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

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==Ligation Process==
==Ligation Strategy==


Step 1: Construct J37023 from Part <bbpart>C0060</bbpart> and an immunotag by PCR
<u><font size="2">'''Ligating the AiiA Testing Construct:'''</font></u>
__NOTOC__
{| border="1" cellpadding="0" width="100%"
|-
!style="background:#E6B3E0" height="25pt" width="80pt" |For !! style="background:#E6B3E0" |Stage 1 !! style="background:#E6B3E0" |Stage 2 !! style="background:#E6B3E0" |Stage 3


Step 2: Construct J37024 by adding a terminator (<bbpart>B0015</bbpart> to J37023
|-
!style="background:#D9F2ED"|<center><bbpart>J37023</bbpart> <br> (PCR'd)</center> !!style="background:#CCEBAD" | <center>[[Image:J37023 logo.png]]</center>
|-
!style="background:#D9F2ED"|<center><bbpart>J37024</bbpart>=<br>(<bbpart>J37023</bbpart>+<bbpart>B0014</bbpart>)</center>|| !!style="background:#CCEBAD" | <center>[[Image:J37023 logo.png]][[Image:Plus sign.png|20px]][[Image:B0014 logo.png]]</center>
|-
!style="background:#D9F2ED"|<center><bbpart>J37022</bbpart>=<br>(<bbpart>J04500</bbpart>+<bbpart>J37024</bbpart>)</center>|| || !!style="background:#CCEBAD" | <center>[[Image:J37022 logo.png]]</center>
|}


Step 3:  Construct J37022 by adding the LacI promoter (<bbpart>R0010</bbpart>) and the RBS <bbpart>B0034</bbpart>, which is contained within the part <bbpart>J04500</bbpart>. 


==<bbpart>J37022</bbpart> (Final AiiA test construct)==
*More information on ligations can be found on the [http://openwetware.org/wiki/IGEM:IMPERIAL/2006/Ligation_Page Ligation Page].
*<bbpart>J04500</bbpart> (16P) + <bbpart>J37024</bbpart>
*J37024 (insert) cut with XbaI & PstI, expected size = 911 bp (on pSB1AK3 = 3189 bp)
*J04500 (16P) (vector) cut with SpeI & PstI, expected size = 220 bp + 3189 (pSB1AK3) = 3409 bp
*Miniprep sizes expected: 1131 bp & 3189 bp (pSB1AK3)


==<bbpart>J37023</bbpart> (for predator cell/test construct)==
==Quality control==
*AiiA with Immunotag
__NOTOC__
*Done by PCR
{| border="1" cellpadding="0" width="100%"
*Status: Complete (awaiting sequencing results)
|-
*Expected Part Length = 816 bp
!height="25pt" width="80pt"| Part !! Logo !! Built !! Sequenced !! Primers Used for Sequencing
*Expected Vector Length = Unknown (Hanging T tail vector)
|-
| <center><bbpart>J37022</bbpart></center> || [[Image:J37022 logo.png]] || No || N/A || N/A
|-
| <center><bbpart>J37023</bbpart></center> || [[Image:J37023 logo.png]] || Yes || [http://openwetware.org/images/b/b3/J37025sequencing.doc Yes] <br> Note: This part was sequenced as a part of J37025 || [http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/parts/Primers Primers used]
|-
| <center><bbpart>J37024</bbpart></center> || [[Image:J37024 logo.png]] || No || N/A || N/A
|}


==<bbpart>J37024</bbpart> (for AiiA predator cell/test construct)==
==WetLab work entries==
*<bbpart>J37023</bbpart> + <bbpart>B0015</bbpart>
 
**J37023 (insert) cut with EcoRI & SpeI, expected size = 816 bp
*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]
**B0014 (1G) (vector) cut with EcoRI & XbaI, expected size = 95 bp + 3189 bp (pSB1AK3) = 3284 bp
*Parts J37024 and J37022 were not built due to time constraints.
*Miniprep sizes expected:  911 bp & 3189 bp (pSB1AK3)

Latest revision as of 08:06, 30 October 2006

Super Parts Predator Construct
Actual Part AiiA Testing Construct <bbpart>J37022</bbpart>
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)
<bbpart>J37024</bbpart>=
(<bbpart>J37023</bbpart>+<bbpart>B0014</bbpart>)
<bbpart>J37022</bbpart>=
(<bbpart>J04500</bbpart>+<bbpart>J37024</bbpart>)


Quality control

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