IGEM:IMPERIAL/2006/project/Oscillator/project browser/Test Killing Predator Construct/Implementation

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Current revision (10:06, 30 October 2006) (view source)
 
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==Ligation Strategy==
==Ligation Strategy==
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Step 1:  Construct <bbpart>J37023</bbpart> from Part <bbpart>C0060</bbpart> and a FLAG immunotag by PCR (sequence from Dr. Mann)
 
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Step 2:  Construct <bbpart>J37024</bbpart> by adding a terminator (<bbpart>B0015</bbpart> to J37023
 
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Step 3:  Construct <bbpart>J37022</bbpart> by adding the LacI promoter (<bbpart>R0010</bbpart>) and the RBS <bbpart>B0034</bbpart>, which is contained within the part <bbpart>J04500</bbpart>.
 
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People Involved with Ligations:
 
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*[[User:Johnsy|John Sy]]
 
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*???
 
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==<bbpart>J37022</bbpart> (Final AiiA test construct)==
 
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*<bbpart>J04500</bbpart> (16P) + <bbpart>J37024</bbpart>
 
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*J37024 (insert) cut with XbaI & PstI, expected size = 911 bp (on pSB1AK3 = 3189 bp)
 
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*J04500 (16P) (vector) cut with SpeI & PstI, expected size = 220 bp + 3189 (pSB1AK3) = 3409 bp
 
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*Miniprep sizes expected: 1131 bp & 3189 bp (pSB1AK3)
 
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==<bbpart>J37023</bbpart> (for predator cell/test construct)==
 
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*AiiA with Immunotag
 
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*Done by PCR
 
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*Status:  Complete (awaiting sequencing results)
 
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*Expected Part Length = 816 bp
 
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*Expected Vector Length = Unknown (Hanging T tail vector)
 
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==<bbpart>J37024</bbpart> (for AiiA predator cell/test construct)==
 
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*<bbpart>J37023</bbpart> + <bbpart>B0015</bbpart>
 
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**J37023 (insert) cut with EcoRI & SpeI, expected size = 816 bp
 
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**B0014 (1G) (vector) cut with EcoRI & XbaI, expected size = 95 bp + 3189 bp (pSB1AK3) = 3284 bp
 
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*Miniprep sizes expected:  911 bp & 3189 bp (pSB1AK3)
 
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<u><font size="2">'''Ligating the AiiA Testing Construct:'''</font></u>
<u><font size="2">'''Ligating the AiiA Testing Construct:'''</font></u>
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==WetLab work entries==
==WetLab work entries==
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*The AiiA gene available from the registry seemed non-functional. We sourced new AiiA to which we then PCR'd LVA-tag and immunotag. See [http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser/Test_Killing_Predator_Construct/Design design section for this part] for more information]
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*Parts J37024 and J37022 were not built due to time constraints.

Current revision

Super Parts Predator Construct
Actual Part AiiA Testing Construct <bbpart>J37022</bbpart>Image:J37022a.png
Sub Parts <bbpart>R0010</bbpart> <bbpart>B0034</bbpart> <bbpart>J37023</bbpart> <bbpart>B0015</bbpart>


Ligation Strategy

Ligating the AiiA Testing Construct:

For Stage 1 Stage 2 Stage 3
<bbpart>J37023</bbpart>
(PCR'd)
Image:J37023 logo.png
<bbpart>J37024</bbpart>=
(<bbpart>J37023</bbpart>+<bbpart>B0014</bbpart>)
Image:J37023 logo.pngImage:B0014 logo.png
<bbpart>J37022</bbpart>=
(<bbpart>J04500</bbpart>+<bbpart>J37024</bbpart>)
Image:J37022 logo.png


  • More information on ligations can be found on the Ligation Page.

Quality control

Part Logo Built Sequenced Primers Used for Sequencing
<bbpart>J37022</bbpart>
Image:J37022 logo.png No N/A N/A
<bbpart>J37023</bbpart>
Image:J37023 logo.png Yes Yes
Note: This part was sequenced as a part of J37025
Primers used
<bbpart>J37024</bbpart>
Image:J37024 logo.png No N/A N/A

WetLab work entries

  • The AiiA gene available from the registry seemed non-functional. We sourced new AiiA to which we then PCR'd LVA-tag and immunotag. See design section for this part for more information]
  • Parts J37024 and J37022 were not built due to time constraints.
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