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 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=

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

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