram positive ● Full genome sequence available Streptococcus ● Small (1.8mbp) ● Gram positive thermophilus ○ Demonstrate system robustness ● GRAS and industrially used
Advantages ● Takes pure gDNA for integration without modification ○ No linear DNA required ● Chassis shown to work with very large amounts of DNA (~4mb) ● Stable, can be sporulated for long periods of time ● Natural competence means simple transformation
Disadvantages ● Its not E. coli ○ No MAGE, no simple point mutations or BACs ● Its not S. cerevisiae ○ Not completely orthogonal from other bacterial cells ○ Not as well characterized as either organism ● Integrations require at least 125bp homology, rather than only 30-40bp ● B subtilis has less efficient DNA transformation
Experimental design
Advantage *Any DNA molecule, if defined by the LP sites, can be integrated. This includes the E. coli genome, S. cerevisiae chromosomes, or the entire biobrick registry into sporulated cells*
Experimental design(cont) 5 plasmids verified All integrate over terminus and employ pos/neg selection for DNA integration (4 below used to integrate yeast DNA as control)
Integration
Excision Cre/lox mediated excision
New genome for cell or purification
Innovations ● Pos/neg selection for integration rather than for deletion (PMID: 16714443) ● Replacing terminus to defeat size limited integration (PMID: 16236728) ● Usage of B. Subtilis rather than yeast for full genome integration (PMID: 18218864) ○ faster growth, easier storage, better stability, easier for bacterial labs to use
Progress ● Verified Integration of 20kb of raw yeast gDNA. ○ No modification of yeast DNA required ● Cloned new modular plasmids for this assembly ● Verified current methods for B. subtilis integration
Future prospects ● Integrate S. thermophilus DNA and demonstrate successful excision ● Integrate other bacterial and yeast DNAs for proof of concepts ● Integrate the entire parts registry into sporulating B. Subtilis ○ Using LP sites and artificial competence ○ Plasmids retrievable with a colony PCR reaction
Future prospects #2 ● Create and verify a robust method for serial integration (as displayed) PMID: 24674868 ● Rigorous testing for optimization of B. subtilis transformation ● Create better plasmids for robust integration
Roadblocks ● S. thermophilus from ATCC would not grow ○ Verified usable cells from yogurt, microscopy + colony PCR ● Yint100kb and Yint1000kb did not successfully transform into B. subtilis , primers for colony PCR of Yint5kb did not function, verified integration of Yint20kb
We were able to identify S. thermophilus in a variety of yogurt starter, but have not yet been able to isolate this species from the yogurt.
Roadblocks cont. ● SCK6 B. subtilis cells had low competency ○ Used protocol by LMU-Munich 2012 ● Gels were messing up ○ Buffer or agarose possibly bad? ● Competent E. coli cells ○ The comp cells do not store at -20, must make new ones every transformation
Roadblocks cont. 2 ● Difficulty with the PCR machine ○ Verified it was not accurately calibrated, this delayed cloning a few weeks for the new constructs. ● Toxin gene in pos/neg selection cassette did not allow the pos/neg cassette to be donated to the registry ○ Other parts were bricked, but they are still in B. subtilis integration vectors.
Acknowledgments LA Biohackers would like to thank the following: Rif and Bridget Hutton , and Black Olive Productions for travel assistance , SnapGene and MatLab for donating software, New England Biolabs for a BioBricks kit, and the Intellectual Property Investment Law Group for travel funds. Without their support, our participation in iGEM 2014 would not have been possible. We would also like to thank all those who have helped with the rent for the last two years, and all who have passed through our doors to simply see what we are all about. Last, thanks to Craigslist, eBay, and every other vehicle by which orphaned equipment has found our lab.
Human Practices/Policies and Procedures - Collaboration Art Center Santa Clara Law School - Open Community Lab Genomikon workshop
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