Synthetic Biology:Vectors/Barcode

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*I didn't bother to try avoiding certain codons like start codons.
*I didn't bother to try avoiding certain codons like start codons.
*These codons may not be optimally spaced from one another?  Tom doesn't think this matters.
*These codons may not be optimally spaced from one another?  Tom doesn't think this matters.
-
*Can we get away with having a case-insensitive code.
+
*Can we get away with having a case-insensitive code?
==Early discussions==
==Early discussions==

Revision as of 12:20, 2 February 2006

Contents

Proposed barcode

Feedback more than welcome!

Codon table

Each codon represents an alphanumeric character (case-insensitive). For convenience, those letters of the alphabet which represent a single letter amino acid code are coded by one of the amino acid's codons (aiming for near 50% GC content).

(Note this table was done by hand so please correct errors!)

Codon Character Rationale
GCA A codon for Ala
GAT B (represents Asx)
TGC C codon for Cys
GAC D codon for Asp
GAA E codon for Glu
TTC F codon for Phe
GGA G codon for Gly
CAC H codon for His
ATC I codon for Ile
AAG J (near lysine)
AAA K codon for Lys
CTA L codon for Leu
ATG M codon for Met
AAC N codon for Asn
CCC O (near proline)
CCA P codon for Pro
CAA Q codon for Gln
AGA R codon for Arg
AGC S codon for Ser
ACA T codon for Thr
GTC U (near valine)
GTA V codon for Val
TGG W codon for Trp
TAG X resembles a stop codon
TAC Y codon for Tyr
CAG Z represents Glx
TGA 0 zero seems to go with stop codon
CCT 1 one starts with an O which is near proline
ACC 2 two starts with a T
ACG 3 three starts with a T
TTT 4 four starts with an F
GTG 5 five has a V in it
TCC 6 six starts with an S
TCG 7 seven starts with an S
GAG 8 eight starts with an E
AAT 9 nine starts with an N

Start and stop sequences

What is a good start and stop sequence for the plasmid barcode?

  • We could possibly use the same sequence that is used for the CDS barcodes (i.e. C TGA TAG TGC TAG TGT AGA T C) without the variable nucleotide. Or would this just confuse any diagnostics people try to run on constructs?
  • Another possibility is to flank both sides with the translational stop sequence.
  • Tom pointed out that the barcode should probably be as GC content neutral (i.e. try to avoid all AT or all GC codons).

Notes

  • I didn't bother to try avoiding certain codons like start codons.
  • These codons may not be optimally spaced from one another? Tom doesn't think this matters.
  • Can we get away with having a case-insensitive code?

Early discussions

Is there a plan for the barcode?

  • Should the barcode only be readable by sequencing or is it sufficient to just look for an amplified band in a PCR reaction.
    • If PCR is sufficient we could build in a unique sequence just before the BB prefix and then design a reverse primer to that sequence to use along with VF.
  • It seems like the most likely short-mid term problem is that a researcher would be uncertain as to which BioBrick vector they had, rather than the doomsday question of trying to work out if there is a BioBrick vector somewhere in the drink that turned Drew's hair pink.
    • Given this assumption, could we choose restriction sites, each of which are found uniquely in one of our BioBrick vectors? A researcher could just prep, digest and run on a gel to tell which vector they had.--BC
      • It might be useful to be able to tell the plasmid (and resistance) by colony PCR rather than a prep. A PCR requires less starting material. -Jkm
  • There is no current plan for the barcode. The intention was just to make the identity of the plasmid obvious from a sequencing reaction but this goal is compatible with making the plasmid identifiable via a colony PCR as well. Choosing a unique restriction site for each vector would be more difficult because that would involve placing additional requirements in the BioBricks standard. i.e. Parts cannot have any of the BioBrick enzymes nor this list of restriction enzymes that are identifiers for vectors. This doesn't seem practical to me. -- RS
    • I'm not in favor of inserting restriction sites but you can probably get away without using any new enzymes under certain assumptions. First let's assume one always inserts into a new plasmid (3-way ligation, either with or without 3 antibiotic selection). Then you can just insert various combinations of BioBrick enzymes into specific locations into the plasmids and look at the pattern of bands when you cut with them. The benefit of this is let's say you cut a part with ES, run on gel, and based on the band pattern from the plasmid, you know immediately which plasmid it's in, and if it's correct, you isolate the part band and can proceed with the assembly. You have the same problem as below if one of the plasmid pieces is the same length as the part, but now you may have more potential conflicting bands. 3-antibiotic assembly without purification shouldn't really be impacted by a couple more pieces of plasmid floating around. You can also take this idea by defining another single enzyme that will be used for this purpose and you can tell plasmids apart again by the differetn lengths generated after digest. So you definitely don't need one enzyme/plasmid.

One plan that I am currently considering is actually encoding the name of the plasmid in DNA. For instance,

AAA = 0; AAC = 1; AAG = 2; AAT = 3; . . . AGC = 9; AGG = A; AGT = B; . . . GAT = Z;

So that you could literally write out pSB5AC4-P1010.I50020 in DNA. Of course, we may want to make this slightly more intelligent to space out characters, include start and stop strings and avoid key codons like ATG, TAA and TGA. Any comments? --RS

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