IGEM:IMPERIAL/2009/Assays Protocols/Testing ConstructsOLD

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):

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

RcsB

• 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

B30237

WaaL

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

OtsB

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

Plambda

Input: HSL (F2620)
Output: Fluorescence

cI and Plambda

Input: HSL (F2620)
Output: (visual) cI physically binding to Plambda

cI and Plambda(GFP reporter)

Input: HSL (F2620)
Output: Change in fluorescence

2) Restriction Enzymes

CHANGE IMAGE - NO LONGER USING pBAD!

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.

DpnI

Input: HSL (F2620)
Output: Change in fluorescence

TaqI

Input: HSL (F2620)
Output: Change in fluorescence

DpnI & TaqI

Input: HSL (F2620)
Output: Change in fluorescence