[iGEM Workshop] Coming up with a Project

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Coming up with a project

Teach the Teachers Workshop2010

Sunday, May 23, 2010

Talk to people

Sunday, May 23, 2010

Previous iGEM projectsigem.org

Sunday, May 23, 2010

New organisms

Sunday, May 23, 2010

New parts and tools for future teams

Most commonly used parts: B0015 - a terminator

F2620 - an inducible promoterB0034 - a RBS

R0011 - lac promoterPlasmid backbones

Sunday, May 23, 2010

Reuse and existing parts

Mechanism & Function

A transcription factor (LuxR) that is active in the presence

of a cell-cell signaling molecule (3OC6HSL) is controlled by

a regulated operator (PLtetO-1). Device input is 3OC6HSL.

Device output is PoPS from a LuxR-regulated operator. If

used in a cell containing TetR then a second input such as

aTc can be used to produce a Boolean AND function.

BBa_F26203OC6HSL PoPS Receiver

Registry of Standard Biological Parts making life better, one part at a time

License: Public

Conditions (abridged)

Output: PoPS measured via BBa_E0240

Culture: Supplemented M9, 37ºC

Plasmid: pSB3K3

Chassis: MG1655

*Equipment: PE Victor3 multi-well fluorimeter

**Equipment: BD FACScan cytometer

1E01E1

1E21E3

1E4

GFP (arbitrary units)

Doublin

gs

20

38

56

74

92

High Input (1E -7 M 3OC6HSL)

92

1E01E1

1E21E3

1E4

Doublin

gs

20

38

56

74

Low Input(0 M 3OC6HSL)

GFP (arbitrary units)

Reliability**

Genetic: >92/>56 culture doublingsPerformance: >92/>56 culture doublings

(low/high input during propagation)

Transcriptional Output Demand (low/high input)

Nucleotides: 0 / 6xNt nucleotides cell-1 s-1

Polymerases: 0 / 1.5E-1xNt RNAP cell-1

(Nt = downstream transcript length)

Chassis: MC4100, MG1655, and DH5

Plasmids: pSB3K3 and pSB1A2

Devices: E0240, E0430 and E0434

BBa_F2620 Response Time: <1 minBBa_T9002 Response Time: 6±1 minInputs: 0 M (Low), 1E-07 M (High) 3OC6HSL

Pmax: 6.6 PoPS cell-1

K: 1.5E-09 M 3OC6HSLn: 1.6

Static Performance*

Pout =Pmax [3OC6HSL]n

Kn+ [3OC6HSL]n

0E+00 1E!10 1E!09 1E!08 1E!07 1E!06 1E!05 1E!04

0

100

200

300

400

500

600

[3OC6HSL] (M)

GF

P s

yn

the

sis

ra

te (

mo

lec

ule

s c

ell!1 s!1)

Population Mean

Colony Range

Hill Equation

0

1

2

3

4

5

6

7

8

Po

PS

ce

ll!1

Part Compatibility (qualitative)

Authors: Barry Canton Ania Labno

Updated: March 2008

Sig

nalin

g D

evic

es

http://p

art

s.m

it.e

du/r

egis

try/index.p

hp/P

art

:BB

a_F

2620

BBa_F2620

R0040 B0034 C0062 B0015 R0062 PLtetO-1 RBS luxR Term. Plux,R

Component Parts

!10 0 10 20 30 40 50

0

100

200

300

400

500

600

+ 3OC6HSL

Time (min)

GF

P s

yn

thesis

rate

(m

ole

cu

les c

ell!1 s!1)

GFP synthesis rate (Low Input)

GFP synthesis rate (High Input)

Polynomial Fit (High Input)

PoPS (High Input)

0

1

2

3

4

5

6

7

8

Po

PS

cell!1

0E+00 1E!10 1E!09 1E!08 1E!07 1E!06 1E!05 1E!04

0

100

200

300

400

500

600

[AHL] (M)

GF

P s

yn

the

sis

ra

te (

mo

lec

ule

s c

ell!1 s!1)

C4HSL

C6HSL

3OC6HSL

C7HSL

C8HSL

3OC8HSL

C10

HSL

C12

HSL

0

1

2

3

4

5

6

7

8

Po

PS

ce

ll!1

Input Compatibility*

Dynamic Performance*

Sunday, May 23, 2010

Let the students choose

Sunday, May 23, 2010

Help them make smart choices

• Figure out what’s practical: How many assembly stages could the team possibly do over the course of the summer? That sets an upper limit to the size of the system.

• Design the project so that different modules can be done in parallel.

• It doesn’t have to be a brand new idea.

Sunday, May 23, 2010

Describe your project on your team wiki

Teach the Teachers Workshop2010

Sunday, May 23, 2010

Sunday, May 23, 2010

Sunday, May 23, 2010

Standard assembly

Teach the Teachers Workshop2010

Sunday, May 23, 2010

BioBrick standard parts

EcoRI XbaI SpeI PstIBioBrick part

Knight, 2003Sunday, May 23, 2010

BioBrick standard assembly

E X S PBioBrick part 1 E X S PBioBrick part 2

Digest with EcoRI and SpeI

Digest with XbaI and PstI

E X SBioBrick part 1 E X S PBioBrick part 2

E X S PBioBrick part 1 BioBrick part 2Mixed

Ligate

Sunday, May 23, 2010

A

B

C

D

E

F

G

H

AB

CD

EF

GH

ABCD

EFGH

Sunday, May 23, 2010

Why use the BioBrick standard?

• It is faster to build multi-part systems

• Assembling every two parts is the same

• You can reuse parts from the Registry

• Other people can reuse your parts

• It is required to win a prize at iGEM!

Sunday, May 23, 2010

Get the enzymes cheaper

Sunday, May 23, 2010

Sunday, May 23, 2010

Sunday, May 23, 2010

Sunday, May 23, 2010

Plasmid backbones

Teach the Teachers Workshop2010

Sunday, May 23, 2010

Sunday, May 23, 2010