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
