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

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:</B> BBa_F2620, RBS, PAH, TT

Cellulase Assay:</B>



Input:</B> HSL (POPs)

Output:</B> Cellulase (Glucose)

Parts Required:</B> BBa_F2620, RBS, Cellulase, TT

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

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

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