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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
10
15
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25
30
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
35
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15
10
5
060 100 140 180 220 260 300 340
m/z
ControlConstitutive ECH and FCS
×104
×106
Abu
ndan
ce
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
0
5
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9 9.5 10 10.5 11 11.5 12 12.5 13
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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25
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5
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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10
8
6
4
20
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
0.05
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0.15
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Time [hours]
OD
600
Growth DataPredicted TotalPredicted InfectedPredicted Healthy
0 1 2 3 4 5 60
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Time [hours]
Rel
ativ
e C
ompo
sitio
n
Cinnamaldehyde - MOI = 0.0001Vanilla - MOI = 0.0001Cinnamaldehyde - MOI = 0.01Vanilla - MOI = 0.01
0
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Cas9 + -
No Arabinose
Arabinose
Red
/Whi
te C
olon
ies
0 10 20 30 40
0.1
0.2
0.3
0.4
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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180
245
Cas
9
Con
trol
Ladd
er
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