Template:SBB10Project-15924
Welcome to your project page!
For each part listed, you should:
- Design oligos to make your part
- Write up proper construction files and put it on the Construction Files page
- Put your oligos on the Oligo Log page
- Put your parts into Clotho
You should design your construction strategy to put your part into plasmid vectorName-Bca1144 (Where vectorName is indicated for each part) using EcoRI and BamHI or just BamHI or BglII for EIPCR. The maps for all plasmids involved in our project can be downloaded here.
Several references have been provided to give you some background on the biology of your part.
Also, use the link above left to upload a picture of yourself, your name, and anything else you'd like.
Finally, you should create a notebook on the main page of the wiki
sbb11: sleeping beauty 5'TR
Source: synthetic Target Sequence: agttgaagtcggaagtttacatacacttaagttggagtcattaaaactcgtttttcaactacaccacaaatttcttgttaacaaacaatagttttggcaagtcagttaggacatctactttgtgcatgacacaagtcatttttccaacaattgtttacagacagattatttcacttataattcactgtatcacaattccagtgggtcagaagtttacatacactaa Vector: pBjk2741 Short description: sleeping beauty 5'TR Family: Terminal Repeat
This part encodes a DNA cis element
DNA cis elements are sequences that are bound by DNA binding domains, replication proteins, or DNA modification enzymes. The proteins sometimes just stick to them, and other times they perform some sort of DNA chemistry on them.
This part is associated with sleeping beauty transposon devices
Sleeping Beauty transposase is a popular enzyme that generates protein-DNA complexes called transposomes from DNAs flanked by "terminal repeats". These terminal repeats, or TR's, are specific cis elements. The transposomes are highly reactive and insert the DNA contained within them randomly into other DNAs (such as a genome).
You should read the following papers
References: DOI 10.1007/s10989-007-9114-z, PMID 19412179
This part requires Gene Synthesis
To make this part, you'll use gene synthesis. You may use the tools at GeneDesign to design your oligos for making this part by the PCA method. The protocol is here.
Proposed synthesis route: Fully Synthetic (GeneDesign)
Proposed construction file (draft): Template:SBB10 ConstructionFiles JimH sbb11
1) sbb11: sleeping beauty 5'TR Pool OJIMH001 through OJIMH010, assemble by PCA PCR OJIMH001/OJIMH010 on PCA reaction (257 bp, EcoRI/BamHI) Sub in pBjk2741-Bca1144 (EcoRI/BamHI, 910+2170, L) Product is pBjk2741-sbb11 ------------ OJIMH001 PCA assembly of sbb11 CCATAGAATTCATGAGATCTAGTTGAAGTCGGAAGTTTACATACACTTAAG OJIMH002 PCA assembly of sbb11 GAAAAACGAGTTTTAATGACTCCAACTTAAGTGTATGTAAACTTCCGACTTC OJIMH003 PCA assembly of sbb11 AAGTTGGAGTCATTAAAACTCGTTTTTCAACTACACCACAAATTTCTTGTTAAC OJIMH004 PCA assembly of sbb11 CTGACTTGCCAAAACTATTGTTTGTTAACAAGAAATTTGTGGTGTAGTTGAAA OJIMH005 PCA assembly of sbb11 TTAACAAACAATAGTTTTGGCAAGTCAGTTAGGACATCTACTTTGTGC OJIMH006 PCA assembly of sbb11 ACAATTGTTGGAAAAATGACTTGTGTCATGCACAAAGTAGATGTCCTAACTG OJIMH007 PCA assembly of sbb11 GACACAAGTCATTTTTCCAACAATTGTTTACAGACAGATTATTTCACTTATAATTC OJIMH008 PCA assembly of sbb11 CCACTGGAATTGTGATACAGTGAATTATAAGTGAAATAATCTGTCTGTAAAC OJIMH009 PCA assembly of sbb11 TAATTCACTGTATCACAATTCCAGTGGGTCAGAAGTTTACATACACTAAG OJIMH010 PCA assembly of sbb11 CTGATGGATCCTTAGTGTATGTAAACTTCTGACCC
JCA Notes
- Correct
sbb13: PhiC31 Integrase
Source: pBca9523-Bca1623, pBca9523-Bca1659
Target Sequence: DTYAGAYDRQSRERENSSAASPATQRSANEDKAADLQREVERDGGRFRFVGHFSEAPGTSAFGTAERPEFERILNECRAGRLNMIIVYDVSRFSRLKVMDAIPIVSELLALGVTIVSTQEGVFRQGNVMDLIHLIMRLDASHKESSLKSAKILDTKNLQRELGGYVGGKAPYGFELVSETKEITRNGRMVNVVINKLAHSTTPLTGPFEFEPDVIRWWWREIKTHKHLPFKPGSQAAIHPGSITGLCKRMDADAVPTRGETIGKKTASSAWDPATVMRILRDPRIAGFAAEVIYKKKPDGTPTTKIEGYRIQRDPITLRPVELDCGPIIEPAEWYELQAWLDGRGRGKGLSRGQAILSAMDKLYCECGAVMTSKRGEESIKDSYRCRRRKVVDPSAPGQHEGTCNVSMAALDKFVAERIFNKIRHAEGDEETLALLWEAARRFGKLTEAPEKSGERANLVAERADALNALEELYEDRAAGAYDGPVGRKHFRKQQAALTLRQQGAEERLAELEAAEAPKLPLDQWFPEDADADPTGPKSWWGRASVDDKRVFVGLFVDKIVVTKSTTGRGQGTPIEKRASITWAKPPTDDDEDDAQDGTEDVAA
Vector: pBjk2741
Short description: <phiC31>
Genbank reference: X59938.1
Flavor: {<part>}
Family: Phage Integrase
A template for making this part has been constructed as two synthons in plasmids pBca9523-Bca1623 and pBca9523-Bca1659. You will need to use SOEing to splice these two synthons together. Take special note that you are creating the correct flavor for this part.
This part encodes a DNA modifying enzyme
Different DNA modification enzymes are needed in different flavors, so take special note of what type this one should be. DNA modification enzymes are often toxic to E. coli, so you may have some difficulty making this part. Make careful note in your notebook about the size distribution of colonies you get on your plates and the ratio of red to white colonies.
This part is associated with phiC31 integration devices
phiC31 is a bacteriophage integrase. It is somewhat similar in mechanism to Cre recombinase, but it acts on the asymmetric sites attB and attP. It causes these sequences to be recombined to generate attL and attR sequences. This system is useful for integrating large DNAs site-specifically into genomes.
You should read the following papers
References: PMID 14996222, PMID 19002165, PMID 19439387, PMID 12034816
Proposed synthesis route: Modified SOE procedure
Proposed construction file (draft): Template:SBB10 ConstructionFiles JimH sbb13
2) sbb13: PhiC31 Integrase
PCR OJIMH011/OJIMH012 on pBca9523-Bca1623 (949 bp, gp = A)
PCR OJIMH013/OJIMH014 on pBca9523-Bca1659 (938 bp, gp = B)
-------------
PCR PJIMH011/OJIMH014 on A+B (1843 bp, EcoRI/BamHI)
Sub in pBjk2741-Bca1144 (EcoRI/BamHI, 910+2170, L)
Product is pBjk2741-sbb13 {<phiC31>}
-------------
OJIMH011 Forward PCR of Part 1 of PhiC31 ccataGAATTCatgAGATCTGACACGTACGCGGGTGCTTA
OJIMH012 SOEing of Part 1 (R) of PhiC31 AAGAAGCCGGACGGCACGCCGACCacgaagattgagggttaccg
OJIMH013 SOEing of Part 2 (F) of PhiC31 cggtaaccctcaatcttcgtGGTCGGCGTGCCGTCCGGCTTCTT
OJIMH014 Reverse PCR of Part 2 of PhiC31 atcagGGATCCCGCCGCTACGTCTTCCGT
JCA Notes
- Oligos 12 and 13 are switched
- Otherwise correct
sbb13: PhiC31 Integrase PCR OJIMH011/OJIMH013 on Bca1623 6-9 (949bp, gp=A) PCR OJIMH012/CA1674 on Bca1559 7-7 (514bp, gp=B) PCR CA1675/OJIMH014 on Bca1559 7-1 (444bp, gp = C) ------------- PCR PJIMH011/OJIMH014 on A+B+C (1843 bp, EcoRI/BamHI) Sub in pBjk2741-Bca1144 (EcoRI/BamHI, 910+2170, L) Product is pBjk2741-sbb13 {<phiC31>} ------------- OJIMH011 Forward PCR of Part 1 of PhiC31 ccataGAATTCatgAGATCTGACACGTACGCGGGTGCTTA OJIMH012 SOEing of Part 1 (R) of PhiC31 AAGAAGCCGGACGGCACGCCGACCacgaagattgagggttaccg OJIMH013 SOEing of Part 2 (F) of PhiC31 cggtaaccctcaatcttcgtGGTCGGCGTGCCGTCCGGCTTCTT CA1674 GGGCGTCGGCGCGCTCCGCAACAAGGTTCGCCCGTTCGCCGCTCTTCTCAG PCA assembly of phiCthreeprime (Bca1659) CA1675 TGCGGAGCGCGCCGACGCCCTGAACGCCCTTGAAGAGCTGTACGAAGACCG PCA assembly of phiCthreeprime (Bca1659) OJIMH014 Reverse PCR of Part 2 of PhiC31 atcagGGATCCCGCCGCTACGTCTTCCGT
