IGEM:MIT/2007/Notebook/2007-6-25

To-Do

1. Sequence inserts OmpC, CPX, with polystyrene-binding (17, 18) and polypyrrole-binding peptides
2. Check sequences
3. Get grads to check sequences
4. 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