Alberta iGEM 2010
22 undergraduates (2nd-4th year) from:
Meet the Team:
the faculties of Science, Engineering, Medicine, Business and Fine Arts
SynBio must provide the means to rapidly design, test and optimize living systems
Acceleration:
Acceleration: Technical and Cultural
Technology: Getting results fast
Minimizing process error
Culture: Access to technology increases understanding of it.
Affordability
Reliability
Process speed
Minimizing user error
Simplicity
But…… What if there was a piece of technology that could answer both of these needs?
That could be revolutionary!
Acceleration can be both Technical and Cultural
A self-contained SynBio kit targeted for high school and first year university students
•Lab manual •Notebook •Info management •Sharing ideas
An online social network resource:
No lab equipment required
Experiments can be completed in an afternoon
Enough parts for 100s of meaningful experiments
Inexpensive!
Introducing
OVERVIEW
Advantages:
3 days
Limitations
Slow Modular Standardized Lots of part choices
3 days
Limitations
Slow Modular Standardized Lots of part choices
Advantages:
BioBricks:
Sx
Px
pSB1C3
BioBricks:
Accelerating Assembly
Ligase + Byte #1
3X Wash resuspend
Magnet
Repeat….
7 minutes
35 min. Iron micro-beads
1st Byte
Accelerating Assembly
The challenge: Fixed orientation Only one byte per cycle
Controlling Byte Order, Orientation and Copy Number
Symmetrical and complementary
BioBrick overhangs:
Mixed products after one cycle
Mixed products after one cycle Nonidentical and non-symmetric
BioBrick overhangs:
Alternating Head-to-Tail
Two Byte Formats
AB
BA
Self-Ligation Self-Ligation Fixed Orientation Single copy addition
Cycle 1 Cycle 2
BioByte overhangs: Nonidentical and non-symmetric
A
B A’
B’
The solution: Special overhangs
Controlling Byte Order, Orientation and Copy Number
Promoters
RBS
Terminators
Coding Non-coding Non-coding Non-coding Coding
Linker ORF ORF Linker Linker
mRNA Linkers
N and C terminal Protein Fusions and Tags
End inverters
AB BA BA BA AB
X Y Met Stp
Linkers Define the 1st and Last Codons
X Y
Functional Nature of Bytes
MOLECULAR DETAIL
BsaI
BsaI
pSB1C3-AB
P S A B
AB Byte
BsaI BsaI
pSB1C3-BA
P S B A
BA Byte
BsaI BsaI
PCR with universal primers
BioByte Production
BsaI: an offset cutter Leaving a 4 base overhang
Recognition
NNNN
dT25
Iron Bead
dT18
Caps
dA18
Anchors
BsaI
Anchor and Cap
Ligate to 1st Byte Remove access anchor
Room Temp
dT25
Iron Bead
5 min@ 75o C
dT25
Iron Bead
Cool & Tranform No ligation!
TESTING
Experiment 1: Fidelity of Ligation
Fidelity & Efficiency confirmed
Experiment 2: 3 Steps of Assembly
25oC 75oC
Anchored Byte
A. Anchor binding and release
B. 2nd Byte addition and release 1 kBp
ligation
Chain initiation, extension & release
Experiment 3: Coupling Efficiency
Building a 12kB Octamer
50% coupling Efficiency final yield: <<<
93% coupling Efficiency 63% final yield BioBytes 2.0
12k 6k
x8
BioBytes 1.0
2009
X10?
Experiment 4: Cap – Anchor Selection
Ori Anchor Cap RFP KanR
AAAAAAAAAAAAA TTTTTTTTTTTT
Cap+
Cap- AAAAAAAAAAAAA
5 part Assembly
Anchor-to-Cap circularization provides strong selection for full-length product
THE KIT
Introduce students to the kit Provide a foundation in
synthetic biology
Empower students to create and complete their own experiments
on the web
Lab Manual: Experiments
Use only materials available in the kit Teach concepts that correspond with a high school biology curriculum Go beyond “learning” and start “doing”
Fluorescent bacteria, Color readouts Genetic circuits
Designed to:
Examples Experiments Include:
The Lab Manual
Look at your colonies of bacteria under a black light.
Use the provided UV flashlight to examine the colonies you grew.
Lab Manual: Interactive features
This experiment was cool, my colonies are glowing!
Take notes Upload images
Lab Manual: Informative Articles
Experiment Designer tool
Automatic Sequence Generation
Generate a Protocol Share your designs
Leading edge of bio lab kits
Natural fit in the high school biology curricula
Introduce 1000s of students to synthetic biology
in the classroom
“I learned the theory already, but I got to apply it today.” –Aymen Saidane, 17
in the classroom
3 rep ORIs 4 SMs 8 reporters 9 promoters X X X
> 864 different plasmids
Enough precision droppers for an entire class
UV flashlight
Iron Microbeads
Magnetic racks
Making Synthetic biology accessible
$3
$15
$4
$7
priceless
Thanks To Our Generous Contributors
Materials Quantity Cost Totals E. coli CC 18 20.00 Magnetic Racks 6 7.50 Precision Dropper 18 2.99 LB Plates 30 1.00 Tubes 18 1.00 Buffer Elution 6 0.50 Buffer Wash 6 0.50 Magnetic Microbeads (ml) 6 4.20 DNA parts 3.00
Labour Stipend Employee (h) .75 @ 15.00 15.00
Variable Overhead Packaging 3.14 Mailing 13.16
Total Incremental Cost 71.99
Cost of the kit
No grant, no problem. Biology on a budget: Total cost of parts 71.99