[iGEM Workshop] Coming up with a Project

Coming up with a project

Teach the Teachers Workshop2010

Sunday, May 23, 2010

Talk to people


Previous iGEM projectsigem.org


New organisms


New parts and tools for future teams

Most commonly used parts: B0015 - a terminator

F2620 - an inducible promoterB0034 - a RBS

R0011 - lac promoterPlasmid backbones


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*


Let the students choose


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.


Describe your project on your team wiki




Measurement• Only some parts in the Registry have

characterization data

• It can be hard to compare the measurements we do have

• Registry tour: BBa_F2620 and Anderson families

• Controls

• Where to put the data: on the Registry


http://partsregistry.org/Measurement




http://partsregistry.org/Part:BBa_F2620

http://partsregistry.org/Part:BBa_F2620

http://partsregistry.org/Promoters/Catalog/Constitutive





Standard assembly



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


A

B

C

D

E

F

G

H

AB

CD

EF

GH

ABCD

EFGH


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!


Get the enzymes cheaper





Plasmid backbones




Education

[iGEM Workshop] Coming up with a Project