IGEM:IMPERIAL/2009/Assays Protocols/Testing ConstructsOLD

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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


Assay Input (Variable PoPs) Protocol (Reporter Quantification) Biobricks Required
PAH AHL concentration Indirect spectrophotometric detection of L-tyrosine F2620, RBS, PAH, TT
Cellulase AHL concentration Indirect spectrophotometric detection of glucose F2620, RBS, Cellulase, TT
Colanic acid AHL concentration Volumetric measurement of packed cell volume (PCV) F2620, RBS, RcsB, TT

F2620, RBS, RcsB, RBS, Waal, TT

F2620, RBS, RcsB, RBS, B3023, TT

F2620, RBS, RcsB, RBS, B3023, RBS, Waal, TT

OtsA AHL concentration Indirect colorimetric measurement of trehalose-6-phosphate F2620, RBS, OtsA, TT
OtsB AHL concentration Measurable by the indirect spectrophotometric detection of trehalose F2620, RBS, OtsB, TT
Trehalose Induction (dependent on promoter) PrMeasurable by the indirect spectrophotometric detection of trehalose Promoter, RBS, OtsA, RBS, OtsB, TT
CI Repressor Temperature (CI conformation) Measure POPs output from λCI promoter by fluorescence detection of GFP expression. J23114, RBS, CI, TT, λCI, E0240
Restriction Enzyme Arabinose concentration Cell death measurable by colony count assay BBa_I0500, RBS, DpnII, TT

BBa_I0500, RBS, Taq1, TT BBa_I0500, RBS, DpnII, RBS, Taq1, TT

BBa_I0500, RBS, DpnII, RBS, Taq1, TT, BBa_J23103,RBS,DAM,TT (in DAM –ve strain)



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:

IMPPromoter.JPG


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.


Module-one.JPG


PAH Assay:

Pah.JPG

Input: HSL (POPs)

Output: PAH (Tyrosine)

Parts Required: BBa_F2620, RBS, PAH, TT



Cellulase Assay:

Cellulase.JPG

Input: HSL (POPs)

Output: Cellulase (Glucose)

Parts Required: BBa_F2620, RBS, Cellulase, TT

Module 2

RcsB

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

B30237

II09 B3203 ClonStrat.png


WaaL

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

OtsB

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

Items.jpg

Total number of ligations: 7

cI repressor

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


Plambda

Plambda.jpg
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

CHANGE IMAGE - NO LONGER USING pBAD!

In total there will be 12 ligation steps.

REligations1.png

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


M3dpnITC1.png
Input: HSL (F2620)
Output: Change in fluorescence

TaqI


M3taqITC1.png
Input: HSL (F2620)
Output: Change in fluorescence

DpnI & TaqI


M3DplusTTC1.png
Input: HSL (F2620)
Output: Change in fluorescence