5. New parts and tools
for future teams
Most commonly used parts:
B0015 - a terminator
F2620 - an inducible promoter
B0034 - a RBS
R0011 - lac promoter
Plasmid backbones
Sunday, May 23, 2010
6. BBa_F2620 BBa_F2620
3OC6HSL PoPS Receiver
Mechanism & Function Component Parts
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 R0040 B0034 C0062 B0015 R0062
used in a cell containing TetR then a second input such as PLtetO-1 RBS luxR Term. Plux,R
aTc can be used to produce a Boolean AND function.
Static Performance* Dynamic Performance*
http://parts.mit.edu/registry/index.php/Part:BBa_F2620
600 8 600 8
GFP synthesis rate (molecules cell!1 s!1)
GFP synthesis rate (molecules cell!1 s!1)
Population Mean
Colony Range 7 + 3OC6HSL 7
500 Hill Equation 500
6 6
400 400
5 5
PoPS cell!1
PoPS cell!1
300 4 300 4
Reuse and
3 3
200 200 GFP synthesis rate (Low Input)
2 GFP synthesis rate (High Input) 2
100 100 Polynomial Fit (High Input)
1 PoPS (High Input) 1
0 0 0 0
0E+00 1E!10 1E!09 1E!08 1E!07 1E!06 1E!05 1E!04 !10 0 10 20 30 40 50
[3OC6HSL] (M) Time (min)
Pmax [3OC6HSL]n Pmax: 6.6 PoPS cell-1 BBa_F2620 Response Time: <1 min
existing parts
Pout = K: 1.5E-09 M 3OC6HSL BBa_T9002 Response Time: 6±1 min
K n + [3OC6HSL]n n: 1.6 Inputs: 0 M (Low), 1E-07 M (High) 3OC6HSL
Input Compatibility* Reliability**
600 8
GFP synthesis rate (molecules cell!1 s!1)
C4HSL
C6HSL 7
500
3OC6HSL
6
C7HSL 92
400
5 74
C8HSL
gs
PoPS cell!1
56
lin
3OC8HSL
300 38 92
ub
4 1E0
Signaling Devices
GFP 1E1
Do
C10HSL 20
(arb1E2 1E3 74
gs
C12HSL 3 itrar 1E4 56
y un
lin
200
its)
ub
38
2 Low Input GFP1E0 1E1
Do
(0 M 3OC6HSL) (arb 1E2 1E3 20
100 itrar
1 y un 1E4
its)
High Input
0 0 (1E -7 M 3OC6HSL)
0E+00 1E!10 1E!09 1E!08 1E!07 1E!06 1E!05 1E!04
[AHL] (M) Genetic: >92/>56 culture doublings
Part Compatibility (qualitative) Performance: >92/>56 culture doublings
(low/high input during propagation)
Chassis: MC4100, MG1655, and DH5
Plasmids: pSB3K3 and pSB1A2
Conditions (abridged)
Devices: E0240, E0430 and E0434
Output: PoPS measured via BBa_E0240
Culture: Supplemented M9, 37ºC
Transcriptional Output Demand (low/high input) Plasmid: pSB3K3
Nucleotides: 0 / 6xNt nucleotides cell-1 s-1 Chassis: MG1655
Polymerases: 0 / 1.5E-1xNt RNAP cell-1 *Equipment: PE Victor3 multi-well fluorimeter
(Nt = downstream transcript length) **Equipment: BD FACScan cytometer
Authors: Barry Canton
Ania Labno Registry of Standard Biological Parts License: Public
Updated: March 2008 making life better, one part at a time
Sunday, May 23, 2010
8. 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
9. Describe your project
on your team wiki
Teach the Teachers Workshop
2010
Sunday, May 23, 2010
11. 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
Sunday, May 23, 2010
13. Standard assembly
Teach the Teachers Workshop
2010
Sunday, May 23, 2010
14. BioBrick standard parts
EcoRI XbaI BioBrick part SpeI PstI
Knight, 2003
Sunday, May 23, 2010
15. BioBrick standard assembly
E X BioBrick part 1 S P E X BioBrick part 2 S P
Digest with Digest with
EcoRI and SpeI XbaI and PstI
E X BioBrick part 1 S E X BioBrick part 2 S P
Ligate
E X BioBrick part 1 Mixed BioBrick part 2 S P
Sunday, May 23, 2010
16. A
AB
B
ABCD
C
CD
D
E
EF
F
EFGH
G
GH
H
Sunday, May 23, 2010
17. 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
