IGEM:Peking/2007/Count-Conjugation-Procedure

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(Make more independent riboregulators)
Current revision (05:26, 31 August 2007) (view source)
(Make more independent riboregulators)
 
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==Make more independent riboregulators==
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==Make more independent riboregulators(Lock & Key section)==
* We choose to use <bbpart>pSB1A2</bbpart> and <bbpart>BBa_R0010</bbpart>(Plac) to express the taRNA(<bbpart>J23066</bbpart>), and <bbpart>pSB3K3</bbpart> and <bbpart>BBa_R0040</bbpart> to express <bbpart>BBa_E0040</bbpart>, whose translation is under the control of crRNA.
* We choose to use <bbpart>pSB1A2</bbpart> and <bbpart>BBa_R0010</bbpart>(Plac) to express the taRNA(<bbpart>J23066</bbpart>), and <bbpart>pSB3K3</bbpart> and <bbpart>BBa_R0040</bbpart> to express <bbpart>BBa_E0040</bbpart>, whose translation is under the control of crRNA.
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* crRNA is synthesized as two partially overlap ~70bp long primers, and 5 cycles PCR can make it to a dsDNA with some BioBrick cloning sites flanked.
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* crRNA is synthesized as two partially overlap ~70bp long primers, and 5 cycles PCR can make it to a dsDNA with some BioBrick cloning sites flanked. ('''"2 primer strategy"'''--sunnyboy 05:26, 31 August 2007 (EDT))
* crRNA should be cut by XbaI at 5' and PstI at 3' and cloned into BBa_R0040 to get the full length BioBrick prefix and suffix.--[[User:YangYifan|YangYifan]] 04:19, 9 July 2007 (EDT)
* crRNA should be cut by XbaI at 5' and PstI at 3' and cloned into BBa_R0040 to get the full length BioBrick prefix and suffix.--[[User:YangYifan|YangYifan]] 04:19, 9 July 2007 (EDT)
* First, we will try out the UC Berkley riboregulators <bbpart>BBa_J23078</bbpart> and <bbpart>BBa_J23066</bbpart> first.
* First, we will try out the UC Berkley riboregulators <bbpart>BBa_J23078</bbpart> and <bbpart>BBa_J23066</bbpart> first.
* This is '''the exact experimental''' [[IGEM:Peking/2007/Count-Procedure-Riboregulator | '''procedure''']].
* This is '''the exact experimental''' [[IGEM:Peking/2007/Count-Procedure-Riboregulator | '''procedure''']].
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===Supplement by Yu Tao===
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*For conveniency, we abbreviate this section as '''''"Lock & Key"'''''. (I learn this from UC Berkeley team.)
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*As is mentioned, the crRNA is the ''Lock'', and we use the '''"2 primer" strategy''' (as indicated by Yang Yifan) to synthesize the ''Lock''.
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*As for ''Key'' which is not mentioned above, we first tried '''"3 primer" strategy'''(similar to '''"2 primer" strategy''', but we divided ''Key'' into 3 overlap parts so that we were able to synthesize the shorter parts at a relatively lower price), but failed.  Now we'd better try the '''"2 primer" strategy'''.
==Construct the tandem oriT region==
==Construct the tandem oriT region==

Current revision

Contents

Conjugation Test

  • We want to do a conjugation efficiency test with wild type F to verify the protocol.
  • And then test the conjugation efficiency with all the plasmids at hand.
  • This is the exact experimental procedure

Helper plasmids construction

  • To construct helper plasmids, we need to knock out the oriT.
  • To put conjugation under control, we need to knock out the relaxase.
  • The Knockout method: (Anting please explain your method here)

Make more independent riboregulators(Lock & Key section)

  • We choose to use <bbpart>pSB1A2</bbpart> and <bbpart>BBa_R0010</bbpart>(Plac) to express the taRNA(<bbpart>J23066</bbpart>), and <bbpart>pSB3K3</bbpart> and <bbpart>BBa_R0040</bbpart> to express <bbpart>BBa_E0040</bbpart>, whose translation is under the control of crRNA.
  • crRNA is synthesized as two partially overlap ~70bp long primers, and 5 cycles PCR can make it to a dsDNA with some BioBrick cloning sites flanked. ("2 primer strategy"--sunnyboy 05:26, 31 August 2007 (EDT))
  • crRNA should be cut by XbaI at 5' and PstI at 3' and cloned into BBa_R0040 to get the full length BioBrick prefix and suffix.--YangYifan 04:19, 9 July 2007 (EDT)
  • First, we will try out the UC Berkley riboregulators <bbpart>BBa_J23078</bbpart> and <bbpart>BBa_J23066</bbpart> first.
  • This is the exact experimental procedure.

Supplement by Yu Tao

  • For conveniency, we abbreviate this section as "Lock & Key". (I learn this from UC Berkeley team.)
  • As is mentioned, the crRNA is the Lock, and we use the "2 primer" strategy (as indicated by Yang Yifan) to synthesize the Lock.
  • As for Key which is not mentioned above, we first tried "3 primer" strategy(similar to "2 primer" strategy, but we divided Key into 3 overlap parts so that we were able to synthesize the shorter parts at a relatively lower price), but failed. Now we'd better try the "2 primer" strategy.

Construct the tandem oriT region

  • For each conjugation plasmids, we want to construct oriT-taRNA-dbTerm-oriT.
  • And Plac to the 5' and GFP to the 3' to test conjugation and deletion.
  • Use pSB1A3 as backbone.
  • This is the exact experimental procedure.

Put the relaxase under the crRNA control

Assemble the tandem oriT together

Hop count experiement

This is our goal.

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