--- /dev/null
+Over the last few days I've been trying to teach myself enough
+genetics to reconstruct [Carrion-Vazquez's poly-I27 synthesis
+procedure][cv99]. I'm not quite there yet, but I feel like I've made
+enough progress that it's worth posting my notes somewhere public in
+case they are useful to others.
+
+Overview
+========
+
+We buy our poly-I27 from [AthenaES][], who market it as [I27O™][I27O].
+Perusing their [technical brief][I27O-tb], makes it clear that I2O7™
+corresponds to Carrion-Vazquez's I27<sup>RS</sup>₈. In
+[Carrion-Vazquez' original paper][cv99] they describe the synthesis of
+both I27<sup>RS</sup>₈ and a variant I27<sup>GLG</sup>₁₂. Their
+I27<sup>RS</sup>₈ procedure is:
+
+* Human cardiac muscle used to generate a cDNA library [Rief 1997]
+* cDNA library amplified with PCR
+ - 5' primer contained a BamHI restriction site that permitted
+ in-frame cloning of the monomer into the expression vector pQE30
+ (Qiagen, Chatsworth, CA).
+ - The 3' primer contained a BglII restriction site, two Cys codons
+ located 3' to the BglII site and in-frame with the I27 domain,
+ and two in-frame stop codons.
+* The PCR product was cloned into pUC19 linearized with BamHI and SmaI.
+* The 8-domain synthetic gene was constructed by iterative cloning
+ of monomer into monomer, dimer into dimer, and tetramer into
+ tetramer.
+* The final construct contained eight direct repeats of the I27
+ domain, an amino-terminal His tag for purification, and two
+ carboxyl-terminal Cys codons used for covalent attachment to the
+ gold-covered coverslips.
+
+They also give the full-length sequence of I27<sup>RS</sup>₈:
+
+ Met-Arg-Gly-Ser-(His)₆-Gly-Ser-(I27-Arg-Ser)₇-I27-...-Cys-Cys
+
+They point out the Arg-Ser (RS) amino acid sequence is the BamHI-BglII
+hybrid site, which makes sense (see below).
+
+Back on the Athena site, they have a [page describing their
+procedure][I27O-syn] (they reference the Carrion-Vazquez paper). They
+claim to use the restriction enzyme KpnI in addition to BamHI, BglII,
+and SmaI.
+
+Carrion-Vazquez points to the following references:
+
+* [Kempe et al. 1985][k85] (CV16), the source of the multi-step cloning technique.
+* [Rief et al.][r97] (CV10), for I27 subcloning.
+
+Rief
+----
+
+In their note 11, Rief et al. explain their synthesis procedure:
+
+* λ cDNA library
+* Titin fragments of interest were amplified by PCR
+* cloned into pET 9d
+* NH₂-terminal domain boundaries were as in [Politou 1996].
+* The clones were fused with an NH₂-terminal His₆ tag and a
+ COOH-terminal Cys₂ tag for immobilization on solid surfaces.
+
+which doesn't help me very much.
+
+Kemp
+----
+
+The [Kempe article][k85] is more informative, focusing entirely on the
+synthesis procedure (albiet for a different gene). Their figure 2
+outlines the general approach, and used the following restriction
+enzymes: PstI, BamHI, PstI, and BglII. I'll walk through their
+procedure in detail below.
+
+Genetic code
+------------
+
+Wikipedia has a good page on the [genetic code][gcode] for converting
+between DNA/mRNA codons and amino acids. I've written up a little
+[[Python]] script, [[mRNAcode.py]], to automate the conversion of
+various sequences, which helped me while I was writing this post. I'm
+sure there are tons of similar programs out there, so don't feel
+pressured to use mine ;).
+
+Restriction enzymes
+-------------------
+
+We'll use the following [restriction enzymes][renz]
+
+[BamHI][]
+
+ 5' G|GATC C 3'
+ 3' C CTAG|G 5'
+
+[BglI][] (N is any nucleotide)
+
+ 5' GCCN NNN|NGGC 3'
+ 3' CGGN|NNN NCCG 5'
+
+[BglII][]
+
+ 5' A|GATC T 3'
+ 3' T CTAG|A 5'
+
+[HindIII][]
+
+ 5' A|AGCT T 3'
+ 3' T TCGA|A 5'
+
+[KpnI][]
+
+ 5' G GTAC|C 3'
+ 3' C|CATG G 5'
+
+[PstI][]
+
+ 5' C TGCA|G 3'
+ 3' G|ACGT C 5'
+
+[SmaI][]
+
+ 5' CCC|GGG 3'
+ 3' GGG|CCC 5'
+
+Details
+=======
+
+Here's my attempt to reconstruct the details of the polymer-cloning
+reactions, where they splice several copies of I27 into the expression
+plasmid.
+
+Kempe procedure
+---------------
+
+Inserted their poly-SP into pHK414 (I haven't been able to find any
+online sources for pHK414. Kempe cites [R.J. Watson et al.
+*Expression of Herpes simplex virus type 1 and type 2 glyco-protein D
+genes using the Escherichia coli lac promoter.* Y. Becker (Ed.),
+*Recombinant DNA Research and Viruses*. Nijhoff, The Hague, 1985,
+pp. 327-352.][w85])
+
+### Synthetic SP
+
+ HindIII. ,BamHI_.
+ | | Met Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met |
+ 5’ GA AGC TTC ATG CGT CCG AAG CCG CAG CAG TTC TTC GGT CTC ATG GAT CCG
+ CT TCG AAG TAC GCA GGC TTC GGC GTC GTC AAG AAG CCA GAG TAC CTA GGC 5’
+
+### pHK414
+
+ _______Linker_sequence______
+ / \
+ HindIII BamHI
+ ,PstI. BglII.| |,SmaI. |
+ CTGCAG...AGATCTAAGCTTCCCGGGGATCCAAGATCC
+ GACGTC...TCTAGATTCGAAGGGCCCCTAGGTTCTAGG
+ . .
+ .......................................
+
+### Synthesizing pSP4-1
+
+#### pHK414 + HindIII + BamHI
+
+ HindIII BamHI.
+ (PstI) BglII,| |
+ CTGCAG...AGATCTA GATCCAAGATCC
+ GACGTC...TCTAGATTCGA GTTCTAGG
+ . .
+ .......................................
+
+#### SP + HindIII + BamHI
+
+ HindIII. ,BamHI_.
+ | | Met Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met |
+ AGC TTC ATG CGT CCG AAG CCG CAG CAG TTC TTC GGT CTC ATG
+ AG TAC GCA GGC TTC GGC GTC GTC AAG AAG CCA GAG TAC CTA G
+
+#### pSP4-1
+
+ HindIII BamHI.
+ ,PstI. BglII.| | MetArgProLysProGlnGlnPhePheGlyLeuMet |
+ CTGCAG...AGATCTAAGCTTCATGCGTCCGAAGCCGCAGCAGTTCTTCGGTCTCATGGATCCAAGATCC
+ GACGTC...TCTAGATTCGAAGTACGCAGGCTTCGGCGTCGTCAAGAAGCCAGAGTACCTAGGTTCTAGG
+ . .
+ ......................................................................
+
+Using `-SP-` to abbreviate the HindIII→Met→Met portion (less the
+terminal G, which is part of the BamHI match sequence).
+
+ ,PstI. BglII. BamHI.
+ CTGCAG...AGATCT-SP-GGATCC
+ GACGTC...TCTAGA-SP-CCTAGG
+ . .
+ .........................
+
+### Synthesizing pSP4-2
+
+pSP4-1 is split in two parallel rections
+
+#### PstI + BamHI
+
+ G...AGATCT-SP-G
+ ACGTC...TCTAGA-SP-CCTAGG
+
+#### PstI + BglII
+
+ CTGCA GATCT-SP-GGATCC
+ G A-SP-CCTAGG
+ . .
+ .........................
+
+#### pSP4-2
+
+Then the SP-containing fragments (shown above) are isolated and mixed
+together to form pSP4-2.
+
+ ,PstI. BglII. other. BamHI.
+ CTGCAG...AGATCT-SP-GGATCT-SP-GGATCC
+ GACGTC...TCTAGA-SP-CCTAGA-SP-CCTAGG
+ . .
+ ...................................
+
+where the "other" sequence is the result of the BamHI/BglII splice.
+Expanding the "-SP-" abbreviation around the SP joint:
+
+ ....SP,other_.HindIII. SP.....
+ Leu Met Asp Leu Ser Phe Met Arg
+ CTC ATG GAT CTA AGC TTC ATG CGT
+ AGA CGT TCG AGC CTA GGA CGT ATG
+
+So the resulting poly-SP will have Asp-Leu-Ser-Phe linking amino
+acids.
+
+By repeating the PstI + BamHI / PstI + BglII split-and-join, you can
+synthesize plasmids with any number of SP repeats.
+
+I27<sup>RS</sup>₈ procedure
+---------------------------
+
+Like Kempe, Carrion-Vazquez et al. flank the I27 gene with BglII and
+BamHI, but they reverse the order. Here's the output of their PCR:
+
+ BamHI-I27-BglII-Cys-Cys-STOP-STOP
+
+From the PDB entry for I27 ([1TIT][]), the amino acid sequence is:
+
+ ,leader_.
+ MHHHHHHSSLIEVEKPLYGVEVFVGETAHFEIELSEPDVHGQWKLKGQPLTASPDCEIIEDGKKHILI
+ LHNCQLGMTGEVSFQAANAKSAANLKVKEL
+
+To translate this into cDNA, I've scanned thorough the sequence of
+[NM_003319.4][], and found a close match from nucleotides 15991
+through 16248.
+
+ 15982 CTAATAAAAG TGGAAAAGCC TCTGTACGGA GTAGAGGTGT TTGTTGGTGA
+ 16032 AACAGCCCAC TTTGAAATTG AACTTTCTGA ACCTGATGTT CACGGCCAGT
+ 16082 GGAAGCTGAA AGGACAGCCT TTGACAGCTT CCCCTGACTG TGAAATCATT
+ 16132 GAGGATGGAA AGAAGCATAT TCTGATCCTT CATAACTGTC AGCTGGGTAT
+ 16182 GACAGGAGAG GTTTCCTTCC AGGCTGCTAA TGCCAAATCT GCAGCCAATC
+ 16232 TGAAAGTGAA AGAATTG
+
+This cDNA match generates an amino acid starting with LIKVEK instead
+of the expected LIEVEK, but the LIKVEK version matches amino acids
+12677-12765 in [Q8WZ42][] (canonical titin), and there is a natural
+variant listed for [12679 K->E][var].
+
+Interestingly, this sequence contains a PstI site at nucleotides 16220
+through 16225. None of our other restriction enzymes have sites in
+the I27 sequence.
+
+Carrion-Vazquez et al. list two vectors in their procedure, but I'm
+not sure about their respective roles.
+
+### pQE30
+
+[pQE30][pQE30-a] ([sequence][pQE30-b]) is listed as the "expression
+vector", but I'm not sure why they would need a non-expression vector,
+as they don't reference cross-vector subcloning after inserting their
+I27 monomer into the plasmid.
+
+From the [Qiagen site][pQE30], the section around the linker
+nucleotides 115 through 203 is:
+
+ ,RGS-His epitope__________________. ,BamHI.
+ Met Arg Gly Ser His His His His His His Gly Ser Ala Cys Glu Leu ...
+ ATG AGA GGA TCG CAT CAC CAT CAC CAT CAC GGA TCC GCA TGC GAG CTC ...
+ CGT CTC TTC GAT ACG ACA ACG ACA ACG ACA TTC GAA TAC GTA TCT AGA ...
+
+ ,SmaI__.
+ ,KpnI_. HindIII
+ Gly Thr Pro Gly Arg Pro Ala Ala Lys Leu Asn STOP
+ GGT ACC CCG GGT CGA CCT GCA GCC AAG CTT AAT TAG CTG AG
+ TTG CAA AAT TTG ATC AAG TAC TAA CCT AGG CCG GCT AGT CT
+
+However, there is no BglII site in this linker. In fact, there is no
+BglII site in the entire pQE30 plasmid, so they'd need to use a third
+restiction enzyme to insert their I27 (which does contain a trailing
+BglII).
+
+### pUC19
+
+From [BCCM/LMBP][pUC19-a] and [GenBank][pUC19-b], the section around
+the linker nucleotides 233 through 289 is:
+
+ ,SmaI_.
+ HindIII. ,PstI__. ,BamHI_. ,KpnI__.
+ Met STOP
+ AA GCT TGC ATG CCT GCA GGT CGA CTC TAG AGG ATC CCC GGG TAC CGA
+
+ GCT CGA ATT C
+
+However, there is no BglII the entire pUC19 plasmid either, so they'd
+need to use a third restiction enzyme to insert their I27.
+
+### Questions
+
+1. Why do Carrion-Vazquez et al. list two different plasmids?
+2. What is the 3'-side restiction enzyme that Carrion-Vazquez et
+ al. use to insert their I27 into their plasmid?
+3. What is the remote restriction enzyme that Carrion-Vazquez et
+ al. use to break their opened plasmids (Kempe PstI equivalent).
+4. The BamHI and SmaI sites in pUC19 overlap, so it is unclear how you
+ could use both to "linearize" pUC19. It would seem that either one
+ would open the plasmid on its own, although I'm not sure you could
+ "heal" the blunt-ended SmaI cut.
+5. Since the Arg-Ser joint is formed by a BglII/BamHI overlap, why are
+ there no BglII-coded amino acids after the last I27 in the I27<sup>RS</sup>₈
+ sequence? If there is, why do Carrion-Vazquez et al. not
+ acknowledge it when they write [3]:
+
+ The full-length construct, I27<sup>RS</sup>₈, results in the following
+ amino acid additions: (i) the amino-terminal sequence is
+ Met-Arg-Gly-Ser-(His)6-Gly-Ser-I27 codons; (ii) the junction
+ between the domains (BamHI-BglII hybrid site) is Arg-Ser; and
+ (iii) the protein terminates in Cys-Cys.
+
+ Since they don't acknowledge an I27-Arg-Ser-Cys-Cys ending, might
+ there be more amino acids in the C terminal addition?
+
+### Working backward
+
+Since I'm stuck trying to get I27 into either plasmid, let's try and
+work backward from
+
+ Met-Arg-Gly-Ser-(His)₆-Gly-Ser-(I27-Arg-Ser)₇-I27-Cys-Cys
+
+The BglII/BamHI overlap would produce the expected Arg-Ser joint.
+
+ BglII BamHI
+ A + GATCC = AGATCC = Arg-Ser
+ TCTAG G TCTAGG
+
+#### Final plasmid (pI27-8)
+
+The beginning of this sequence looks like the start of pQE30's linker,
+so we'll assume the final plasmid was:
+
+ Met-Arg-Gly-Ser-(His)₆-Gly-Ser-(I27-Arg-Ser)₇-I27-Arg-Ser-Cys-Cys
+
+ remote ... ,RGS-His epitope__________________. ,BamHI. I27...
+ ... Met Arg Gly Ser His His His His His His Gly Ser Leu Ile ...
+ ??? ... ATG AGA GGA TCG CAT CAC CAT CAC CAT CAC GGA TCC CTA ATA ...
+ ??? ... CGT CTC TTC GAT ACG ACA ACG ACA ACG ACA TTC GAA GAT TAT ...
+
+ ........I27 joint_. I27 ... final I27 ,BglII. continuation of pQE30?
+ ... Glu Leu Leu ... Leu Arg Ser Cys Cys STOPSTOP...
+ ... GAA TTG AGA TCC CTA ... TTG AGA TCT TGC TGC TAG TAG ...
+ ... CTT AAC TCT AGG GAT ... GAT CTC GAG GTA GTA GCT GCT ...
+
+#### Penultimate plasmid (pI27-4)
+
+ remote ... ,RGS-His epitope__________________. ,BamHI. I27...
+ Met Arg Gly Ser His His His His His His Gly Ser Leu Ile ...
+ ??? ... ATG AGA GGA TCG CAT CAC CAT CAC CAT CAC GGA TCC CTA ATA ...
+ ??? ... CGT CTC TTC GAT ACG ACA ACG ACA ACG ACA TTC GAA GAT TAT ...
+
+ ... I27 joint_. I27 ... fourth I27 ,BglII. continuation of pQE30?
+ ... Glu Leu Leu ... Leu Arg Ser Cys Cys STOPSTOP...
+ ... GAA TTG AGA TCC CTA ... TTG AGA TCT TGC TGC TAG TAG ...
+ ... CTT AAC TCT AGG GAT ... GAT CTC GAG GTA GTA GCT GCT ...
+
+##### pI27-4 + BamHI + remote
+
+ remote ,BamHI. I27...
+ Leu Ile ...
+ ? GA TCC CTA ATA ...
+ ?? A GAT TAT ...
+
+ ....... I27 joint_. I27 ... fourth I27 ,BglII. continuation of pQE30?
+ ... Glu Leu Leu ... Leu Arg Ser Cys Cys STOPSTOP...
+ ... GAA TTG AGA TCC CTA ... TTG AGA TCT TGC TGC TAG TAG ...
+ ... CTT AAC TCT AGG GAT ... GAT CTC GAG GTA GTA GCT GCT ...
+
+##### pI27-4 + BglII + remote
+
+ remote ... ,RGS-His epitope__________________. ,BamHI. I27...
+ Met Arg Gly Ser His His His His His His Gly Ser Leu Ile ...
+ ?? ... ATG AGA GGA TCG CAT CAC CAT CAC CAT CAC GGA TCC CTA ATA ...
+ ? ... CGT CTC TTC GAT ACG ACA ACG ACA ACG ACA TTC GAA GAT TAT ...
+
+ ....... I27 joint_. I27 ... fourth I27 ,BglII.
+ ... Glu Leu Leu ... Leu
+ ... GAA TTG AGA TCC CTA ... TTG A
+ ... CTT AAC TCT AGG GAT ... GAT CTC GA
+
+##### pI27-8
+
+ remote ... ,RGS-His epitope__________________. ,BamHI. I27...
+ Met Arg Gly Ser His His His His His His Gly Ser Leu Ile ...
+ ??? ... ATG AGA GGA TCG CAT CAC CAT CAC CAT CAC GGA TCC CTA ATA ...
+ ??? ... CGT CTC TTC GAT ACG ACA ACG ACA ACG ACA TTC GAA GAT TAT ...
+
+ ....... I27 joint_. I27 ... fourth I27 ,other. I27...
+ ... Glu Leu Leu ... Leu Gly Ser Leu Ile ...
+ ... GAA TTG AGA TCC CTA ... TTG AGA TCC CTA ATA ...
+ ... CTT AAC TCT AGG GAT ... GAT CTC GAA GAT TAT ...
+
+ ....... I27 joint_. I27 ... fourth I27 ,BglII. continuation of pQE30?
+ ... Glu Leu Leu ... Leu Arg Ser Cys Cys STOPSTOP...
+ ... GAA TTG AGA TCC CTA ... TTG AGA TCT TGC TGC TAG TAG ...
+ ... CTT AAC TCT AGG GAT ... GAT CTC GAG GTA GTA GCT GCT ...
+
+#### Continuing to the first plasmid, pI27-1 must have been
+
+ remote ... ,RGS-His epitope__________________. ,BamHI. I27...
+ ... Met Arg Gly Ser His His His His His His Gly Ser Leu Ile ...
+ ??? ... ATG AGA GGA TCG CAT CAC CAT CAC CAT CAC GGA TCC CTA ATA ...
+ ??? ... CGT CTC TTC GAT ACG ACA ACG ACA ACG ACA TTC GAA GAT TAT ...
+
+ ........I27 ,BglII. continuation of pQE30?
+ ... Glu Leu Arg Ser Cys Cys STOPSTOP...
+ ... GAA TTG AGA TCT TGC TGC TAG TAG ...
+ ... CTT AAC CTC GAG GTA GTA GCT GCT ...
+
+### Potential pQE30 insertion points
+
+* Kpn1 (present after BamHI in both plasmids)
+
+### Potential remote restriction enzymes
+
+* BglI (pQE30 nucleotides 2583-2593 (GCCGGAAGGGC), Amp-resistance
+ 3256-2396; pUC19 has two BglI sites (bad idea))
+
+
+[cv99]: http://dx.doi.org/10.1073/pnas.96.7.3694
+[AthenaES]: http://www.athenaes.com/
+[I27O]: http://www.athenaes.com/I27OAFMReferenceProtein.php
+[I27O-tb]: http://www.athenaes.com/tech_brief_I27O_protein.php
+[I27O-syn]: http://www.athenaes.com/Projects_Polyproteins.php
+[k85]: http://dx.doi.org/10.1016/0378-1119(85)90318-X
+[gcode]: http://en.wikipedia.org/wiki/Genetic_code
+[renz]: http://en.wikipedia.org/wiki/Restriction_enzyme
+[BamHI]: http://en.wikipedia.org/wiki/BamHI
+[BglI]: http://en.wikipedia.org/wiki/List_of_restriction_enzyme_cutting_sites:_Bd-Bp#Bd_-_Bp
+[BglII]: http://en.wikipedia.org/wiki/BglII
+[HindIII]: http://en.wikipedia.org/wiki/HindIII
+[KpnI]: http://en.wikipedia.org/wiki/List_of_restriction_enzyme_cutting_sites:_G-K#K
+[PstI]: http://en.wikipedia.org/wiki/PstI
+[SmaI]: http://en.wikipedia.org/wiki/List_of_restriction_enzyme_cutting_sites:_S#S
+[w85]: http://books.google.com/books?id=eA6iSmR0I4wC
+[1TIT]: http://www.pdb.org/pdb/explore/explore.do?structureId=1TIT
+[NM_003319.4]: http://www.ncbi.nlm.nih.gov/nuccore/NM_003319
+[Q8WZ42] http://www.uniprot.org/blast/?about=Q8WZ42[12677-12765]
+[var] http://web.expasy.org/cgi-bin/variant_pages/get-sprot-variant.pl?VAR_040140
+[pQE30-a] http://www.qiagen.com/literature/vectors_pqe.aspx
+[pQE30-b] http://www.qiagen.com/literature/pqesequences/pqe-30w.txt
+[pUC19-a] http://bccm.belspo.be/db/lmbp_plasmid_details.php?NM=pUC19
+[pUC19-b] http://www.ncbi.nlm.nih.gov/nucleotide/M77789?report=genbank
+
+[[!tag tags/theory]]
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