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)

Running total = 10-11

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

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
Parts: BBa_F2620, RBS, OtsA, TT
Assay Method

• OtsB