Difference between revisions of "IGEM:IMPERIAL/2009/Assays Protocols/Testing ConstructsOLD"

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m (Ligation Summary)
m (Ligation Summary)
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5) Restriction Enzymes (12 ligations)
5) Restriction Enzymes (12 ligations)
6)Encapsulation (16 ligations)
6)Encapsulation (15 ligations)
<b>Running total</b> = 29-30
<b>Running total</b> = 29-30

Revision as of 01:28, 4 August 2009

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 = 29-30

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): II09 E0240.png

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


II09 RcsB ClonStrat.png

  • 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.

II09 RcsB Assay.png

Inputs : HSL (POPs)
Outputs : Colanic Acid Production
Parts: BBa_F2620, RBS, RcsB, TT


II09 B3203 ClonStrat.png


II09 WaaL ClonStrat.png

II09 WaaL Assay.png

Inputs : HSL (POPs)
Outputs : WaaL Ligase
Parts: BBa_F2620, RBS, WaaL, TT

OtsA : Trehalose–6-Phosphate Synthase Assay

II09 OtsA ClonStrat.png

II09 OtsA Assay.png

Inputs : HSL (POPs)
Outputs : Trehalose 6-phosphate Synthase
Parts: BBa_F2620, RBS, OtsA, TT
Assay Method


II09 OtsB ClonStrat.png

II09 OtsB Assay.png

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


Input: HSL (F2620)
Output: Fluorescence

cI and Plambda

CI and Plambda.jpg
Input: HSL (F2620)
Output: (visual) cI physically binding to Plambda

cI and Plambda(GFP reporter)

CI Plambda GFP.jpg

Input: HSL (F2620)
Output: Change in fluorescence

2) Restriction Enzymes

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.


Input: HSL (F2620)
Output: Change in fluorescence


Input: HSL (F2620)
Output: Change in fluorescence

DpnI & TaqI

Input: HSL (F2620)
Output: Change in fluorescence