IGEM:IMPERIAL/2009/Assays Protocols/Testing ConstructsOLD: Difference between revisions
Royah Vaezi (talk | contribs) |
Royah Vaezi (talk | contribs) |
||
| Line 169: | Line 169: | ||
[[Image:REligations1.png |500px]] | [[Image:REligations1.png |500px]] | ||
<br> | <br> | ||
We will test using a non-leaky pBAD promoter in order to utilise the GFP reporter cell death assay (see [http://openwetware.org/wiki/IGEM:IMPERIAL/2009/Assays_Protocols/M3_Assays here]).<br> | |||
An alternative to the GFP assay is to measure [http://openwetware.org/wiki/IGEM:IMPERIAL/2009/Assays_Protocols/M3_Assays colony forming units].<br> | |||
===DpnI=== | ===DpnI=== | ||
Revision as of 16:40, 3 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)
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):
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
Module 3
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
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
