IGEM:Peking/2007/Count-Conjugation-Procedure: Difference between revisions
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=Make more independent riboregulators= | ==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''' [[IGEM:Peking/2007/Count-Procedure-Conjugation | '''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== | |||
* We choose to use <bbpart>pSB1A3</bbpart> and <bbpart>BBa_R0010</bbpart>(Plac) to express the taRNA, 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>pSB1A3</bbpart> and <bbpart>BBa_R0010</bbpart>(Plac) to express the taRNA, 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 ~50bp long primers, and one cycle PCR can make it to a dsDNA with some BioBrick cloning sites flanked. | * crRNA is synthesized as two partially overlap ~50bp long primers, and one cycle PCR can make it to a dsDNA with some BioBrick cloning sites flanked. | ||
Line 5: | Line 16: | ||
* 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''']]. | ||
==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''' [[IGEM:Peking/2007/Count-Procedure-tandem-oriT | '''procedure''']]. | |||
==Put the relaxase under the crRNA control== | |||
==Assemble the tandem oriT together== | |||
==Hop count experiement== | |||
This is our goal. |
Revision as of 01:41, 9 July 2007
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
- We choose to use <bbpart>pSB1A3</bbpart> and <bbpart>BBa_R0010</bbpart>(Plac) to express the taRNA, 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 ~50bp long primers, and one cycle PCR can make it to a dsDNA with some BioBrick cloning sites flanked.
- crRNA should be cut by NotI at 5' and SpeI at 3' and cloned into pSB1A3 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.
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.