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