IGEM:IMPERIAL/2009/Assays Protocols/Cloning strategy M2: Difference between revisions

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[[Image:cloningstrategym2.jpg]]
=Legend=
 
 
*'''[[User:James Chappell|James Chappell]] 09:18, 29 July 2009 (EDT)''':General feedback - Have a look at last years cloning strategy, it can really help to plan wet lab [http://openwetware.org/wiki/Image:Imperial_2008_Critical_Pathway.png CLICK HERE TO SEE PNG]. Also would be good to put names on biobrick pictures.
 
1. Ligate cI responsive promoter e.g. Bba_R0051 (P3) to RcsB (G4)
 
2. Test for colonic acid production
*'''[[User:James Chappell|James Chappell]] 09:16, 29 July 2009 (EDT)''':Normally for testing we use well characterised inducible promoters, not sure the cI responsive promoter is the best opition for this. how about using a lac promoter for testing of RscB.
 
3. Remove terminators from the P3+RcsB construct
*'''[[User:James Chappell|James Chappell]] 09:16, 29 July 2009 (EDT)''':As we said yesterday once you have built you biobrick devices you cannot delete any of the parts it contains only add to them either in front or behind.


4. Ligate B3023 construct. Test for increased colonic acid production
[[Image:m2legend.jpg]]
*'''[[User:James Chappell|James Chappell]] 09:16, 29 July 2009 (EDT)''':Whats B3023? wouldnt you want to decouple its affect on colonic acid production from RscB?


5. Remove terminators from the P3+RcsB+B3023 construct
=Test for colanic acid=


6. Ligate Waal ligase and perform the colonic acid assay
*'''[[User:James Chappell|James Chappell]] 15:14, 29 July 2009 (EDT)''':'''Overall Feedback''' be careful about copying last years cloning strategy too much, we also do not need the 5' and 3' biobricks. Also please say what G2, G3 etc is some where.  




=Test for colonic acid=
Legend: ABR = Antibiotic resistance gene. We don't actually need it!!!
*'''[[User:James Chappell|James Chappell]] 15:14, 29 July 2009 (EDT)''':be careful about copying last years cloning strategy too much, we also do not need the 5' and 3' biobricks. Also please say what G2, G3 etc is some where.
<u>Phase 1: Testing of promoters</u>
<u>Phase 1: Testing of promoters</u>


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2. Testing of P2: inhibition of P2 by G2
2. Testing of P2: inhibition of P2 by G2
[[Image:testp2a.jpg]]


[[Image:testp2.jpg]]
[[Image:testp2.jpg]]


3. Testing of P3: inhibition of P3 by G3
3. Testing of P3: inhibition of P3 by G3
[[Image:testp3a.jpg]]


[[Image:testp3.jpg]]
[[Image:testp3.jpg]]
Line 40: Line 28:
<u>Phase 2: Testing of genes</u>  
<u>Phase 2: Testing of genes</u>  


1. Testing to see if RcsB works:
1. Characterisating RcsB:


Colonic acid assay will be performed. Production of colonic acid is a proof of a positive test.
Colanic acid assay will be performed. Production of colanic acid is a proof of a positive test.


[[Image:m2genet1.jpg]]
[[Image:m2genet1.jpg]]
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[[Image:m2genet1m.jpg]]
[[Image:m2genet1m.jpg]]


2. Testing to see if the RcsB+B3023 works:
2. Characterising B3023:
 
Perform colanic acid assay to determine if colanic acid is produced. A positive test would show the production of colanic acid.
 
[[Image:m2genet1b.jpg]]
 
3. Characterising Waal ligase:
 
Perform colanic acid assay to determine if colanic acid is produced. A positive test would show the production of colanic acid that remains after washing with distilled water.
 
[[Image:m2genet1c.jpg]]
 
4. Characterising the construct RcsB+B3023:


Colonic acid assay will be performed. Increased production of colonic acid is a proof of a positive test.
B3023 is a transcription factor that increases the amount of colanic acid that is produced.
 
Colanic acid assay will be performed. Increased production of colanic acid is a proof of a positive test.


[[Image:m2genet2.jpg]]
[[Image:m2genet2.jpg]]


3. Testing to see if RcsB+B3023+Waal ligase works.
5. CharacterisationR of the construct RcsB+B3023+Waal ligase.


A different form of colonic acid will be produced. Try washing colonic acid off. Colonic acid still remaining is a proof of a positive test.
A different form of colanic acid will be produced. Try washing colanic acid off. Colanic acid still remaining is a proof of a positive test.


[[Image:m2genet3.JPG]]
[[Image:m2genet3.JPG]]
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= Test for trehalose production =
= Test for trehalose production =


1. Ligate P3 to OtsA.
1. Test for trehalose production. A positive test means trehalose is produced.


2. Test for trehalose production.
[[Image:m2genet4.jpg]]


3. Remove terminators from P3+OtsA construct
2. Test for trehalose production. A positive test means trehalose is produced.


4. Ligate OtsB to P3+OtsA construct
[[Image:m2genet4a.jpg]]


5. Test for enhanced trehalose production
3. Test for enhanced trehalose production.


[[Image:m2genet5.jpg]]


=Test for both trehalose and colonic acid production=
=Putting all together=


1. Ligate +RcsB+B3023+Waal Ligase construct with terminators, and P3+OtsA+OtsB construct with terminators.
1. Ligate +RcsB+B3023+Waal Ligase construct with terminators, and P3+OtsA+OtsB construct with terminators.


2. Test for both colonic acid and trehalose production
2. Test for both colanic acid and trehalose production
 
[[Image:m2genet6.jpg]]
 
=Draft 1=
 
[[Image:cloningstrategym2.jpg]]
 
 
*'''[[User:James Chappell|James Chappell]] 09:18, 29 July 2009 (EDT)''':General feedback - Have a look at last years cloning strategy, it can really help to plan wet lab [http://openwetware.org/wiki/Image:Imperial_2008_Critical_Pathway.png CLICK HERE TO SEE PNG]. Also would be good to put names on biobrick pictures.
 
1. Ligate cI responsive promoter e.g. Bba_R0051 (P3) to RcsB (G4)
 
2. Test for colanic acid production
*'''[[User:James Chappell|James Chappell]] 09:16, 29 July 2009 (EDT)''':Normally for testing we use well characterised inducible promoters, not sure the cI responsive promoter is the best opition for this. how about using a lac promoter for testing of RscB.
 
3. Remove terminators from the P3+RcsB construct
*'''[[User:James Chappell|James Chappell]] 09:16, 29 July 2009 (EDT)''':As we said yesterday once you have built you biobrick devices you cannot delete any of the parts it contains only add to them either in front or behind.
 
4. Ligate B3023 construct. Test for increased colanic acid production
*'''[[User:James Chappell|James Chappell]] 09:16, 29 July 2009 (EDT)''':Whats B3023? wouldnt you want to decouple its affect on colanic acid production from RscB?
 
5. Remove terminators from the P3+RcsB+B3023 construct
 
6. Ligate Waal ligase and perform the colanic acid assay

Latest revision as of 02:41, 31 July 2009

Legend

Test for colanic acid

  • James Chappell 15:14, 29 July 2009 (EDT):Overall Feedback be careful about copying last years cloning strategy too much, we also do not need the 5' and 3' biobricks. Also please say what G2, G3 etc is some where.


Phase 1: Testing of promoters

1. Testing of P1:how varying arabinose causes variation in GFP

2. Testing of P2: inhibition of P2 by G2

3. Testing of P3: inhibition of P3 by G3

Phase 2: Testing of genes

1. Characterisating RcsB:

Colanic acid assay will be performed. Production of colanic acid is a proof of a positive test.

Perhaps we could do this to simplify matters???

2. Characterising B3023:

Perform colanic acid assay to determine if colanic acid is produced. A positive test would show the production of colanic acid.

3. Characterising Waal ligase:

Perform colanic acid assay to determine if colanic acid is produced. A positive test would show the production of colanic acid that remains after washing with distilled water.

4. Characterising the construct RcsB+B3023:

B3023 is a transcription factor that increases the amount of colanic acid that is produced.

Colanic acid assay will be performed. Increased production of colanic acid is a proof of a positive test.

5. CharacterisationR of the construct RcsB+B3023+Waal ligase.

A different form of colanic acid will be produced. Try washing colanic acid off. Colanic acid still remaining is a proof of a positive test.

Test for trehalose production

1. Test for trehalose production. A positive test means trehalose is produced.

2. Test for trehalose production. A positive test means trehalose is produced.

3. Test for enhanced trehalose production.

Putting all together

1. Ligate +RcsB+B3023+Waal Ligase construct with terminators, and P3+OtsA+OtsB construct with terminators.

2. Test for both colanic acid and trehalose production

Draft 1


  • James Chappell 09:18, 29 July 2009 (EDT):General feedback - Have a look at last years cloning strategy, it can really help to plan wet lab CLICK HERE TO SEE PNG. Also would be good to put names on biobrick pictures.

1. Ligate cI responsive promoter e.g. Bba_R0051 (P3) to RcsB (G4)

2. Test for colanic acid production

  • James Chappell 09:16, 29 July 2009 (EDT):Normally for testing we use well characterised inducible promoters, not sure the cI responsive promoter is the best opition for this. how about using a lac promoter for testing of RscB.

3. Remove terminators from the P3+RcsB construct

  • James Chappell 09:16, 29 July 2009 (EDT):As we said yesterday once you have built you biobrick devices you cannot delete any of the parts it contains only add to them either in front or behind.

4. Ligate B3023 construct. Test for increased colanic acid production

  • James Chappell 09:16, 29 July 2009 (EDT):Whats B3023? wouldnt you want to decouple its affect on colanic acid production from RscB?

5. Remove terminators from the P3+RcsB+B3023 construct

6. Ligate Waal ligase and perform the colanic acid assay

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