IGEM:IMPERIAL/2009/Assays Protocols/Testing ConstructsOLD

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Ligation Summary

- Running total of number of ligations required for the assembly of testing constructs.

1) Promoter Testing (4-5 ligations)

2) PAH (3 ligations)

3) Cellulase (3 ligations)

4) Thermoinduction (7 ligations)

5) Restriction Enzymes (12 ligations)

6)Encapsulation (15 ligations)

Running total = 45-46

Testing Construct Summary

Determine input protocol by looking at this paper: http://www.nature.com/nbt/journal/v26/n7/extref/nbt1413-S1.pdf

Assay Input (Variable PoPs) Protocol (Reporter Quantification) Biobricks Required
PAH AHL concentration Indirect spectrophotometric detection of L-tyrosine F2620, RBS, PAH, TT
Cellulase AHL concentration Indirect spectrophotometric detection of glucose F2620, RBS, Cellulase, TT
Colanic acid AHL concentration Volumetric measurement of packed cell volume (PCV) F2620, RBS, RcsB, TT

F2620, RBS, RcsB, RBS, Waal, TT

F2620, RBS, RcsB, RBS, B3023, TT

F2620, RBS, RcsB, RBS, B3023, RBS, Waal, TT

OtsA AHL concentration Indirect colorimetric measurement of trehalose-6-phosphate F2620, RBS, OtsA, TT
OtsB AHL concentration Measurable by the indirect spectrophotometric detection of trehalose F2620, RBS, OtsB, TT
Trehalose Induction (dependent on promoter) PrMeasurable by the indirect spectrophotometric detection of trehalose Promoter, RBS, OtsA, RBS, OtsB, TT
CI Repressor Temperature (CI conformation) Measure POPs output from λCI promoter by fluorescence detection of GFP expression. J23114, RBS, CI, TT, λCI, E0240
Restriction Enzyme Arabinose concentration Cell death measurable by colony count assay BBa_I0500, RBS, DpnII, TT

BBa_I0500, RBS, Taq1, TT BBa_I0500, RBS, DpnII, RBS, Taq1, TT

BBa_I0500, RBS, DpnII, RBS, Taq1, TT, BBa_J23103,RBS,DAM,TT (in DAM –ve strain)

Promoter Testing

We aim to use a generic promoter testing system, however we are unsure of the exact promoters that we are using at this stage becuase of the timer situation.

At this present moment in time, we are estimating using 4-5 different promoters in our system. Since each will need to be characterised, 4-5 ligations will be required to faciliate the assembly of the testing constructs.

Testing Construct Assembly:


Input: POPs

Output: GFP

Parts Required: BBa_E0240, Each promoter to be characterised.

BBa_E0240 (RBS-GFP-TT): Image:II09_E0240.png

Module 1

Testing Construct Assembly

We must test both cellulase and PAH, both testing constructs can be made using a total of 6 ligation steps.


PAH Assay:


Input: HSL (POPs)

Output: PAH (Tyrosine)

Parts Required: BBa_F2620, RBS, PAH, TT

Cellulase Assay:


Input: HSL (POPs)

Output: Cellulase (Glucose)

Parts Required: BBa_F2620, RBS, Cellulase, TT

Module 2



  • James Chappell 04:26, 4 August 2009 (EDT):I would join the RBS-Gx to the T first, think about the type of modules we want to submit to the registry. Think having a module that takes pops in and gives out G4 output is a good module to submit.


Inputs : HSL (POPs)
Outputs : Colanic Acid Production
Parts: BBa_F2620, RBS, RcsB, TT






Inputs : HSL (POPs)
Outputs : WaaL Ligase
Parts: BBa_F2620, RBS, WaaL, TT

OtsA : Trehalose–6-Phosphate Synthase Assay



Inputs : HSL (POPs)
Outputs : Trehalose 6-phosphate Synthase
Parts: BBa_F2620, RBS, OtsA, TT
Assay Method




Inputs : HSL (POPs)
Outputs : Trehalose 6-phosphate Phosphatase
Parts: BBa_F2620, RBS, OtsB, TT

Module 3

1)The thermoinduction system

Total number of ligations: 7

cI repressor

Input: HSL (F2620)
Output: Amount of cI protein


Input: HSL (F2620)
Output: Fluorescence

cI and Plambda

Image:cI and Plambda.jpg
Input: HSL (F2620)
Output: (visual) cI physically binding to Plambda

cI and Plambda(GFP reporter)

Image:cI Plambda GFP.jpg

Input: HSL (F2620)
Output: Change in fluorescence

2) Restriction Enzymes


In total there will be 12 ligation steps.

We will test using a non-leaky pBAD promoter in order to utilise the GFP reporter cell death assay (see here).
An alternative to the GFP assay is to measure colony forming units.


Input: HSL (F2620)
Output: Change in fluorescence


Input: HSL (F2620)
Output: Change in fluorescence

DpnI & TaqI

Input: HSL (F2620)
Output: Change in fluorescence

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