From 2010.igem.org
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| == Fusion Protein == | | == Fusion Protein == |
- | We need to fusion protein blah blah blah
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| {{:Team:NYMU-Taipei/NStyle}} | | {{:Team:NYMU-Taipei/NStyle}} |
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| === eIF4A === | | === eIF4A === |
- | We split eIF4A ({{:Team:NYMU-Taipei/BBa|K411100}}) at 215&216aa based on [http://igem.ym.edu.tw/pv2010/images/b/be/File:NYMU_Nmeth1023-S5.pdf 2.protocol file from Natalia E. Broude]. But first we needed to mutate the two PstI cutting sites. | + | We split eIF4A ({{:Team:NYMU-Taipei/BBa|K411100}}) at 215&216aa based on Natalia E. Broude's study. But first we needed to mutate the two PstI cutting sites. |
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| We took the protein coding region from the [http://www.ncbi.nlm.nih.gov/nuccore/NM_144958 eIF4A mRNA transcript sequence from Mouse (from NCBI)] and found it had 2 PstI cutting sites: | | We took the protein coding region from the [http://www.ncbi.nlm.nih.gov/nuccore/NM_144958 eIF4A mRNA transcript sequence from Mouse (from NCBI)] and found it had 2 PstI cutting sites: |
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| === Linker === | | === Linker === |
- | We took the linker from 2.protocol file from Natalia E. Broude:[http://igem.ym.edu.tw/pv2010/images/b/be/File:NYMU_Nmeth1023-S5.pdf ] | + | We took the linker from 2.protocol file from Natalia E. Broude. |
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| {{:Team:NYMU-Taipei/Seq|linker GSSGSSGSGS|30| | | {{:Team:NYMU-Taipei/Seq|linker GSSGSSGSGS|30| |
Latest revision as of 02:55, 28 October 2010
Fusion Protein
Legend
Effect | Class
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Primers: (changes overline/underline and background)
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Forward Primer | FP
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Overlapping Primer | OP
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Reverse Primer | RP
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Parts: (changes text color)
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Promoter | P
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Ribosome Binding site | R
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RNA | rna
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Coding region | C
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Terminator | T
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Special | S
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Cutting sites: (changes background)
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Biobrick cutting site | cut
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Biobrick cutting site mutation | cutmut
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GFP
We based splitting GFP (BBa_E0040) based on the split point at 157&158aa used by iGEM07_Davidson_Missouri's BBa_I715019 and BBa_I715020. The A-part split is the same as BBa_I715019, but the B-part has one base difference from BBa_I715020.
atgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggaga
gggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactt
tcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaa
agaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaagg
tattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaaga
atggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgat
ggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttga
gtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataa
atgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggaga
gggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactt
tcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaa
agaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaagg
tattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaa
FP (including start codon): atgcgtaaaggagaagaacttttc (55c,24bp,38gc)
RP: ttgtttgtctgccatgatgtatac (55c,24bp,38gc)
aagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattg
gcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtcctt
cttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataa
FP: aagaatggaatcaaagttaacttcaaaa (55c,28bp,25gc)
RP (excluding stop codon): tttgtatagttcatccatgccatg (55c,24bp,38gc)
RFP
We based splitting RFP (BBa_E1010) based on the split point at 154&155aa used by iGEM07_Davidson_Missouri's BBa_I715022 and BBa_I715023. The A-part split is the same as BBa_I715022, but the B-part has one base difference from BBa_I715023.
atggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcgaaatcgaaggtgaaggtg
aaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagta
cggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaacgtgttatgaacttcgaa
gacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtc
cggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaatcaaaatgcgtctgaaact
gaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaaaccgacatcaaactggac
atcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgcttaataa
atggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcgaaatcgaaggtgaaggtg
aaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagta
cggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaacgtgttatgaacttcgaa
gacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtc
cggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagac
FP (including start codon): atggcttcctccgaagac (55C,18bp,56%)
RP: gtcttccgggtacatacgtt (55C,20bp,50%)
ggtgctctgaaaggtgaaatcaaaatgcgtctgaaactgaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttc
agctgccgggtgcttacaaaaccgacatcaaactggacatcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtca
ctccaccggtgcttaataa
FP: ggtgctctgaaaggtgaaatc (55C,21bp,48%)
RP (excluding stop codon): agcaccggtggagtga (56C,16bp,63%)
eIF4A
We split eIF4A (BBa_K411100) at 215&216aa based on Natalia E. Broude's study. But first we needed to mutate the two PstI cutting sites.
We took the protein coding region from the eIF4A mRNA transcript sequence from Mouse (from NCBI) and found it had 2 PstI cutting sites:
atggagccggaaggcgtcatcgagagtaactggaacgagattgtggatagctttgatgacatgaatctctcagagtccctcctccgtggtatttatgcct
atggttttgagaagccctctgccatccagcagcgagctattcttccttgtatcaagggttatgatgtgattgctcaagcccagtctgggactgggaaaac
agctacatttgccatatcaattctgcagcagattgaattagatctaaaggccactcaggctttggttctggcacccacacgtgaattggctcagcagata
caaaaggtggttatggcattaggagactacatgggtgcctcttgtcatgcctgcattgggggcaccaatgtgcgtgctgaggtgcagaagctgcagatgg
aagctccccatatcatcgtgggtacccctggccgggtgtttgacatgcttaaccggagatacctgtcccccaaatacatcaagatgttcgtactggatga
agcagatgaaatgttaagccgagggttcaaggatcagatctatgacatattccagaagctcaacagcaacacacaggtagttttgttgtctgctacaatg
ccttctgatgtccttgaggtgaccaagaaatttatgagagaccctattcggattcttgtcaagaaggaagaattgaccctggagggtatccgccaattct
acatcaatgtggaacgagaggagtggaagcttgacacattgtgtgacttgtatgagacgctgaccatcacccaggcagtcatctttatcaacaccagaag
gaaggtggactggctcaccgagaagatgcatgcccgagatttcactgtttctgccatgcacggagatatggaccaaaaggaacgagatgtgatcatgagg
gagttccggtctggctctagcagagtattaattaccactgacctgttggccagaggcattgatgtgcagcaggtctccttagtcatcaactatgaccttc
ccaccaacagggaaaactacatccacagaatcggtcgaggtggtcggtttggtcgtaagggtgtggctattaacatggtgaccgaagaagacaagaggac
tcttcgagacattgagactttctacaacacctccattgaagagatgcccctcaacgttgctgacctcatttga
We need to mutate out the two PstI cutting sites. The template of eIF4A on a pGEX-4TI vector was kindly provided by C.Proud. We used these primers to mutate the cutting site:
2 of pstI cutting site
mut1(24bp,55C,42%)
FP: ccatatcaattctccagcagattga
RP: caatctgctggagaattgatatggc
mut2(18bp,55C,56%)
FP: gcagaagctccagatggaa
RP: tccatctggagcttctgca
The new sequence after mutation:
atggagccggaaggcgtcatcgagagtaactggaacgagattgtggatagctttgatgacatgaatctctcagagtccctcctccgtggtatttatgcct
atggttttgagaagccctctgccatccagcagcgagctattcttccttgtatcaagggttatgatgtgattgctcaagcccagtctgggactgggaaaac
agctacatttgccatatcaattctccagcagattgaattagatctaaaggccactcaggctttggttctggcacccacacgtgaattggctcagcagata
caaaaggtggttatggcattaggagactacatgggtgcctcttgtcatgcctgcattgggggcaccaatgtgcgtgctgaggtgcagaagctccagatgg
aagctccccatatcatcgtgggtacccctggccgggtgtttgacatgcttaaccggagatacctgtcccccaaatacatcaagatgttcgtactggatga
agcagatgaaatgttaagccgagggttcaaggatcagatctatgacatattccagaagctcaacagcaacacacaggtagttttgttgtctgctacaatg
ccttctgatgtccttgaggtgaccaagaaatttatgagagaccctattcggattcttgtcaagaaggaagaattgaccctggagggtatccgccaattct
acatcaatgtggaacgagaggagtggaagcttgacacattgtgtgacttgtatgagacgctgaccatcacccaggcagtcatctttatcaacaccagaag
gaaggtggactggctcaccgagaagatgcatgcccgagatttcactgtttctgccatgcacggagatatggaccaaaaggaacgagatgtgatcatgagg
gagttccggtctggctctagcagagtattaattaccactgacctgttggccagaggcattgatgtgcagcaggtctccttagtcatcaactatgaccttc
ccaccaacagggaaaactacatccacagaatcggtcgaggtggtcggtttggtcgtaagggtgtggctattaacatggtgaccgaagaagacaagaggac
tcttcgagacattgagactttctacaacacctccattgaagagatgcccctcaacgttgctgacctcatttga
atggagccggaaggcgtcatcgagagtaactggaacgagattgtggatagctttgatgacatgaatctctcagagtccctcctccgtggtatttatgcct
atggttttgagaagccctctgccatccagcagcgagctattcttccttgtatcaagggttatgatgtgattgctcaagcccagtctgggactgggaaaac
agctacatttgccatatcaattctccagcagattgaattagatctaaaggccactcaggctttggttctggcacccacacgtgaattggctcagcagata
caaaaggtggttatggcattaggagactacatgggtgcctcttgtcatgcctgcattgggggcaccaatgtgcgtgctgaggtgcagaagctccagatgg
aagctccccatatcatcgtgggtacccctggccgggtgtttgacatgcttaaccggagatacctgtcccccaaatacatcaagatgttcgtactggatga
agcagatgaaatgttaagccgagggttcaaggatcagatctatgacatattccagaagctcaacagcaacacacaggtagttttgttgtctgctacaatg
ccttctgatgtccttgaggtgaccaagaaatttatgagagaccct
FP: atggagccggaaggcgtcatcga (66c,24bp,61gc) [design error -- was suppose to design it to be 55c]
RP: agggtctctcataaatttcttgg (54C,23bp,39%)
attcggattcttgtcaagaaggaagaattgaccctggagggtatccgccaattct
acatcaatgtggaacgagaggagtggaagcttgacacattgtgtgacttgtatgagacgctgaccatcacccaggcagtcatctttatcaacaccagaag
gaaggtggactggctcaccgagaagatgcatgcccgagatttcactgtttctgccatgcacggagatatggaccaaaaggaacgagatgtgatcatgagg
gagttccggtctggctctagcagagtattaattaccactgacctgttggccagaggcattgatgtgcagcaggtctccttagtcatcaactatgaccttc
ccaccaacagggaaaactacatccacagaatcggtcgaggtggtcggtttggtcgtaagggtgtggctattaacatggtgaccgaagaagacaagaggac
tcttcgagacattgagactttctacaacacctccattgaagagatgcccctcaacgttgctgacctcatttga
FP: attcggattcttgtcaagaagg (54C,22bp,41%)
RP (including stop codon): tcaaatgaggtcagcaacgttgag (59c,24bp,46gc) [design error -- was suppose to design it to be 55c]
Linker
We took the linker from 2.protocol file from Natalia E. Broude.
ggcagcagcggcagcagcggcagcggcagc
We decided to use primers to create the linker:
cggcagcagcggcagcggcagc
ggcagcagcggcagcagcggcagcggcagc
ggcagcagcggcagcagcggcagc
overlapping region: cggcagcagcggcagc (63c,16bp,81gc)
FP: cggcagcagcggcagcggcagc (22bp)
RP: gctgccgctgctgccgctgctgcc (24bp)
Fusioning it together
GFP fusion part
(note the extra taa stop codon at the end)
atgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtg
aaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatgg
tgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttc
aaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatg
gaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaggcagcagcggcagcagcggcagcggcag
catggagccggaaggcgtcatcgagagtaactggaacgagattgtggatagctttgatgacatgaatctctcagagtccctcctccgtggtatttatgcc
tatggttttgagaagccctctgccatccagcagcgagctattcttccttgtatcaagggttatgatgtgattgctcaagcccagtctgggactgggaaaa
cagctacatttgccatatcaattctccagcagattgaattagatctaaaggccactcaggctttggttctggcacccacacgtgaattggctcagcagat
acaaaaggtggttatggcattaggagactacatgggtgcctcttgtcatgcctgcattgggggcaccaatgtgcgtgctgaggtgcagaagctccagatg
gaagctccccatatcatcgtgggtacccctggccgggtgtttgacatgcttaaccggagatacctgtcccccaaatacatcaagatgttcgtactggatg
aagcagatgaaatgttaagccgagggttcaaggatcagatctatgacatattccagaagctcaacagcaacacacaggtagttttgttgtctgctacaat
gccttctgatgtccttgaggtgaccaagaaatttatgagagacccttga
GFP_split_A_FP: gaattcgcggccgcttctagag atgcgtaaaggagaagaacttttc (46bp) OK
GFP_split_A_RP: gctgccgctgctgccgctgctgcc ttgtttgtctgccatgatgtatac (48bp) RE DING
eIF4A_split_A_FP: cggcagcagcggcagcggcagc atggagccggaaggcgtcatcga (45bp) OK
eIF4A_split_A_RP: ctgcagcggccgctactagta tca agggtctctcataaatttcttgg (47bp) OK
(note that we added as start codon and removed the stop codon of GFP_B)
atgaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcg
atggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtt
tgtaacagctgctgggattacacatggcatggatgaactatacaaaggcagcagcggcagcagcggcagcggcagcattcggattcttgtcaagaaggaa
gaattgaccctggagggtatccgccaattctacatcaatgtggaacgagaggagtggaagcttgacacattgtgtgacttgtatgagacgctgaccatca
cccaggcagtcatctttatcaacaccagaaggaaggtggactggctcaccgagaagatgcatgcccgagatttcactgtttctgccatgcacggagatat
ggaccaaaaggaacgagatgtgatcatgagggagttccggtctggctctagcagagtattaattaccactgacctgttggccagaggcattgatgtgcag
caggtctccttagtcatcaactatgaccttcccaccaacagggaaaactacatccacagaatcggtcgaggtggtcggtttggtcgtaagggtgtggcta
ttaacatggtgaccgaagaagacaagaggactcttcgagacattgagactttctacaacacctccattgaagagatgcccctcaacgttgctgacctcat
ttga
GFP_split_B_FP: gaattcgcggccgcttctagag atg aagaatggaatcaaagttaacttcaaaa (53bp)
GFP_split_B_RP: gctgccgctgctgccgctgctgcc tttgtatagttcatccatgccatg (48bp)
eIF4A_split_B_FP: cggcagcagcggcagcggcagc attcggattcttgtcaagaagg (44bp)
eIF4A_split_B_RP: ctgcagcggccgctactagta tcaaatgaggtcagcaacgttgag (45bp)
RFP fusion part
(note the extra taa stop codon at the end)
atggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcgaaatcgaaggtgaaggtg
aaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagta
cggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaacgtgttatgaacttcgaa
gacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtc
cggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggcagcagcggcagcagcggcagcggcagcatggagcc
ggaaggcgtcatcgagagtaactggaacgagattgtggatagctttgatgacatgaatctctcagagtccctcctccgtggtatttatgcctatggtttt
gagaagccctctgccatccagcagcgagctattcttccttgtatcaagggttatgatgtgattgctcaagcccagtctgggactgggaaaacagctacat
ttgccatatcaattctccagcagattgaattagatctaaaggccactcaggctttggttctggcacccacacgtgaattggctcagcagatacaaaaggt
ggttatggcattaggagactacatgggtgcctcttgtcatgcctgcattgggggcaccaatgtgcgtgctgaggtgcagaagctccagatggaagctccc
catatcatcgtgggtacccctggccgggtgtttgacatgcttaaccggagatacctgtcccccaaatacatcaagatgttcgtactggatgaagcagatg
aaatgttaagccgagggttcaaggatcagatctatgacatattccagaagctcaacagcaacacacaggtagttttgttgtctgctacaatgccttctga
tgtccttgaggtgaccaagaaatttatgagagacccttga
RFP_split_A_FP: gaattcgcggccgcttctag atggcttcctccgaagac (38bp)
RFP_split_A_RP: gctgccgctgctgccgctgctgcc gtcttccgggtacatacgtt (44bp)
eIF4A_split_A_FP and RP are the same as the GFP part above.
(note that we added as start codon and removed the stop codon of RFP_B)
atgggtgctctgaaaggtgaaatcaaaatgcgtctgaaactgaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccgg
ttcagctgccgggtgcttacaaaaccgacatcaaactggacatcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcg
tcactccaccggtgctggcagcagcggcagcagcggcagcggcagcattcggattcttgtcaagaaggaagaattgaccctggagggtatccgccaattc
tacatcaatgtggaacgagaggagtggaagcttgacacattgtgtgacttgtatgagacgctgaccatcacccaggcagtcatctttatcaacaccagaa
ggaaggtggactggctcaccgagaagatgcatgcccgagatttcactgtttctgccatgcacggagatatggaccaaaaggaacgagatgtgatcatgag
ggagttccggtctggctctagcagagtattaattaccactgacctgttggccagaggcattgatgtgcagcaggtctccttagtcatcaactatgacctt
cccaccaacagggaaaactacatccacagaatcggtcgaggtggtcggtttggtcgtaagggtgtggctattaacatggtgaccgaagaagacaagagga
ctcttcgagacattgagactttctacaacacctccattgaagagatgcccctcaacgttgctgacctcatttga
RFP_split_B_FP: gaattcgcggccgcttctag atg ggtgctctgaaaggtgaaatc (44bp)
RFP_split_B_RP: gctgccgctgctgccgctgctgcc agcaccggtggagtga (40bp)
eIF4A_split_B_FP and RP are the same as the GFP part above.
Aptamer
- Aptamer in front of non-coding sequence:
-
RNAfold prediction of Aptamer Structure with Biobrick scars in front of a non-coding region. The scars bond together, but the overall structure does not change.
- Check to see if the addition of the biobrick scars affect the secondary structure in front of coding sequence:
-
RNAfold prediction of Aptamer Structure with Biobrick scars in front of a coding region. The scars bond together, but the overall structure does not change.
- Structure interacting with the split proteins:
Aptamer Structure interacting with split protein [6]