Synthetic Biology:Vectors/Barcode

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(Proposed barcode)
Current revision (10:45, 11 December 2012) (view source)
(Overview)
 
(24 intermediate revisions not shown.)
Line 1: Line 1:
-
==Proposed barcode==
+
==Overview==
-
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 their first codon (in alphabetical order).
+
The MIT SBWG has been discussing the barcoding of engineered biological systems for a while.  An initial attempt (described at [[Barcodes]]) was made to implement barcodes on many BioBrick coding regions.  Here is a quick (and likely incomplete) overview on some of the issues surrounding barcodes.
 +
 
 +
As Drew has [[Talk:Synthetic Biology:Vectors/Barcode | initially suggested]], there are three basic purposes for barcoding synthetic systems. 
 +
 
 +
#'''Detection''': to enable detection of standard biological parts in arbitrary DNA samples.  For instance, users wish to detect cases of misuse of parts.
 +
#'''Identification''': to enable identification of biological parts, devices and systems.  Such identification may also involve determining the original designer.  For instance, users wish to quickly identify the vector in which their part resides in a typical sequencing reaction.
 +
#'''Authentication''': to enable verification of the integrity of a DNA sequence.  Such a barcode would allow users to check for naturally-occuring or human-induced mutations.
 +
 
 +
There are a couple schemes for barcoding that have been implemented on a trial basis or proposed.
 +
 
 +
#The [[Barcodes | original barcodes scheme]] enabled quick detection of BioBricks via PCR methods.  It is not clear that detection-based barcodes are necessary given the ease with which sequencing can be done.
 +
#The barcode schemes described below are primarily for the purpose of identifying BioBricks.  Since the scheme proposed by Austin is able to encode arbitrary bit strings, such a barcode would permit inline documentation of anything include
 +
#*BioBrick part number
 +
#*URL or doi number
 +
#*designer
 +
#*inline comments
 +
#There is no available proposal to accomplish the third goal of authentication.
 +
 
 +
A universal barcoding scheme is difficult to implement for several reasons.
 +
 
 +
#'''Barcodes should be biologically innocuous.'''  Ideally, barcodes should not be DNA sequences that will encode a biological function including but not limited to the following.
 +
#*initiate transcription (be a promoter)
 +
#*contain coding sequences
 +
#*initiate translation
 +
#*have secondary structure
 +
#*have restriction enzyme cut sites
 +
#*have too many repetitive elements
 +
#*have too many strings of a single nucleotide or strings of purines/pyrimidines.
 +
#'''Barcodes should not be sequences that interfere with system function.'''  There must be a mechanism to insert "escape sequences" to interrupt system-specific meaningful sequences.
 +
#'''Barcodes should not be too long.'''  Long sequences can add to fabrication costs and may impact system function.
 +
#'''Barcodes should be sequenceable.'''
 +
#'''Barcodes should have some mechanism for detecting and/or correcting for errors due to mutation.'''  Additional bases are needed for error detection and correction which can lengthen barcode length.
 +
#*Mutations are relatively straightforward to cope with using standard coding theory.  However, frameshift mutations are far more tricky to deal with.  See [[Talk:Synthetic Biology:Vectors/Barcode]] for details.
 +
 
 +
Austin's barcode scheme to encode bit strings below represents an attempt to address many of these issues.
 +
 
 +
==Scheme to encode bit strings==
 +
 
 +
Enable encoding of arbitrary bit strings into DNA without introducing "biologically bad" sequences.  (Tom and Austin).
 +
 
 +
[http://www.roubaixinteractive.com/PlayGround/Binary_Conversion/Binary_To_Text.asp Text to bit string converter] (ASCII not Unicode)
 +
 
 +
Compression algorithms for DNA sequences: X. Chen, S. Kwong, M. Li, Genome Informatics (GIW'99), Tokyo, Japan, pp.51-61, 1999.
 +
 
 +
 
 +
I've put up a test page for playing with encoding binary into DNA at http://synbio.mit.edu/tools/encoder.cgi
 +
 
 +
Check out the world's first [[Illegal DNA sequence]].
 +
 
 +
The general encoding/decoding method: Each byte of 8 bits is split into 4x2 bits. Each pair of bits at each location is mapped to some nucleotide. For example 00 at position 0 could be mapped to A, 01 at position 1 to T, 00 at position 1 to T, etc. To be decodable, there must be a 1 to 1 mapping at each position from 2 bits to 4 nucleotides. But this leaves <math>24^4</math> different ways to do this type of encoding. Given a string to encode, all possible encodings are looked at and the encoding with the best following properties are used:
 +
* %GC close to 50%
 +
* %GT as high as possible (biased nucleotide use).
 +
 
 +
Biasing the nucleotides makes it less likely for restriction sites or other secondary structures to appear. At the beginning of the encoded string, we attach the encoding table as a simple fixed 12nt (4x3 nt as the 4th nucleotide can be derived from the first 3).
 +
 
 +
The current escape sequence is simply 'AC'. As the encoded string is GT biased, the occurrence of AC turns out to be fairly low. If AC occurs in the encoded sequence, it gets escaped itself with another copy, e.g. AC->ACAC. All other escape sequences begin with AC followed by some sequence. For example, currently we have an escape sequence to represent the beginning and end of the code. There are also escape sequences that allows insertion of arbitrary sequence ('comments?') into the encoding. This allows you to modify the %GC content if desired, break up bad sequences, or whatever else by inserting arbitrary non-coding sequence.
 +
 
 +
Randy suggested some form of compression. Not sure how much space we save or how much more complex it would make the algorithm.
 +
 
 +
Adding the ability to correct base mutations in the DNA (on the computer not in vivo) is possible and a Reed-Solomon code is the most promising candidate code (not implemented yet). Being resistant to frameshifts appears to be more difficult.
 +
 
 +
This algorithm provides the ability to encode anything such as Unicode, pictures, or anything else under the sun. Is the complexity and increase in size worth this capability?
 +
 
 +
==Scheme to encode text only==
 +
 
 +
===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!)
(Note this table was done by hand so please correct errors!)
 +
===Encoding table===
{| border="1"
{| border="1"
|-
|-
! Codon  
! Codon  
 +
! Character
 +
! Rationale
 +
! Codon
! Character
! Character
! Rationale
! Rationale
Line 13: Line 84:
| GCA  
| GCA  
| A
| A
 +
| codon for Ala
 +
| GCT
 +
| a
| codon for Ala
| codon for Ala
|-
|-
-
| AAT
+
| GCC
| B
| B
-
| (represents Asx)
+
| (near alanine)
 +
| GCG
 +
| b
 +
| (near alanine)
|-
|-
| TGC
| TGC
| C
| C
 +
| codon for Cys
 +
| TGT
 +
| c
| codon for Cys
| codon for Cys
|-
|-
| GAC
| GAC
| D
| D
 +
| codon for Asp
 +
| GAT
 +
| d
| codon for Asp
| codon for Asp
|-
|-
| GAA
| GAA
| E
| E
 +
| codon for Glu
 +
| GAG
 +
| e
| codon for Glu
| codon for Glu
|-
|-
| TTC
| TTC
| F
| F
 +
| codon for Phe
 +
| TTT
 +
| f
| codon for Phe
| codon for Phe
|-
|-
| GGA
| GGA
| G
| G
 +
| codon for Gly
 +
| GGC
 +
| g
| codon for Gly
| codon for Gly
|-
|-
| CAC
| CAC
| H
| H
 +
| codon for His
 +
| CAT
 +
| h
| codon for His
| codon for His
|-
|-
-
| ATA
+
| ATC
| I
| I
 +
| codon for Ile
 +
| ATA
 +
| i
| codon for Ile
| codon for Ile
|-
|-
-
| AAG
+
| GGT
| J
| J
-
| (near lysine)
+
| (no reason)
 +
| GGG
 +
| j
 +
| (no reason)
|-
|-
-
| AAA
+
| AAG
| K
| K
 +
| codon for Lys
 +
| AAA
 +
| k
| codon for Lys
| codon for Lys
|-
|-
| CTA
| CTA
| L
| L
 +
| codon for Leu
 +
| CTC
 +
| l
| codon for Leu
| codon for Leu
|-
|-
Line 62: Line 169:
| M
| M
| codon for Met
| codon for Met
 +
| CTG
 +
| m
 +
| sometimes codes for Met
|-
|-
| AAC
| AAC
| N
| N
 +
| codon for Asn
 +
| AAT
 +
| n
| codon for Asn
| codon for Asn
|-
|-
| CCC
| CCC
| O
| O
 +
| (near proline)
 +
| CCU
 +
| o
| (near proline)
| (near proline)
|-
|-
-
| CCA
+
| CCG
| P
| P
 +
| codon for Pro
 +
| CCA
 +
| p
| codon for Pro
| codon for Pro
|-
|-
| CAA
| CAA
| Q
| Q
 +
| codon for Gln
 +
| CAG
 +
| q
| codon for Gln
| codon for Gln
|-
|-
-
| CGA
+
| AGA
| R
| R
 +
| codon for Arg
 +
| AGG
 +
| r
| codon for Arg
| codon for Arg
|-
|-
-
| TCA
+
| AGC
| S
| S
 +
| codon for Ser
 +
| AGT
 +
| s
| codon for Ser
| codon for Ser
|-
|-
| ACA
| ACA
| T
| T
 +
| codon for Thr
 +
| ACT
 +
| t
| codon for Thr
| codon for Thr
|-
|-
| GTC
| GTC
| U
| U
 +
| (near valine)
 +
| GTG
 +
| u
| (near valine)
| (near valine)
|-
|-
| GTA
| GTA
| V
| V
 +
| codon for Val
 +
| GTT
 +
| v
| codon for Val
| codon for Val
|-
|-
Line 102: Line 239:
| W
| W
| codon for Trp
| codon for Trp
 +
| TGA
 +
| w
 +
| (no reason)
|-
|-
-
| TAA
+
| TAG
| X
| X
 +
| resembles a stop codon
 +
| TAA
 +
| x
| resembles a stop codon
| resembles a stop codon
|-
|-
| TAC
| TAC
| Y
| Y
 +
| codon for Tyr
 +
| TAT
 +
| y
| codon for Tyr
| codon for Tyr
|-
|-
-
| CAG
+
| TTG
| Z
| Z
-
| represents Glx
+
| (no reason)
 +
| TTA
 +
| z
 +
| (no reason)
|-
|-
-
| TGA
+
| ATT
| 0
| 0
| zero seems to go with stop codon
| zero seems to go with stop codon
|-
|-
-
| CCG
+
| CTT
| 1
| 1
-
| one starts with an O which is near proline
+
| (looks like an l)
|-
|-
| ACC
| ACC
Line 131: Line 280:
| three starts with a T
| three starts with a T
|-
|-
-
| TTT
+
| CGA
| 4
| 4
-
| four starts with an F
+
| has an R in it
|-
|-
-
| GTG
+
| TCT
| 5
| 5
-
| five has a V in it
+
| (no reason)
|-
|-
| TCC
| TCC
Line 147: Line 296:
| seven starts with an S
| seven starts with an S
|-
|-
-
| GAG
+
| TCA
| 8
| 8
-
| eight starts with an E
+
| (no reason)
|-
|-
-
| AAT
+
| CGT
| 9
| 9
-
| nine starts with an N
+
| (no reason)
|}
|}
-
==Early discussions==
+
===Lookup table===
-
Is there a plan for the barcode?
+
{| border="1"
-
*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 [[Barcodes|pink]].
+
! T
-
**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.--[[User:Bcanton|BC]]
+
! C
-
***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. -[[User:Jkm|Jkm]]
+
! A
-
*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.  --[[Reshma Shetty | RS]]
+
! G
-
**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.
+
! rowspan=4 | T
-
For instance,
+
| f
 +
| 5
 +
| y
 +
| c
 +
! T
 +
|-
 +
| F
 +
| 6
 +
| Y
 +
| C
 +
! C
 +
|-
 +
| z
 +
| 8
 +
| x
 +
| w
 +
! A
 +
|-
 +
| Z
 +
| 7
 +
| X
 +
| W
 +
! G
 +
|-
 +
! rowspan=4 | C
 +
| 1
 +
| o
 +
| h
 +
| 9
 +
! T
 +
|-
 +
| l
 +
| O
 +
| H
 +
| spacer
 +
! C
 +
|-
 +
| L
 +
| p
 +
| Q
 +
| 4
 +
! A
 +
|-
 +
| m
 +
| P
 +
| q
 +
| spacer
 +
! G
 +
|-
 +
! rowspan=4 | A
 +
| 0
 +
| t
 +
| n
 +
| s
 +
! T
 +
|-
 +
| I
 +
| 2
 +
| N
 +
| S
 +
! C
 +
|-
 +
| i
 +
| T
 +
| k
 +
| R
 +
! A
 +
|-
 +
| M
 +
| 3
 +
| K
 +
| r
 +
! G
 +
|-
 +
! rowspan=4 | G
 +
| v
 +
| a
 +
| d
 +
| J
 +
! T
 +
|-
 +
| U
 +
| B
 +
| D
 +
| g
 +
! C
 +
|-
 +
| V
 +
| A
 +
| E
 +
| G
 +
! A
 +
|-
 +
| u
 +
| b
 +
| e
 +
| j
 +
! G
 +
|}
-
<font face="courier">
+
===Start and stop sequences===
-
AAA = 0;
+
What is a good start and stop sequence for the plasmid barcode? 
-
AAC = 1;
+
*We could possibly use the same sequence that is used for the [[Barcodes | 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?
-
AAG = 2;
+
*Another possibility is to flank both sides with the translational stop sequence.
-
AAT = 3;
+
*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)?
-
.
+
-
.
+
-
AGC = 9;
+
-
AGG = A;
+
-
AGT = B;
+
-
.
+
-
.
+
-
.
+
-
GAT = Z;
+
-
</font>
+
-
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?  --[[User:Rshetty|RS]]
+
===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).

Current revision

Contents

Overview

The MIT SBWG has been discussing the barcoding of engineered biological systems for a while. An initial attempt (described at Barcodes) was made to implement barcodes on many BioBrick coding regions. Here is a quick (and likely incomplete) overview on some of the issues surrounding barcodes.

As Drew has initially suggested, there are three basic purposes for barcoding synthetic systems.

  1. Detection: to enable detection of standard biological parts in arbitrary DNA samples. For instance, users wish to detect cases of misuse of parts.
  2. Identification: to enable identification of biological parts, devices and systems. Such identification may also involve determining the original designer. For instance, users wish to quickly identify the vector in which their part resides in a typical sequencing reaction.
  3. Authentication: to enable verification of the integrity of a DNA sequence. Such a barcode would allow users to check for naturally-occuring or human-induced mutations.

There are a couple schemes for barcoding that have been implemented on a trial basis or proposed.

  1. The original barcodes scheme enabled quick detection of BioBricks via PCR methods. It is not clear that detection-based barcodes are necessary given the ease with which sequencing can be done.
  2. The barcode schemes described below are primarily for the purpose of identifying BioBricks. Since the scheme proposed by Austin is able to encode arbitrary bit strings, such a barcode would permit inline documentation of anything include
    • BioBrick part number
    • URL or doi number
    • designer
    • inline comments
  3. There is no available proposal to accomplish the third goal of authentication.

A universal barcoding scheme is difficult to implement for several reasons.

  1. Barcodes should be biologically innocuous. Ideally, barcodes should not be DNA sequences that will encode a biological function including but not limited to the following.
    • initiate transcription (be a promoter)
    • contain coding sequences
    • initiate translation
    • have secondary structure
    • have restriction enzyme cut sites
    • have too many repetitive elements
    • have too many strings of a single nucleotide or strings of purines/pyrimidines.
  2. Barcodes should not be sequences that interfere with system function. There must be a mechanism to insert "escape sequences" to interrupt system-specific meaningful sequences.
  3. Barcodes should not be too long. Long sequences can add to fabrication costs and may impact system function.
  4. Barcodes should be sequenceable.
  5. Barcodes should have some mechanism for detecting and/or correcting for errors due to mutation. Additional bases are needed for error detection and correction which can lengthen barcode length.
    • Mutations are relatively straightforward to cope with using standard coding theory. However, frameshift mutations are far more tricky to deal with. See Talk:Synthetic Biology:Vectors/Barcode for details.

Austin's barcode scheme to encode bit strings below represents an attempt to address many of these issues.

Scheme to encode bit strings

Enable encoding of arbitrary bit strings into DNA without introducing "biologically bad" sequences. (Tom and Austin).

Text to bit string converter (ASCII not Unicode)

Compression algorithms for DNA sequences: X. Chen, S. Kwong, M. Li, Genome Informatics (GIW'99), Tokyo, Japan, pp.51-61, 1999.


I've put up a test page for playing with encoding binary into DNA at http://synbio.mit.edu/tools/encoder.cgi

Check out the world's first Illegal DNA sequence.

The general encoding/decoding method: Each byte of 8 bits is split into 4x2 bits. Each pair of bits at each location is mapped to some nucleotide. For example 00 at position 0 could be mapped to A, 01 at position 1 to T, 00 at position 1 to T, etc. To be decodable, there must be a 1 to 1 mapping at each position from 2 bits to 4 nucleotides. But this leaves 244 different ways to do this type of encoding. Given a string to encode, all possible encodings are looked at and the encoding with the best following properties are used:

  •  %GC close to 50%
  •  %GT as high as possible (biased nucleotide use).

Biasing the nucleotides makes it less likely for restriction sites or other secondary structures to appear. At the beginning of the encoded string, we attach the encoding table as a simple fixed 12nt (4x3 nt as the 4th nucleotide can be derived from the first 3).

The current escape sequence is simply 'AC'. As the encoded string is GT biased, the occurrence of AC turns out to be fairly low. If AC occurs in the encoded sequence, it gets escaped itself with another copy, e.g. AC->ACAC. All other escape sequences begin with AC followed by some sequence. For example, currently we have an escape sequence to represent the beginning and end of the code. There are also escape sequences that allows insertion of arbitrary sequence ('comments?') into the encoding. This allows you to modify the %GC content if desired, break up bad sequences, or whatever else by inserting arbitrary non-coding sequence.

Randy suggested some form of compression. Not sure how much space we save or how much more complex it would make the algorithm.

Adding the ability to correct base mutations in the DNA (on the computer not in vivo) is possible and a Reed-Solomon code is the most promising candidate code (not implemented yet). Being resistant to frameshifts appears to be more difficult.

This algorithm provides the ability to encode anything such as Unicode, pictures, or anything else under the sun. Is the complexity and increase in size worth this capability?

Scheme to encode text only

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
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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).
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