SBB10Ntbk-MichelNofal

Construction Files

sbb02: N15 Protelomerase

sbb02: N15 Protelomerase

PCR MNF11-F/MNF12-R on pBca9523-Bca1455		(945 bp, gp = A) 
PCR MNF13-F/MNF14-R on pBca9523-Bca1485		(1033 bp, gp = B) 
------------- 
PCR MNF11-F/MNF14-R on A + B			(1925 bp, EcoRI/BamHI) 
Sub in pBjk2741-Bca1144 	(EcoRI/BamHI, 910+2170, L) 
Product is pBjk2741-sbb02	{<protel!} 
------------- 
MNF11-F	Forward PCR of Part 1 of sbb02	ccagtGAATTCgtccAGATCTAGCAAGGTAAAAATCGGTGAGTTG
MNF12-R	SOEing of Part1 of sbb02	ggttacgcttttatcttcactgcgTTTTTTAGCTTGCCCTGAGAAATTAACCG
MNF13-F	SOEing of Part2 of sbb02	CGGTTAATTTCTCAGGGCAAGCTAAAAAAcgcagtgaagataaaagcgtaacc
MNF14-R	Reverse PCR of Part 2 of sbb02	gcagtggatccTCAGCTGTAGTACGTTTCCCATGC

JCA Notes

• Have gtcc instead of atg in prefix location
• Need to put part on Clotho
• Otherwise correct
• Internal oligos are longer than necessary, but would work
• Due to similarity to other part, some oligos are being replaced:

Construction of sbb02: N15 Protelomerase

PCR MNF11-F/CA1472 on Bca1455 1-3               (569bp, gp=A)
PCR ca1461/BDBn15001R on Bca1455 1-1		(762bp, gp=B)
PCR BDBn15002F/BDBn15002R on Bca1485 2-1	(1023 bp, gp=C)
--------------------------------------------------------------
PCR BDBn15001F/BDBn15002R on A+B+C		(1924 bp, EcoRI/BamHI)
Sub in pBjk2741-Bca1144				(EcoRI/BamHI, 910+2170, L)
Product is pBjk2741-sbb01	{rbs.part!}

MNF11-F	Forward PCR of Part 1 of sbb02	
ccagtGAATTCatgAGATCTAGCAAGGTAAAAATCGGTGAGTTG
CA1461	GGAAAGGATTGCAGAAAAGAATAACCGCGAATACTTTTAACGCCTATATGA	
PCA assembly of protelomerasefiveprime (Bca1455)
CA1472	TTGTTGGAATAACTTATAAAGATAGTCACGGCGCTCCTTCCAATCATCACT	
PCA assembly of protelomerasefiveprime (Bca1455)
BDBn15001R	Reverse retrieval and SOE-site introduciton on Bca1455
CGCTTTTATCTTCACTGCGTTTTTTAGCTTGCCCTGAG
BDBn15002F	Forward retrieval and SOE-site introduciton on Bca1485
CTCAGGGCAAGCTAAAAAACGCAGTGAAGATAAAAGCG
BDBn15002R	Reverse retrieval and BioBricking on Bca1485
CTGATggatccttaGCTGTAGTACGTTTCCCATGCGG

part:  
GATCTAGCAAGGTAAAAATCGGTGAGTTGATCAACACGCTTGTGAATGAGGTAGAGGCCATTGATGCCTCAGACCGCCCACAAGGCGACAAAACGAAGAGAATTAAAGCCGCAGCCGCACGGTATAAGAACGCGTTATTTAATGATAAAAGAAAGTTCCGTGGGAAAGGATTGCAGAAAAGAATAACCGCGAATACTTTTAACGCCTATATGAGCAGGGCAAGAAAGCGGTTTGATGATAAATTACATCATAGCTTTGATAAAAATATTAATAAATTATCGGAAAAGTATCCTCTTTACAGCGAAGAATTATCTTCATGGCTTTCTATGCCTACGGCTAATATTCGCCAGCACATGTCATCGTTACAATCTAAATTGAAAGAAATAATGCCGCTTGCCGAAGAGTTATCAAATGTAAGAATAGGCTCTAAAGGCAGTGATGCAAAAATAGCAAGACTAATAAAAAAATATCCAGATTGGAGTTTTGCTCTTAGTGATTTAAACAGTGATGATTGGAAGGAGCGCCGTGACTATCTTTATAAGTTATTCCAACAAGGCTCTGCGTTGTTAGAAGAACTACACCAGCTCAAGGTCAACCATGAGGTTCTGTACCATCTCCAGCTAAGCCCTGCGGAGCGTACATCTATACAGCAACGATGGGCCGATGTTCTGCGCGAGAAGAAGCGTAATGTTGTGGTTATTGACTACCCAACATACATGCAGTCTATCTATGATATTTTGAATAATCCTGCGACTTTATTTAGTTTAAACACTCGTTCTGGAATGGCACCTTTGGCCTTTGCTCTGGCTGCGGTATCAGGGCGAAGAATGATTGAGATAATGTTTCAGGGTGAATTTGCCGTTTCAGGAAAGTATACGGTTAATTTCTCAGGGCAAGCTAAAAAACGCAGTGAAGATAAAAGCGTAACCAGAACGATTTATACTTTATGCGAAGCAAAATTATTCGTTGAATTATTAACAGAATTGCGTTCTTGCTCTGCTGCATCTGATTTCGATGAGGTTGTTAAAGGATATGGAAAGGATGATACAAGGTCTGAGAACGGCAGGATAAATGCTATTTTAGCAAAAGCATTTAACCCTTGGGTTAAATCATTTTTCGGCGATGACCGTCGTGTTTATAAAGATAGCCGCGCTATTTACGCTCGCATCGCTTATGAGATGTTCTTCCGCGTCGATCCACGGTGGAAAAACGTCGACGAGGATGTGTTCTTCATGGAGATTCTCGGACACGACGATGAGAACACCCAGCTGCACTATAAGCAGTTCAAGCTGGCCAACTTTTCCAGAACCTGGCGACCGGAAGTTGGGGATGAAAACACCAGGCTGGTGGCTCTCCAGAAACTGGACGATGAAATGCCAGGCTTTGCCAGAGGTGACGCTGGCGTCCGTCTCCATGAAACCGTTAAGCAGCTGGTGGAGCAGGACCCATCAGCAAAAATAACCAACAGCACTCTCCGGGCCTTTAAATTTAGCCCGACGATGATTAGCCGGTACCTGGAGTTTGCCGCTGATGCATTGGGGCAGTTCGTTGGCGAGAACGGGCAGTGGCAGCTCAAGATAGAGACACCTGCAATCGTCCTGCCTGATGAAGAATCCGTTGAGACCATCGACGAACCGGATGATGAGTCCCAAGACGACGAGCTGGATGAAGATGAAATTGAGCTCGACGAGGGTGGCGGCGATGAACCAACCGAAGAGGAAGGGCCAGAAGAACATCAGCCAACTGCTCTAAAACCCGTCTTCAAGCCTGCAAAAAATAACGGGGACGGAACGTACAAGATAGAGTTTGAATACGATGGAAAGCATTATGCCTGGTCCGGCCCCGCCGATAGCCCTATGGCCGCAATGCGATCCGCATGGGAAACGTACTACAGCTAAG

sbb06: piggieBac 3'TR

sbb06: piggieBac 3'TR

Pool MNF001 through MNF008, assemble by PCA 	
PCR MNF01/MNF10 on PCA reaction		(267 bp, EcoRI/BamHI)
Sub in pBjk2741-Bca1144	(EcoRI/BamHI, 910+2170, L)
Product is pBjk2741-sbb06 {<piggieBac-3'TR>}
-------------
MNF01	PCA Assembly of sbb06	CCAGTGAATTCGTCCAGATCTTTTGTTACTTTATAGAAGAAATT
MNF02	PCA Assembly of sbb06	TTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAA
MNF03	PCA Assembly of sbb06	TTTTGTTTTTTTTTAATAAATAAATAAACATAAATAAATTGTTTG
MNF04	PCA Assembly of sbb06	CACTTACATACTAATAATAAATTCAACAAACAATTTATTTATGTT
MNF05	PCA Assembly of sbb06	TATTATTAGTATGTAAGTGTAAATATAATAAAACTTAATATCTAT
MNF06	PCA Assembly of sbb06	ATATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATT
MNF07	PCA Assembly of sbb06	AATAAATAAACCTCGATATACAGACCGATAAAACACATGCGTCAA
MNF08	PCA Assembly of sbb06	TACGTTAAAGATAATCATGCGTAAAATTGACGCATGTGTTTTATC
MNF09	PCA Assembly of sbb06	CATGATTATCTTTAACGTACGTCACAATATGATTATCTTTCTAGG
MNF10	PCA Assembly of sbb06	GCAGTGGATCCTTAACCCTAGAAAGATAATCATAT

JCA Notes

• Correct except for prefix sequence should be atg

sbb06: piggieBac 3'TR

Pool MNF001 through MNF008, assemble by PCA 	
PCR MNF01/MNF10 on PCA reaction		(267 bp, EcoRI/BamHI)
Sub in pBjk2741-Bca1144	(EcoRI/BamHI, 910+2170, L)
Product is pBjk2741-sbb06 {<piggieBac-3'TR>}
-------------
MNF01	PCA Assembly of sbb06	CCAGTGAATTCatgAGATCTTTTGTTACTTTATAGAAGAAATT
MNF02	PCA Assembly of sbb06	TTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAA
MNF03	PCA Assembly of sbb06	TTTTGTTTTTTTTTAATAAATAAATAAACATAAATAAATTGTTTG
MNF04	PCA Assembly of sbb06	CACTTACATACTAATAATAAATTCAACAAACAATTTATTTATGTT
MNF05	PCA Assembly of sbb06	TATTATTAGTATGTAAGTGTAAATATAATAAAACTTAATATCTAT
MNF06	PCA Assembly of sbb06	ATATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATT
MNF07	PCA Assembly of sbb06	AATAAATAAACCTCGATATACAGACCGATAAAACACATGCGTCAA
MNF08	PCA Assembly of sbb06	TACGTTAAAGATAATCATGCGTAAAATTGACGCATGTGTTTTATC
MNF09	PCA Assembly of sbb06	CATGATTATCTTTAACGTACGTCACAATATGATTATCTTTCTAGG
MNF10	PCA Assembly of sbb06	GCAGTGGATCCTTAACCCTAGAAAGATAATCATAT

Part Assembly (Wet Lab)

2.17.2010

sbb02

Note: I'll be sharing oligos with Ben for this part.

• Oligo samples were diluted to 100uM in the tubes they came in, then diluted again to 10uM in separate tubes to be used in the experiment.
• Three PCR reactions were prepared (water added first, protein last) in separate tubes with these oligos/templates:

1. MNF11-F/CA1472 on Bca1455 1-3
2. ca1461/BDBn15001R on Bca1455 1-1
3. BDBn15002F/BDBn15002R on Bca1455 1-2

24uL ddH2O
3.3uL 10x Expand Buffer "2"
3.3uL dNTPs (2mM in each)
1uL Oligo 1, 10uM
1uL Oligo 2, 10uM
0.5uL Expand polymerase "1"
0.5uL Template DNA

• The predicted products for all three are below 2000kb, so the reaction mixtures should be PCRed up using the 2k55 temperature program.
• The GSIs get to do the actual PCRing.. (aka putting the tubes in the thermocycler and choosing the program, pressing the button, and waiting.)

sbb06

Completed initial assembly step (as described in the PCA Gene Synthesis protocol) in the construction of sbb06 by PCA.

OK, so you've got a bunch of oligos, now what? First, use this recipe and program to do initial assembly of the oligos (do a separate one of these reactions for each chunk you're assembling):
Recipe

   1. 38 uL ddH2O
   2. 5 ul 10x expand buffer
   3. 5 ul 2mM dNTPs
   4. 1 ul oligo mixture (100uM total, mixture of oligos after combination of 100uM stocks)
   5. 0.75 ul Expand polymerase 

Program (can run JCA/PCA1)

   1. 2 min initial denature at 94oC
   2. 30 sec denature at 94oC
   3. 30 sec anneal at 55oC [This should be the overlap temp of your oligos - vary as needed]
   4. 30 sec extension at 72oC
   5. repeat 2-4 30 times total