Synthetic Biology:Vectors/Barcode

Alternative barcode

Actually encode a bit string into the DNA (Tom and Austin).

Proposed barcode

Feedback more than welcome!

Case-sensitive codon tables

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!)

Encoding table

Codon	Character	Rationale	Codon	Character	Rationale
GCA	A	codon for Ala	GCT	a	codon for Ala
GCC	B	(near alanine)	GCG	b	(near alanine)
TGC	C	codon for Cys	TGT	c	codon for Cys
GAC	D	codon for Asp	GAT	d	codon for Asp
GAA	E	codon for Glu	GAG	e	codon for Glu
TTC	F	codon for Phe	TTT	f	codon for Phe
GGA	G	codon for Gly	GGC	g	codon for Gly
CAC	H	codon for His	CAT	h	codon for His
ATC	I	codon for Ile	ATA	i	codon for Ile
GGT	J	(no reason)	GGG	j	(no reason)
AAG	K	codon for Lys	AAA	k	codon for Lys
CTA	L	codon for Leu	CTC	l	codon for Leu
ATG	M	codon for Met	CTG	m	sometimes codes for Met
AAC	N	codon for Asn	AAT	n	codon for Asn
CCC	O	(near proline)	CCU	o	(near proline)
CCG	P	codon for Pro	CCA	p	codon for Pro
CAA	Q	codon for Gln	CAG	q	codon for Gln
AGA	R	codon for Arg	AGG	r	codon for Arg
AGC	S	codon for Ser	AGT	s	codon for Ser
ACA	T	codon for Thr	ACT	t	codon for Thr
GTC	U	(near valine)	GTG	u	(near valine)
GTA	V	codon for Val	GTT	v	codon for Val
TGG	W	codon for Trp	TGA	w	(no reason)
TAG	X	resembles a stop codon	TAA	x	resembles a stop codon
TAC	Y	codon for Tyr	TAT	y	codon for Tyr
TTG	Z	(no reason)	TTA	z	(no reason)
ATT	0	zero seems to go with stop codon
CTT	1	(looks like an l)
ACC	2	two starts with a T
ACG	3	three starts with a T
CGA	4	has an R in it
TCT	5	(no reason)
TCC	6	six starts with an S
TCG	7	seven starts with an S
TCA	8	(no reason)
CGT	9	(no reason)

Lookup table

	T	C	A	G
T	f	5	y	c	T
	F	6	Y	C	C
	z	8	x	w	A
	Z	7	X	W	G
C	1	o	h	9	T
	l	O	H	spacer	C
	L	p	Q	4	A
	m	P	q	spacer	G
A	0	t	n	s	T
	I	2	N	S	C
	i	T	k	R	A
	M	3	K	r	G
G	v	a	d	J	T
	U	B	D	g	C
	V	A	E	G	A
	u	b	e	j	G

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.
• Maybe a start and stop sequence isn't necessary?
• One problem with this codon table it that it becomes possible to accidentally encode BioBricks sites in the barcode. A case-insensitive code might reduce the likelihood of that happening? Any possible fixes to this problem? Use one of the codons that doesn't encode a alphanumeric character as a "spacer" in this eventuality (i.e. CGC or CGG)?

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.
• Tom pointed out that the barcode should probably be as GC content neutral (i.e. try to avoid all AT or all GC codons).

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