Synthetic Biology:BioBricks/Part fabrication: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
No edit summary
Line 9: Line 9:
#[[Silver: BB Strategy | Silver lab strategy]]
#[[Silver: BB Strategy | Silver lab strategy]]


The exact approach used when fabricating a BioBrick part depends on the fabrication approach being used as well as the type of part being constructed.
The exact approach used when fabricating a BioBrick part depends on the fabrication method as well as the type of part being constructed.


==Constructing a BioBrick part via PCR==
==Constructing a BioBrick part via PCR==
A BioBrick can be constructed via PCR if there already exists template DNA from which the BioBricks can be amplified or if the part is short enough that it can be created by [[Annealing and primer extension | primer annealing and extension]].  When constructing a BioBrick part via PCR, specific sequences corresponding to the BioBrick ends must be included on the 5' end of each of the two PCR primers.  The exact sequence of that primer tail depends on whether the desired BioBrick parts is a protein coding sequence or some other kind of BioBrick part.


===Standard part fabrication===
===Standard part fabrication===
Use this approach for promoters, ribosome binding sites, terminators and most other BioBricks parts.


====Prefix====
====Prefix====
Line 51: Line 55:
##promote addition of an A base on the opposite strand by Taq polymerase for high efficiency TA cloning if desired.  See [[Knight:TOPO TA cloning#Notes | notes on TOPO TA cloning]].
##promote addition of an A base on the opposite strand by Taq polymerase for high efficiency TA cloning if desired.  See [[Knight:TOPO TA cloning#Notes | notes on TOPO TA cloning]].
#Random extra bases
#Random extra bases
====Quick reference====
For those who already are familiar with constructing BioBricks parts, the sequences you need are included here for your copying pleasure.
Copy and paste the following 30 bp sequence onto the 5' end of your upstream primer:
5' ---> 3'
CCG TTT CTG AAT TCG CGG CCG CTT CTA GAG
Copy and paste the following 29 bp sequence onto the 5' end of your downstream primer:
5' ---> 3'
CCG TTT CTC TGC AGC GGC CGC TAC TAG TA


===Protein coding sequence fabrication===
===Protein coding sequence fabrication===
Line 56: Line 71:
Construction of protein coding sequences in BioBricks form requires slightly specialized BioBricks prefixes and suffixes for two reasons.
Construction of protein coding sequences in BioBricks form requires slightly specialized BioBricks prefixes and suffixes for two reasons.
#The prefix is slightly altered to ensure appropriate spacing between the ribsome binding site and the start codon.
#The prefix is slightly altered to ensure appropriate spacing between the ribsome binding site and the start codon.
#BioBricks coding sequences standardly end with two TAA stop codons.   
#BioBricks coding sequences standardly end with two sequential TAA stop codons.   


====Prefix====
====Prefix====
Line 93: Line 108:
##promote addition of an A base on the opposite strand by Taq polymerase for high efficiency TA cloning if desired.  See [[Knight:TOPO TA cloning#Notes | notes on TOPO TA cloning]].
##promote addition of an A base on the opposite strand by Taq polymerase for high efficiency TA cloning if desired.  See [[Knight:TOPO TA cloning#Notes | notes on TOPO TA cloning]].
#Random extra bases
#Random extra bases
====Quick reference====
For those who already are familiar with constructing BioBricks parts, the sequences you need are included here for your copying pleasure.
Copy and paste the following 31 bp sequence onto the 5' end of your upstream primer for your coding sequence:
includes the ATG start codon!
5' ---> 3'
CCG TTT CTG AAT TCG CGG CCG CTT CTA GAT G
Copy and paste the following 35 bp sequence onto the 5' end of your upstream primer for your coding sequence:
includes the TAATAA double stop codon!
5' ---> 3'
CCG TTT CTC TGC AGC GGC CGC TAC TAG TAT TAT TA


==Constructing a BioBrick part via direct synthesis==
==Constructing a BioBrick part via direct synthesis==
Line 101: Line 129:
#[http://hdl.handle.net/1721.1/21168 Idempotent Vector Design for Standard Assembly of BioBricks] by [[Tom Knight]]
#[http://hdl.handle.net/1721.1/21168 Idempotent Vector Design for Standard Assembly of BioBricks] by [[Tom Knight]]
#http://parts.mit.edu/r/parts/htdocs/Assembly/rbs_cds.cgi
#http://parts.mit.edu/r/parts/htdocs/Assembly/rbs_cds.cgi
==Contacts==
The BioBricks idempotent assembly scheme was designed by [[Tom Knight]].
This page was developed by [[Reshma Shetty]] for instructional purposes.

Revision as of 12:49, 4 January 2006

UNDER CONSTRUCTION: use at your own risk. This information may contain errors.


This page is intended as a how to guide for constructing novel BioBrick parts for submission to the Registry of Standard Biological Parts.

For help in assembling two preexisting BioBricks parts together, see one of the following pages.

  1. BioBricks construction tutorial
  2. 3A assembly
  3. Silver lab strategy

The exact approach used when fabricating a BioBrick part depends on the fabrication method as well as the type of part being constructed.

Constructing a BioBrick part via PCR

A BioBrick can be constructed via PCR if there already exists template DNA from which the BioBricks can be amplified or if the part is short enough that it can be created by primer annealing and extension. When constructing a BioBrick part via PCR, specific sequences corresponding to the BioBrick ends must be included on the 5' end of each of the two PCR primers. The exact sequence of that primer tail depends on whether the desired BioBrick parts is a protein coding sequence or some other kind of BioBrick part.

Standard part fabrication

Use this approach for promoters, ribosome binding sites, terminators and most other BioBricks parts.

Prefix

5' CC  GTTTCT GAATTC GCGGCCGC  T  TCTAGA  G   [part] 3'
3' GG  CAAAGA CTTAAG CGCCGGCG  A  AGATCT  C   [part] 5'
   (1) (2)    (3)    (4)      (5) (6)    (7)  (8)
  1. Random extra bases
  2. Extra bases designed to both
    1. permit cutting of the PCR product with EcoRI by providing extra "spacer" bases. See notes on cutting near the ends of linear DNA fragments.
    2. promote addition of an A base on the opposite strand by Taq polymerase for high efficiency TA cloning if desired. See notes on TOPO TA cloning.
  3. EcoRI recognition site
  4. NotI recognition site
  5. Extra base to prevent inadvertent creation of EcoBI or EcoKI methylation sites which could inhibit efficient digestion by the BioBricks enzymes.
  6. SpeI recognition site
  7. Extra G base to prevent inadvertent creation of either
    1. a GATC site (which can undergo methylation in some strains thereby inhibiting digestion by the BioBricks enzymes.)
    2. an ATG start codon
  8. Approximately 20 bp of sequence that matches the 5' end of the part you wish to construct.

Suffix

5' [part] T ACTAGT  A  GCGGCCG CTGCAG AGAAAC GG  3'
3' [part] A TGATCA  T  CGCCGGC GACGTC TCTTTG CC  5'
   (1)      (2)    (3) (4)     (5)    (6)    (7)
  • The above sequence assumes that your part is on the forward strand running in the 5' to 3' direction. To construct a PCR primer, you will need to use the bottom strand in the reverse direction.
  1. Approximately 20 bp of sequence that matches the 3' end of the part you wish to construct.
  2. SpeI recognition site
  3. Extra base to prevent inadvertent creation of EcoBI or EcoKI methylation sites which could inhibit efficient digestion by the BioBricks enzymes.
  4. NotI recognition site
  5. PstI recognition site
  6. Extra bases designed to both
    1. permit cutting of the PCR product with PstI by providing extra "spacer" bases. See notes on cutting near the ends of linear DNA fragments.
    2. promote addition of an A base on the opposite strand by Taq polymerase for high efficiency TA cloning if desired. See notes on TOPO TA cloning.
  7. Random extra bases

Quick reference

For those who already are familiar with constructing BioBricks parts, the sequences you need are included here for your copying pleasure. 

Copy and paste the following 30 bp sequence onto the 5' end of your upstream primer:
5' ---> 3'
CCG TTT CTG AAT TCG CGG CCG CTT CTA GAG

Copy and paste the following 29 bp sequence onto the 5' end of your downstream primer:
5' ---> 3'
CCG TTT CTC TGC AGC GGC CGC TAC TAG TA

Protein coding sequence fabrication

Construction of protein coding sequences in BioBricks form requires slightly specialized BioBricks prefixes and suffixes for two reasons.

  1. The prefix is slightly altered to ensure appropriate spacing between the ribsome binding site and the start codon.
  2. BioBricks coding sequences standardly end with two sequential TAA stop codons.

Prefix

5' CC  GTTTCT GAATTC GCGGCCGC  T  TCTAG [ATG Remaining CDS] 3'
3' GG  CAAAGA CTTAAG CGCCGGCG  A  AGATC [TAC Remaining CDS] 5'
   (1) (2)    (3)    (4)      (5) (6)    (7) (8)
  1. Random extra bases
  2. Extra bases designed to both
    1. permit cutting of the PCR product with EcoRI by providing extra "spacer" bases. See notes on cutting near the ends of linear DNA fragments.
    2. promote addition of an A base on the opposite strand by Taq polymerase for high efficiency TA cloning if desired. See notes on TOPO TA cloning.
  3. EcoRI recognition site
  4. NotI recognition site
  5. Extra base to prevent inadvertent creation of EcoBI or EcoKI methylation sites which could inhibit efficient digestion by the BioBricks enzymes.
  6. SpeI recognition site
  7. An ATG start codon
  8. Approximately 20 bp of sequence that matches the 5' end of the coding sequence you wish to construct (excluding the start codon).

Suffix

5' [part] TAATAA T ACTAGT  A  GCGGCCG CTGCAG AGAAAC GG  3'
3' [part] ATTATT A TGATCA  T  CGCCGGC GACGTC TCTTTG CC  5'
   (1)    (2)      (3)    (4) (5)     (6)    (7)    (8)
  • The above sequence assumes that your part is on the forward strand running in the 5' to 3' direction. To construct a PCR primer, you will need to use the bottom strand in the reverse direction.
  1. Approximately 20 bp of sequence that matches the 3' end of the CDS you wish to construct (excluding the stop codon).
  2. Two sequential stop codons. TAA is the default stop codon used in all BioBricks coding sequences.
  3. SpeI recognition site
  4. Extra base to prevent inadvertent creation of EcoBI or EcoKI methylation sites which could inhibit efficient digestion by the BioBricks enzymes.
  5. NotI recognition site
  6. PstI recognition site
  7. Extra bases designed to both
    1. permit cutting of the PCR product with PstI by providing extra "spacer" bases. See notes on cutting near the ends of linear DNA fragments.
    2. promote addition of an A base on the opposite strand by Taq polymerase for high efficiency TA cloning if desired. See notes on TOPO TA cloning.
  8. Random extra bases

Quick reference

For those who already are familiar with constructing BioBricks parts, the sequences you need are included here for your copying pleasure. 

Copy and paste the following 31 bp sequence onto the 5' end of your upstream primer for your coding sequence:
includes the ATG start codon!
5' ---> 3'
CCG TTT CTG AAT TCG CGG CCG CTT CTA GAT G

Copy and paste the following 35 bp sequence onto the 5' end of your upstream primer for your coding sequence:
includes the TAATAA double stop codon!
5' ---> 3'
CCG TTT CTC TGC AGC GGC CGC TAC TAG TAT TAT TA

Constructing a BioBrick part via direct synthesis

Coming soon.

References

  1. Idempotent Vector Design for Standard Assembly of BioBricks by Tom Knight
  2. http://parts.mit.edu/r/parts/htdocs/Assembly/rbs_cds.cgi

Contacts

The BioBricks idempotent assembly scheme was designed by Tom Knight.

This page was developed by Reshma Shetty for instructional purposes.

Retrieved from "https://openwetware.org/mediawiki/index.php?title=Synthetic_Biology:BioBricks/Part_fabrication&oldid=16136"