LA Biohackers presents: A Strategy to Create a Chassis to Boot an - - PowerPoint PPT Presentation

A Strategy to Create a Chassis to Boot an Artificial Genome LA Biohackers presents:

Our Mission:

The mission of the Los Angeles Biohackers is to make science accessible to people of all ages and educational backgrounds. We do this by providing laboratory equipment and workspace, resources which are typically out of the reach of the amateur, to anyone passionate about learning.

Authors and their Affiliated Institution(s)

Keoni Gandall, UCI, Edison High School Cory Tobin, Caltech D

• u

g F

• s

t e r

C S U N

Sophia Hewlitt Dan Wright David McDuffee Tony Manzo Wolfy Hutton

Milken Community Middle School

Not shown: Joseph Ayar and Cambell Yore, of Santa Clara

University School of Law

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

• ver.

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

• rganism?

Casting call: pool of known life forms...

We are

HERE

• E. coli
• B. subtilis

&

• S. thermophilus

are HERE

Closer look

• S. thermophilus

&

• B. subtilis

are HERE

• E. coli is

HERE

Bacteria of course!

Strains

Trait Bacteria Species

• E. coli
• B. subtilis
• S. thermophilus

rod/spheres/spirals rod rod sphere gram +/-

• +

+ base pairs in chromosome 5 mb 4.2 mb 1.8 mb plasmids naturally present yes yes yes Comparison of bacterial species

• E. coli (K-12 strain)

Streptococcus thermophilus Bacillus subtilis

Strains cont.

• Used for cloning
• Full genome sequence available
• Small (1.8mbp)
• Gram positive

○ Demonstrate system robustness

• GRAS and industrially used
• Well studied, robust, large integrations

used in past

• GRAS
• Model organism
• Sporulates
• Gram positive

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*

5 plasmids verified All integrate over terminus and employ pos/neg selection for DNA integration

(4 below used to integrate yeast DNA as control)

Experimental design(cont)

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)

• Rigorous testing for optimization of B.

subtilis transformation

• Create better plasmids for robust integration

PMID: 24674868

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

• nes 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

• ther 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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www.biohackers.la Southern California Los Angeles

1340 E. 6th

• St. L.A., CA

90021 #603

Thank you!