Template:SBB12Project stdt1200

Spencer Scott

Welcome to your project page!

I've given you two parts to make.

• Design oligos to make your part
• Write up a proper construction file on the wiki template associated with your part
• Enter your Features (REMEMBER: It's not a bug, it's a feature. We just don't know what it does yet), Oligos, Parts, and Plasmids into Clotho

Also, use the link above left to upload a picture of yourself, your name, and anything else you'd like, and rename the link from "Your Name Here" to your name.

Finally, you should create a notebook on the on the main class page of the wiki

Create a LexA extravaganza.

LexA Fusion Two-Hybrid System Construction File:

Construction of w.t. LexA
PCR srs177f/srs178r on MG1655 genome   (640 bp, pcrprdct1)
Digest pcrprdct1              	    (Eco/Bam, 624, L, pcrdig)
Digest pBca9145-part			    (Eco/Bam, L, vectdig)
Ligate pcrdig, vectdig			    (9145-Bsrs038)

srs177f	CCATAgaattcATGagatctatgaaagcgttaacggccaggc	
srs178r	gctagGGATCCttacagccagtcgccgttgcg	

Construction of LexA(1-87) with nhe1 end
PCR srs177f/srs188r on w.t. LexA       (292 bp, pcrprdct1)
Digest pcrprdct1              	    (Eco/Bam, 276 L, pcrdig)
Digest pBca9145-part			    (Eco/Bam, L, vectdig)
Ligate pcrdig, vectdig			    (9145-Bsrs044)

srs188r	catagGCTAGCtggttcaccggcagccacacg


Constructin of LexA408 (PA40, NS41, AS42) SOEing
w.t.: cca-aac-gcg
codon optimized PNA->ASS: gct-tct-tct **

PCR ca998/srs187r on 9145-Bsrs038		(222 bp, A)
PCR g00101/srs186f on 9145-Bsrs038	     (609 bp, B)
PCR ca998/g00101 on A+B		 	     (783 bp, pcrprdct)

Digest pcrprdct              		     (Eco/Bam, 624, L, pcrdig)
Digest pBca9145-part		          (Eco/Bam, L, vectdig)

Ligate pcrdig, vectdig			     (9145-Bsrs039)

srs186f	GCGTTTGGGGTTCCGTTCCgcttcttctGCTGAAGAACATCTGAAGGC
srs187r	GCCTTCAGATGTTCTTCAGCagaagaagcGGAACGGAACCCCAAACGC


Wobble to create sulA promoter with cononical RBS
Wobble srs189f/srs190r            		(104bp, sulA1)
Wobble srs191f/srs192r				(104 bp, sulA2)

Digest sulA1 					(Eco/Bam 87+10+6, L, dig1)
Digest sulA2				    	(Eco/Bam 87+10+6, L, dig2)
Digest 9525					(Eco/Bam, L, vectdig)

Ligate sulA1+9525				(Bsrs040)
Ligate sulA1+9525				(Bsrs041)
Bsrs040 is w.t. sulA promoter {op+/op+}
Bsrs041 is mutant sulA promoter {408/op+}


w.t sulA promoter (op+/op+):
srs189f CCATAgaattcATGagatctaatagggttgatctttgttgtcaCTGGATGTACtGTACATCC
Srs190r CTGATggatccaatcaatccagcccctgtgagttactgtatGGATGTACaGTACATCCAG

Hybrid sulA promoter (408/op+):
Srs191f CCATAgaattcATGagatctaatagggttgatctttgttgtcaCTGGATGTACCGTACATCC
Srs192r CTGATggatccaatcaatccagcccctgtgagttactgtatGGATGTACGGTACATCCAG