TEAM ALBERTA

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Creating Parts

PCRing Antibiotic Resistance Markers

• May 25, 2010

PCRed the antibiotic resistance marker biobytes (Ampicillin, Tetracycline and Chlormaphenicol) that are analogous to the kanamycin PCR fragements.

• Ampicillin:

Primers = Pr.A.SB4A5.Apr+ and Pr.B. SB4A5.Apr -

Template = pSB4A5

• Chloramphenical:

Primers = Pr. A. SB4C5.Chr + and Pr.B.SB4A5.Chr -

Template = pSB4C5

• Tetracycline:

Primers = Pr.A.SB3T5.Tr+ and Pr.B.PSB3T5.Tr -

Template = pSB3T5

Ampillicin Resistance AB in pSB1C3

• June 3, 2010:

PCR Ampillicin Resistance (AmpR) AB BioByte.

• June 9, 2010:

Digested AmpR AB with BsaI.

• June 10, 2010:

Digested pSB1C3 Vector with BsaHF.

Ligated and transformed AmpR AB with pSB1C3 Vector.

• June 17, 2010:

Miniprepped AmpR + pSB1C3.

AB chlor and tet parts

Strategy:

Digest both AB kan PCR product and pSB1A3 with NotI, and ligate. Then, cut chlor and tet AB PCR products with BsaI and cut out the kan from the plasmid using BsaI, and ligate chlor or tet into it.

• June 30, 2010:

Digested PCR product kan AB and pSB1A3 with NotI. Ligated the kan and pSB1A3 together. Gel “29.06.10.alina 2”

• July 5, 2010:

Transformed from the ligation of kan and pSB1A3.

• July 6, 2010:

White colonies. Set up overnights.

• July 7, 2010:

Made minipreps from the overnights and digested them with XbaI and PstI to check for proper orientation. Gel “07.07.10.alina”? Digested those in correct orientation with BsaI to remove kan. Digested PCR product chlor AB and tet AB with BsaI. Ligated chlor and tet separately with the PSB1A3 backbone. Gel “07.07.10.karinaalina2”?

• July 8, 2010:

Transformed from ligations of chlor and tet AB with pSB1A3 backbone.

• July 9, 2010:

White colonies. Set up overnights.

• July 10, 2010:

Minipreps were done and were digested with EcoRI to test for size. Gel “07.10.10.alina”?

July 13, 2010:

Digested kan, tet and amp AB’s with BsaIHF. Gel “13.07.10.alina2”

• July 14, 2010:

Digested kan, chlor, tet and amp AB’s with XbaI and PstI. Gel “14.07.10.alina” **********ARE THESE Bfu ones? Probably not.

• July 20, 2010:

Glycerol stocks were made of chlor and tet AB.

• August 9, 2010:

Sequenced AB amp (tube A) (from Anh’s previous work), tet (tube B1) and chlor (tube D1). All look good.

• August 13, 2010:

Transformed from the successful, sequenced tubes.

• August 16-17, 2010:

White colonies. Made maxipreps, after inoculation from transformed plates, of AB chlor (pC.s.A.005) and amp (pC.s.A.003)

• August 18, 2010:

Digested the maxipreps with BsaI. Gel “18.08.10.alina”

Bsa BA amp and tet

August 19, 2010 Performed PCR using AB amp in pSB1C3, AB chlor in pSB1A3, and AB tet in pSB1A3 as templates to obtain BA amp, tet, chlor products. August 20, 2010 Gel “20.08.10.aina”. Digested BA amp and tet products and BA pSB1C3 backbone with BsaI. Ligated. Transformed. Gel “20.08.10.alina2” (bad gel) August 21, 2010 5 white colonies on tet, lot of colonies on amp. August 22, 2010 Set up overnights from colonies. August 23, 2010 Miniprepped BA amp and chlor. August 24, 2010 Digested BA amp and chlor with BsaI gel “24.08.10.alina” August 25, 2010 Digested BA amp and chlor with EcoRI gel “25.08.10.alina*”, XbaI and PstI “25.08.10.alina2” August 26, 2010 Sequenced BA amp. ***Resequenced on September 7th - Looks good. Transformed AB tet on [1/4 tet]. August 27, 2010 White colonies, streaked on [1/2 tet] August 28, 2010 White streaks with individual colonies. Set up overnights of AB tet. August 31, 2010 Minipreps of AB tet. Digested them with BsaI. Great gel “31.08.10 Alina” September 1, 2010 Performed PCR (like Aug 19) using AB amp in pSB1C3, AB chlor in pSB1A3, and AB tet in pSB1A3 as templates. One tube was from sequenced tet and one from the re-transformed sequenced tet. Gel “01.09.10.alina”. Digest PCR products and BA kan in pSB1C3 with BsaI. LIgated. Transformed. September 6, 2010 White colonies. Set up overnights of tet BA. September 7, 2010 Miniprepped the overnights of tet Ba. September 13, 2010 Digested tet BA with BsaI gel “13.09.10.alina*” September 15, 2010 Sequenced tet BA. Looks good.

Chlor BA

First, we have to make a Bsa BA pSB1A3 backbone by digesting pSB1A3 with XbaI and PstI and inserting Bsa BA kan. Then, the kan is cut out with BsaI and chlor BA is placed in. September 17, 2010 Digested pSB1A3 and BA kan with XbaI and PstI gel “17.09.10.alina”. Ligated. Transformed. September 18, 2010 White colonies. Set up overnights of BA kan in pSB1A3 backbone. September 20, 2010 Miniprepped overnights of BA kan in pSB1A3 backbone. September 24, 2010 Digested the BA kan in pSB1A3 backbone and BA chlor PCR product (from Sept. 1) with BsaI. Ligated. September 27, 2010 Transformed. September 29, 2010 White colonies. Set up overnights. September 30, 2010 Made minipreps of BA chlor. October 1, 2010 White Colonies. Digested BA chlor in pSB1A3 backbone with BsaI gel “08.10.10.alina”