CRISPR Mediated Automated Design Engineered to Bring You...

CRISPR MADE BY UCRISPR Mediated Automated Design Engineered to Bring You Ultrabiotics

CRISPRs are loci found in some prokaryotes that function as an adaptive immune system.

Exogenous DNA is processed into short “spacers” which are inserted into the host genome between repeat sequences. The repeat-spacer-repeat array is expressed and processed to form small RNAs (crRNAs) that guide the specific cleavage of target sequences by Cas9.

Spacer SelectionWe wrote a Perl script to find spacers that were found in several phage genomes, not in the host genome and not containing any illegal restriction sites. By combining these broadly neutralizing spacers, it is possible to immunize against 22 T4-like phages with only 5 unique spacers.

System validationThe expression of Cas9 under both constitutive and arabinose inducible promoters was verified by SDS-PAGE.

We next assembled the three necessary components [Cas9, tracrRNA (tr) and the repeat-spacer-repeat array (sp)]. From this we assayed resistance to toxic plasmids and growth kinetics under T4 phage predation.

Growth of minimal CRISPR system under toxic plasmid and phage selection. Left. Ratio of colony counts of a control to toxic plasmid co-transformation in strains with inducible tracrRNA and repeat-spacers, with or without constitutively expressed Cas9. Right. Growth kinetics of T4 phage predation on inducible minimal CRISPR-Cas9 construct with or without Cas9, compared to empty vector.

We confirmed cinnamaldehyde production from phenylalanine!

Vanillin is a valuable aromatic compound used in flavouring, preservatives, pharmaceuticals, and fragrances.

Our goal was to produce vanillin in E. coli from tyrosine through five enzymatic steps.

We combined enzymes available from the registry (*CMH: KU Leuven 2009 and *COMT: Edinburgh 2007) with our own biobricks to assemble the above pathway. Intermediate and final products were characterized by GC-MS.

We characterized compound generation for all steps in the pathway from p-coumaric acid to vanillin!

We consulted with a wide variety of experts to develop and carry out a plan to design a marketing strategy for GM yogurt.

GMO PerspectivesTo understand the potential issues of using GMOs in the dairy products, we interviewed dairy industry and academic professionals.

Public Opinion SurveyWe surveyed the public to evaluate the major factors that consumers associate with genetically modified foods.

Summary of Our Survey Results• Health benefits and taste are strongly considered for both types of yogurt.

• Price was influential, but not a driving factor in purchasing GM yogurt.

• Ecological impact proved to be the least influential factor.

• Potential health hazards of GM yogurt were a concern, but individuals were more willing to purchase labeled products.

Transparency emerged as a key issue in genetically modified foods. We are currently using this information to refine a marketing strategy for GM yogurt.

Introduction CRISPR-Cas9 Immunity

GMO Yogurt Marketing Strategy

Compound detection by GC-MS. Vanillin detection from culture containing ferulic acid and strains with Fcs and Ech.

0

5

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35

9 9.5 10 10.5 11 11.5 12

10.4

86

OCH3

OH

CHO

Vanillin

10.486

Abu

ndan

ce

Retention Time (min)

45.1

73.1

104.1137.1163.0

224.1

254.0 299.1

194.1

276.1 326.2348.2

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m/z

ControlConstitutive ECH and FCS

×104

×106

Abu

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Vanillin Biosynthesis

OCH3

OH

CHO

Vanillin

OCH3

CoA

OH

OSO

Feruoyl-CoA

OCH3

OH

OH

Ferulic Acid

O

OHOH

OH

Caffeic Acid

O

OH

OH

p-Coumaric Acid

O

NH2

OH

OH

Tyrosine

O

TALBBa_K1129000

CMH*BBa_K1129046

COMT*BBa_K1129041

FCSBBa_K1129024

ECHBBa_K1129022

Get more details, interview responses and our marketing strategy!

Cinnamaldehyde provides the characteristic aroma and flavour of cinnamon products and is anti-microbial and anti-diabetic.

Our goal was to produce cinnamaldehyde in E. coli from phenylalanine through three enzymatic steps.

We combined enzymes available from the registry (*4CL: TU Munich 2012) with our own biobricks. We confirmed both the conversion of phenylalanine to cinnamic acid through EncP and the conversion of phenylalanine to cinnamaldehyde through EncP, 4CL and AtCCR1 with GC-MS.

Cinnamaldehyde Biosynthesis

Cinnamaldehyde

CoAOSO

Cinnamoyl-CoACinnamic acid

OHO HO

NH2

OH

Phenylalanine

O

PAL (EncP)BBa_K1129026

(BBa_K1129003)4CL*

BBa_K1129042AtCCR1

BBa_K1129039

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10.5

80

11.8

19

Constitutive EncP, 4CL and AtCRR1Control

Abu

ndan

ce

Retention Time (min)

Cinnamic acid

45.1

75.1103.1 161.1

131.1

229.1263.1

299.1348.2376.2

205.145

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Abu

ndan

ce

10.580

60 140 220 300 3800

OHO

×103

×106

Cinnamaldehyde

HO11.819

60 140 220 300 380

45.1

73.1

147.1

118.1175.1220.1265.1

293.1

320.1359.2

409.1

Abu

ndan

ce

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m/z

×105

Compound detection by GC-MS. Cinnamic acid and cinnamaldehyde detection from culture supernatant containing phenylalanine with strains harbouring EncP, 4CL and AtCCR1.

We assembled, expressed and are characterizing a minimal CRISPR-Cas9 system in E. coli!

Gather Information

Develop Public Survey

Conduct Public Survey

Professional Interviews

Industry

Apply for Ethics

Approval

Academic

Telus World of Science

Marketing Professionals

Developing iGEM Project

Consult

Partner

Analyze Survey Data

Design Marketing Strategy

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HealthBenefits

Environmental Impact

Price HealthHazards

Taste

Rel

ativ

e Im

port

ance

(5

hig

hest

)

Spacer PAM

Exogeno

u s D NA

Cas9 crRNA

... leader repeat repeat ...repeat

pre-crRNA

cas

UBC2013

Team Members: Fisal Elstone, Liz Geum, Joe Ho, Dan Korvin, Joel Kumlin, Anna Müller, Michael Peters, Frances Russell, Cameron Strachan, Negin Tousi, Dave VanInsberghe, Grace Yi, Tony ZhaoGraduate Student Advisors: Christopher Lawson, James Round, Ray Socha, Michael VanInsbergheFaculty Advisors: Joanne Fox, Steven Hallam

There are an estimated 1031 phage on Earth

Outnumbering prokaryotes about 10:1Bacterial processes are thus under

the constant threat of

For example, 10% of dairy fermentations collapse due to phage infections

dxdt

We refactored the CRISPR-Cas9 bacterial immune system to the minimum components in order to

engineer broad immunity to phage

We realized that our system could also be used to apply selective

pressure to tune mixed bacterial populations

Comprehensive models were

used to help us understand how mixed populations grow under dynamic selective pressure

We envision the next generation of microbes in yogurt fermentation being able to produce natural prod-ucts in vivo. We assembled the

biosynthetic pathways for vanillin and cinnamaldehyde

Through consultations with a wide variety of experts, we developed and executed a plan to design a

marketing strategy for GMO yogurt

Host genome

T4

AR1HX10

RB14...

...

...

...

Broadly neutralizing repeat spacer repeat array

Virus speci�c spacers

We realized that strain balance could be modulated in independently immunized cultures using phages to apply dynamic selection pressure. An example of mixed culture fermentation is yogurt, where the quality of the final product requires optimal strain balance.

Deterministic Model: Growth Kinetics under phage predation

Monod growth kinetics were used to describe growth and the Poisson distribution was used to determine the infected proportion. We trained the model on experimental growth curves and validated it against another set. As can be seen in the above figure, predicted growth closely matches the actual.

Compound production from a mixed culture

This basic growth model was then extended to co-cultures. Yield terms were added to account for the production of two different flavours.

As can be seen in the adjacent figure, by using different virus inoculums, it is possible to achieve different final amounts of compounds from a mixed culture.

Probabilistic Model:

We numerically simulated the co-cultures of two flavour producing strains using Gro1. The model considers two strains on a two-dimensional surface under phage predation. One of the strains is immune to infection, whereas the other is susceptible.

1. gro the cell programming language http://depts.washington.edu/soslab/gro/index.html

Population Dynamicsdxdt

Find a team member to see the Gro animation of our numerical model!

Cinnamaldehyde VanillaCRISPR

CRISPR

Both strains are resistant to the environmental phage

Only one is immunized against the control phage

t = 3.4 min t = 24.5 min t = 64.0 min

Susceptible to phage Resistant to phage Infected Phage

0 4 8 12 160

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Time [hours]

OD

600

Growth DataPredicted TotalPredicted InfectedPredicted Healthy

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ompo

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Cinnamaldehyde - MOI = 0.0001Vanilla - MOI = 0.0001Cinnamaldehyde - MOI = 0.01Vanilla - MOI = 0.01

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Cas9 + -

No Arabinose

Arabinose

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/Whi

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olon

ies

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sp tr, Cas9 +sp tr, Cas9 -Vector control

OD

600

Time [h]

Thank you to our sponsors and those who helped us along the way!• UBC Life Sciences Institute • UBC Department of Microbiology & Immunology • UBC Michael Smith Laboratories• UBC Department of Chemical & Biological Engineering • UBC Faculty of Applied Science • UBC Faculty of Science• UBC Walter H. Gage Memorial Fund• UBC Engineering Undergraduate Society• Pfizer Canada Inc.• Genome British Columbia

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2025

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Cas

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Con

trol

Ladd

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