IGEM:Peking/2007/Count-Conjugation-Procedure

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>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.
• 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.

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.

Hop count experiement

This is our goal.