IGEM:MIT/2007/Notebook/2007-6-25
To-Do
- Sequence inserts OmpC, CPX, with polystyrene-binding (17, 18) and polypyrrole-binding peptides
- Check sequences
- Get grads to check sequences
- Ask about UCSB eCPX
DNA Sequences
NOTE: it would be better if we could order these as oligos and stitch them together rather than ordering a huge protein and waiting a month.
CPX with polystyrene (18) binding peptide as passenger peptide)
Random bp and XbaI (TCTAGA)
1 CATTAGTCTA GA
OmpX Native signal sequence
13 ATGAAAAA AATTGCATGT CTTTCAGCAC 41 TGGCCGCAGT TCTGGCTTTC ACCGCAGGTA 71 CTTCCGTAGC T
Linker Sequence, GGCCNNNNNGGCC SfiI Restriction Site
82 GGCCAGTCT GGCCAG
His6 Tag
97 CATC ACCATCACCA TCAC
Trypsin Cleavage Site
115 AAACGT
HindIII
121 AAGCTT
Polystyrene-binding Peptide 18
127 TTCT TCTCTTTCTT CTTCCCGGCT 151 TCTGCTTGGG GTTCT
sALI
166 GTCGA C
Linker with embedded SfiI
172 GGTGGCCAA TCAGGCCAG
OmpX S54-F148
189 T CTGGTGACTA CAACAAAAAC 211 CAGTACTACG GCATCACTGC TGGTCCGGCT 241 TACCGCATTA ACGACTGGGC AAGCATCTAC 271 GGTGTAGTGG GTGTGGGTTA TGGTAAATTC 301 CAGACCACTG AATACCCGAC CTACAAACAC 331 GACACCAGCG ACTACGGTTT CTCCTACGGT 361 GCGGGTCTGC AGTTCAACCC GATGGAAAAC 391 GTTGCTCTGG ACTTCTCTTA CGAGCAGAGC 421 CGTATTCGTA GCGTTGACGT AGGCACCTGG 451 ATTGCCGGTG TTGGTTACCG CTTC
Linker for OmpX C & N Terminus
475 GGTGGT TCTGGT
OmpX A1-S53
487 GCGA CTTCTACTGT AACTGGCGGT 511 TACGCACAGA GCGACGCTCA GGGCCAAATG 541 AACAAAATGG GCGGTTTCAA CCTGAAATAC 571 CGCTATGAAG AAGACAACAG CCCGCTGGGT 601 GTGATCGGTT CTTTCACTTA CACCGAGAAA 631 AGCCGTACTG CAAGC
Stop Codons
646 TAGTA G
EcoRI (GAATTC) and Random bps
652 GAATTCCAT TAG
OmpC with polystyrene (18) binding peptide as passenger peptide)
Random bps and BamHI (GGATCC)
1 CATTAGGGAT CC
Front Half of OmpC
13 ATGAAAGT TAAAGTTCTG TCTCTGCTGG 41 TTCCGGCTCT GCTGGTTGCT GGTGCTGCTA 71 ACGCTGCTGA AGTTTACAAC AAAGACGGTA 101 ACAAACTGGA CCTGTACGGT AAAGTTGACG 131 GTCTGCACTA CTTCTCTGAC GACAAATCTG 161 TTGACGGTGA CCAGACCTAC ATGCGTCTGG 191 GTTTCAAAGG TGAAACCCAG GTTACCGACC 221 AGCTGACCGG TTACGGTCAG TGGGAATACC 251 AGATCCAGGG TAACTCTGCT GAAAACGAAA 281 ACAACTCTTG GACCCGTGTT GCTTTCGCTG 311 GTCTGAAATT CCAGGACGTT GGTTCTTTCG 341 ACTACGGTCG TAACTACGGT GTTGTTTACG 371 ACGTTACCTC TTGGACCGAC GTTCTGCCGG 401 AGTTCGGTGG TGACACCTAC GGTTCTGACA 431 ACTTCATGCA GCAGCGTGGT AACGGTTTCG 461 CTACCTACCG TAACACCGAC TTCTTCGGTC 491 TGGTTGACGG TCTGAACTTC GCTGTTCAGT 521 ACCAGGGTAA AAACGGTTCT GTTTCTGGTG 551 AAGGTATGAC CAACAACGGT CGTGAAGCTC 581 TGCGTCAGAA CGGTGACGGT GTTGGTGGTT 611 CTATCACCTA CGACTACGAA GGTTTCGGTA 641 TCGGTGCTGC TGTTTCTTCT TCTAAACGTA 671 CCGACGACCA GAACTCTCCG CTGTACATCG 701 GTAACGGTGA CCGTGCTGAA ACCTACACCG 731 GTGGTCTGAA ATACGACGCT AACAACATCT 761 ACCTGGCTGC TCAGTACACC CAGACCTACA 791 ACGCTACCCG TGTTGGTTCT CTGGGTTGGG 821 CTAACAAAGC TCAGAACTTC GAAGCTGTTG 851 CTCAGTACCA GTTCGACTTC GGTCTGCGTC 881 CGTCTCTGGC TTACCTGCAG
His6 Tag
901 CATCACCATC ACCATCAC
Trypsin Cleavage site
919 AA ACGT
ggsg AA linker
925 GGTGGT TCTGGT
HindIII
937 AAGC TT
Polystyrene-binding peptide (18)
943 TTCTTCTC TTTCTTCTTC CCGGCTTCTG 971 CTTGGGGTTC T
SalI
982 GTCGAC
Back Half of OmpC"
988 TCT AAAGGTAAAA ACCTGGGTGT 1011 TATCAACGGT CGTAACTACG ACGACGAAGA 1041 CATCCTGAAA TACGTTGACG TTGGTGCTAC 1071 CTACTACTTC AACAAAAACA TGTCTACCTA 1101 CGTTGACTAC AAAATCAACC TGCTGGACGA 1131 CAACCAGTTC ACCCGTGACG CTGGTATCAA 1161 CACCGACAAC ATCGTTGCTC TGGGTCTGGT 1191 TTACCAGTTC
Stop Codons
1201 TAGTAG
XbaI (TCTAGA) and Random bps
1207 TCTA GACATTAG
Passenger Peptide Oligos w/ Restriction Cuts Built In
Sense/Coding strand: 5'- HindIII cut + Peptide + SalI cut -3'
Polystyrene-binding peptide 18:
5’ AGCTT tttttttctttcttcttcccggcttctgcttggggttct G 3’ 3’ A CAGCT 5’
Reverse of anti-sense/non-coding strand: 5’ TCGAC agaaccccaagcagaagccgggaagaagaaagaaaaaaa A 3’
Polystyrene-binding peptide 17:
Sense: 5’ AGCTT ttcttcccgtcttcttggtactctcacctgggtgttctg G
Anti-sense: 5’ TCGAC cagaacacccaggtgagagtaccaagaagacgggaagaa A
Polypyrrole-binding peptide:
Sense: 5’ AGCTT acccaccgtacctctaccctggactacttcgttatc G
Anti-sense: 5’ TCGAC gataacgaagtagtccagggtagaggtacggtgggt A
USING SEQUENCHER (Demo)
- Copy desired gene sequence (to check)
- Sequence tab
- Create New Sequence
- Give name
- Paste sequence
- View tab
- Translation – different reading frames (gives AAs)
- Fifth button over – check presence of all common enzyme sites
- Cut map tab – show where different restriction sites
- Select enzymes – look for different enzymes and click ok to see where that site is (Ensure presence/absence of restriction enzymes)
- Bases
- View tab
- Reverse and Com – gives reverse complementary sequence
- Can look at sequencing data (ambiguities?)
- Take whole sequence (front, middle, back) and ensure all restriction enzymes are correct and present
