Team:NYMU-Taipei/Project/Speedy reporter/Materials and Methods

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Contents

Fusion Protein

Legend

Effect Class
Primers: (changes overline/underline and background)
Forward Primer FP
Overlapping Primer OP
Reverse Primer RP
Parts: (changes text color)
Promoter P
Ribosome Binding site R
RNA rna
Coding region C
Terminator T
Special S
Cutting sites: (changes background)
Biobrick cutting site cut
Biobrick cutting site mutation cutmut

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.

>GFP (BBa_E0040)720bp
atgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggaga gggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactt tcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaa agaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaagg tattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaaga atggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgat ggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttga gtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataa

>GFP_A (BBa_I175019)471bp
atgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggaga gggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactt tcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaa agaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaagg tattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaa

FP (including start codon): atgcgtaaaggagaagaacttttc (55c,24bp,38gc)
RP: ttgtttgtctgccatgatgtatac (55c,24bp,38gc)

>GFP_B249bp
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.

>RFP (BBa_E1010)681bp
atggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcgaaatcgaaggtgaaggtg aaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagta cggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaacgtgttatgaacttcgaa gacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtc cggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaatcaaaatgcgtctgaaact gaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaaaccgacatcaaactggac atcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgcttaataa

>RFP_A (BBa_I715022)462bp
atggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcgaaatcgaaggtgaaggtg aaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagta cggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaacgtgttatgaacttcgaa gacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtc cggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagac

FP (including start codon): atggcttcctccgaagac (55C,18bp,56%)
RP: gtcttccgggtacatacgtt (55C,20bp,50%)

>RFP_B219bp
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:

>eIF4A_original(BBa_K411100)1173bp
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:

>eIF4A (BBa_K411100)1173bp
atggagccggaaggcgtcatcgagagtaactggaacgagattgtggatagctttgatgacatgaatctctcagagtccctcctccgtggtatttatgcct atggttttgagaagccctctgccatccagcagcgagctattcttccttgtatcaagggttatgatgtgattgctcaagcccagtctgggactgggaaaac agctacatttgccatatcaattctccagcagattgaattagatctaaaggccactcaggctttggttctggcacccacacgtgaattggctcagcagata caaaaggtggttatggcattaggagactacatgggtgcctcttgtcatgcctgcattgggggcaccaatgtgcgtgctgaggtgcagaagctccagatgg aagctccccatatcatcgtgggtacccctggccgggtgtttgacatgcttaaccggagatacctgtcccccaaatacatcaagatgttcgtactggatga agcagatgaaatgttaagccgagggttcaaggatcagatctatgacatattccagaagctcaacagcaacacacaggtagttttgttgtctgctacaatg ccttctgatgtccttgaggtgaccaagaaatttatgagagaccctattcggattcttgtcaagaaggaagaattgaccctggagggtatccgccaattct acatcaatgtggaacgagaggagtggaagcttgacacattgtgtgacttgtatgagacgctgaccatcacccaggcagtcatctttatcaacaccagaag gaaggtggactggctcaccgagaagatgcatgcccgagatttcactgtttctgccatgcacggagatatggaccaaaaggaacgagatgtgatcatgagg gagttccggtctggctctagcagagtattaattaccactgacctgttggccagaggcattgatgtgcagcaggtctccttagtcatcaactatgaccttc ccaccaacagggaaaactacatccacagaatcggtcgaggtggtcggtttggtcgtaagggtgtggctattaacatggtgaccgaagaagacaagaggac tcttcgagacattgagactttctacaacacctccattgaagagatgcccctcaacgttgctgacctcatttga


>eIF4A_A645bp
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%)

>eIF4A_B528bp
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.

>linker GSSGSSGSGS30bp
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)

>GFP-linker-eIF4A_A (BBa_K411101)1149bp
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)

>GFP-linker-eIF4A_B (BBa_K411102)804bp
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)

>RFP-linker-eIF4A_A (BBa_K411103)1140bp
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)

>RFP-linker-eIF4A_B (BBa_K411104)774bp
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]