Building Base Plasmids
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Base Plasmid Version 1
Our original base plasmids contain a Kanamycin resistance cassette (p1003) bracketed by BsaI or BfuAI or BbsI cut sites. If cut with either BsaI or BfuAI or BbsI, the Kanamycin cassette is release with sticky ends characteristic of an A or a B BioByte.
- May 10, 2010: PCRed p1003 (Kanamycin cassette)with primers PrA_p1003+ and PrB'_p1003
- May 10, 2010 PCRed p1003 (Kanamycin cassette)with primers PrB_p1003+ and PrA'_p1003
- May 27, 2010 Digested pSB1C3 and our PCR products of p1003 (Kanamycin cassette)with Not1
- May 27, 2010 Ligated PCR products of p1003 (Kanamycin cassette) and pSB1C3
- May 27, 2010 Transformed Ligation.
- May 28, 2010 Success! We got colonies that grew on Kan/Chlor plates
- May 30, 2010-June 9,2010 Miniprep of colonies, Digested with EcoRI and XbaI to determine orientation of Kanamycin Cassette until found plasmids in which the BioBrick prefix and suffix remained intact
- June 10, 2010 PCRed p1003 (Kanamycin cassette)with primers PrA.Bbs_p1003+ and PrB'Bbs_p1003
- June 10, 2010 PCRed p1003 (Kanamycin cassette)with primers PrB.Bbs_p1003+ and PrA'Bbs_p1003
- June 14, 2010 Digested PCR product and pSB1C3 with NotI, ligated
- June 16, 2010 Transformed from ligation
- June 17, 2010 Success! We did manage to get colonies that grew on Kan/Chlor plates
- June 18-21, 2010 Miniprep of colonies, Digested with EcoRI and XbaI to determine orientation of Kanamycin Cassette until found plasmids in which the BioBrick prefix and suffix remained intact
- June 15, 2010 PCRed p1003 (Kanamycin cassette)with primers PrA.Bfu_p1003+ and PrB'Bfu_p1003
- June 15, 2010 PCRed p1003 (Kanamycin cassette)with primers PrB.Bfu_p1003+ and PrA'Bfu_p1003
- June 22,2010 Digested both kanamycin fragments and pSB1C3 with NotI. Ligated pSB1C3 to each of the kanamycin fragments. Transformed the ligations into cells and plated on kanamycin and chloramphenicol plates for selection.
- June 23,2010 Got colonies on plates from the transformations. Set up overnight liquid cultures of 8 white colonies from each ligation. (red colonies are psb1C3)
- June 24,2010 Minipreped the liquids set up the night before. Digested each miniprep with XbaI and PstI. Also digested each miniprep with just Pst.
- June 25, 2010 Ran a gel of yesterday’s digests. For the plasmid to be correct it needed a 3kb for the single digest and a 2kb and 1kb band for the double digests. 1 of the AB kanamycin part minipreps was correct and three of BA kanamycin part minipreps.
- June 28 2010 The correct minipreps were nanodropped and the two with the highest concentrations were kept.
Base Plasmid Version 1.2
We had thought to make a base plasmid v1.2 in which instead of Kanamycin resistance we would have the CcdB gene. The CcdB gene is a gene that causes most E.coli to die. We would amplify the Base plasmid in a strain that could survive with CcdB. When we wanted to amplify our parts, we would use the base plasmid and insert our part where CcdB was, and transform into a strain that could not survive with CcdB. In this manner we would have a positive selection for plasmids containing our parts, regardless of the part’s other characteristics. We abandoned this project because we could not seem to get a plasmid that stably kept the CcdB gene.
Base Plasmid Version 1.3
We had also thought to make a base plasmid v.1.3 in which instead of Kanamycin resistance we would have Blue Fluorescent Protein. This selection would work exactly the same as our current Base plasmid v.2.0, but the color was not strong enough.
Base Plasmid Version 2
Our second base plasmids contain a RFP coding device cassette (J044450) bracketed by BsaI or BfuAI or BbsI cut sites. If cut with either BsaI or BfuAI or BbsI, the J04450 cassette is release with sticky ends characteristic of an A or a B BioByte. It was created by PCRing the RFP coding device with flanking restriction sites that left characteristic A or B BioByte overhangs. It was then put into the Base Plasmids v.1 using the BioByte overhangs.
- July 19,2010 PCRed J04450 (RFP coding device cassette) with primers rfp.A.Bfu+ and rfp.B’.Bfu-
- July 21, 2010 PCRed J04450 (RFP coding device cassette) with primers rfp.B.Bfu+ and rfp.A’.Bfu-
- July 21, 2010 Digested J04450 PCR products with BfuAI
- July 22, 2010 Digested BsaI, BbsI, and BfuAI Base plasmids v.1 with respective BsaI, BbsI and BfuAI respectively
- July 22, 2010 Ligated J04450 PCR product into BsaI, BbsI and Bfu AI Base plasmids v.1
- July 25, 2010 Transformed DH5α with Ligation, plated on Chlor plates
- July 26, 2010 Success! We did manage to get pink colonies on all plates.
- July 27-August 7 2010: Minipreps of colonies and Digests with EcoRI and XbaI and BsaI, BbsI and BfuAI to confirm orientation and identity of plamsids.
Selecting Colonies with our Plasmid
This tested whether when using Base Plasmids V.2 we could selectively identify plasmids that had the part of interest in it.
- July 27,2010 PCR mWasabi ORF with primers mwfp.A.Bbs+ and mwfp.B.Bbs-
- July 29,2010 Digest mWasabi ORF with BbsI
- July 29,2010 Digest BfuAI Base Plasmid V.2 with BfuAI
- July 30, 2010 Ligated mWasabi ORF into digested BfuAI Base Plasmid V.2
- July 30,2010 Transformed Ligation
- July 31, 2010 Success the ligation had both white and pink colonies! (white colonies should contain mWasabi ORF)
- Aug 5, 2010 Digest mWasabi plasmid with BfuAI, the expected band lengths were observed when ran on a gel. Considered a success.
Base Plasmid Version 2.1
The purpose of this plasmid was to test and isolate different types of ori bytes for our kit (low copy origins, different high copy oris, etc). We digested base plasmid Version 2 with BsaI and ligated it with the ori byte. We then removed the pSB1C3's ori with PstI and re-ligated to circularize the plasmid. When it was transformed, the desired colonies with our oris were white because the ori we tested replaced the original RFP byte.
- Sept 9, 2010: PCR pMBori and RFP + ChlorR Backbone.
- Sept 10, 2010: Digested pMBori and RFP + ChlorR Backbone with PstI.
- Sept 13, 2010: Ligated pMBori with RFP + ChlorR Backbone.
- Sept 14, 2010: Transformed pMBori with RFP + ChlorR Backbone.
- Sept 16, 2010: Miniprepped pMBori + RFP + ChlorR.
- Sept 21, 2010: Digest with PstI to check if pMBori can be cut out of the plasmid.