IGEM:IMPERIAL/2009/Assays Protocols/Testing ConstructsOLD

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(2) Restriction Enzymes)
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=== Ligation Summary===
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== Ligation Summary==
- Running total of number of ligations required for the assembly of testing constructs.
- Running total of number of ligations required for the assembly of testing constructs.
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<b>Running total</b> = 29-30
<b>Running total</b> = 29-30
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=== Promoter Testing===
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== 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.
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.
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[[Image:II09_E0240.png]]
[[Image:II09_E0240.png]]
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=== Module 1===
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== Module 1==
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<B>Parts Required:</B> BBa_F2620, RBS, Cellulase, TT
<B>Parts Required:</B> BBa_F2620, RBS, Cellulase, TT
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===Module 2 ===
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==Module 2 ==
====RcsB  ====
====RcsB  ====
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Parts: BBa_F2620, RBS, OtsB, TT <br>
Parts: BBa_F2620, RBS, OtsB, TT <br>
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===Module 3 ===
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==Module 3 ==
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====1)The thermoinduction system=====
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====1)The thermoinduction system====
[[Image:items.jpg |700px]]
[[Image:items.jpg |700px]]

Revision as of 03:23, 4 August 2009

Contents

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)

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:

Image:IMPPromoter.JPG


Input: POPs

Output: GFP

Parts Required: BBa_E0240, Each promoter to be characterised.


BBa_E0240 (RBS-GFP-TT): Image: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.


Image:Module-one.JPG


PAH Assay:

Image:pah.JPG

Input: HSL (POPs)

Output: PAH (Tyrosine)

Parts Required: BBa_F2620, RBS, PAH, TT



Cellulase Assay:

Image:cellulase.JPG

Input: HSL (POPs)

Output: Cellulase (Glucose)

Parts Required: BBa_F2620, RBS, Cellulase, TT

Module 2

RcsB

Image:II09_RcsB_ClonStrat.png


Image:II09_RcsB_Assay.png


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

B30237

Image:II09_B3203_ClonStrat.png


WaaL

Image:II09_WaaL_ClonStrat.png


Image:II09_WaaL_Assay.png

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

OtsA : Trehalose–6-Phosphate Synthase Assay

Image:II09_OtsA_ClonStrat.png


Image:II09_OtsA_Assay.png

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

OtsB

Image:II09_OtsB_ClonStrat.png


Image: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

Image:cIrepressor.jpg
Input: HSL (F2620)
Output: Amount of cI protein


Plambda

Image:Plambda.jpg
Input: HSL (F2620)
Output: Fluorescence


cI and Plambda

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


cI and Plambda(GFP reporter)

Image: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.

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

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