Synthetic Biology:BioBricks/Part fabrication: Difference between revisions
No edit summary |
No edit summary |
||
Line 3: | Line 3: | ||
'''This page is intended as a how-to guide for constructing novel BioBrick parts for submission to the [http://parts.mit.edu Registry of Standard Biological Parts].''' | '''This page is intended as a how-to guide for constructing novel BioBrick parts for submission to the [http://parts.mit.edu Registry of Standard Biological Parts].''' | ||
'''For those already familiar with making BioBrick parts, see the [[Synthetic Biology:BioBricks/Part fabrication quick reference | quick reference guide]].''' | '''For those already familiar with making BioBrick parts, see the [[Synthetic Biology:BioBricks/Part fabrication/quick reference | quick reference guide]].''' | ||
'''For help in assembling two preexisting BioBricks parts together, see one of the following pages.''' | '''For help in assembling two preexisting BioBricks parts together, see one of the following pages.''' | ||
Line 26: | Line 26: | ||
====Prefix==== | ====Prefix==== | ||
5' CC | 5' GTTTCT CC GAATTC GCGGCCGC T TCTAGA G [part] 3' | ||
3' GG | 3' CAAAGA GG CTTAAG CGCCGGCG A AGATCT C [part] 5' | ||
(1) (2) | (1) (2)(3) (4) (5) (6) (7) (8) | ||
#Extra bases designed to both | #Extra bases designed to both | ||
##permit cutting of the PCR product with EcoRI by providing extra "spacer" bases. See [[Restriction digest#Notes | notes on cutting near the ends of linear DNA fragments]]. | ##permit cutting of the PCR product with EcoRI by providing extra "spacer" bases. See [[Restriction digest#Notes | notes on cutting near the ends of linear DNA fragments]]. | ||
##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 spacer bases | |||
#EcoRI recognition site | #EcoRI recognition site | ||
#NotI recognition site | #NotI recognition site | ||
Line 45: | Line 45: | ||
====Suffix==== | ====Suffix==== | ||
5' [part] T ACTAGT A GCGGCCG CTGCAG AGAAAC | 5' [part] T ACTAGT A GCGGCCG CTGCAG GG AGAAAC 3' | ||
3' [part] A TGATCA T CGCCGGC GACGTC TCTTTG | 3' [part] A TGATCA T CGCCGGC GACGTC CC TCTTTG 5' | ||
(1) (2) (3) (4) (5) (6) | (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. | *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. | ||
Line 56: | Line 56: | ||
#NotI recognition site | #NotI recognition site | ||
#PstI recognition site | #PstI recognition site | ||
#Random extra spacer bases | |||
#Extra bases designed to both | #Extra bases designed to both | ||
##permit cutting of the PCR product with PstI by providing extra "spacer" bases. See [[Restriction digest#Notes | notes on cutting near the ends of linear DNA fragments]]. | ##permit cutting of the PCR product with PstI by providing extra "spacer" bases. See [[Restriction digest#Notes | notes on cutting near the ends of linear DNA fragments]]. | ||
##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]]. | ||
====Quick reference==== | ====Quick reference==== | ||
Line 66: | Line 66: | ||
Copy and paste the following 30 bp sequence onto the 5' end of your upstream primer: | Copy and paste the following 30 bp sequence onto the 5' end of your upstream primer: | ||
5' ---> 3' | 5' ---> 3' | ||
CCG | GTT TCT CCG AAT TCG CGG CCG CTT CTA GAG | ||
Copy and paste the following 29 bp sequence onto the 5' end of your downstream primer: | Copy and paste the following 29 bp sequence onto the 5' end of your downstream primer: | ||
5' ---> 3' | 5' ---> 3' | ||
GTT TCT CCC TGC AGC GGC CGC TAC TAG TA | |||
===Protein coding sequence fabrication=== | ===Protein coding sequence fabrication=== | ||
Line 80: | Line 80: | ||
====Prefix==== | ====Prefix==== | ||
5' CC | 5' GTTTCT CC GAATTC GCGGCCGC T TCTAG [ATG Remaining CDS] 3' | ||
3' GG | 3' CAAAGA GG CTTAAG CGCCGGCG A AGATC [TAC Remaining CDS] 5' | ||
(1) (2) | (1) (2)(3) (4) (5) (6) (7) (8) | ||
#Extra bases designed to both | #Extra bases designed to both | ||
##permit cutting of the PCR product with EcoRI by providing extra "spacer" bases. See [[Restriction digest#Notes | notes on cutting near the ends of linear DNA fragments]]. | ##permit cutting of the PCR product with EcoRI by providing extra "spacer" bases. See [[Restriction digest#Notes | notes on cutting near the ends of linear DNA fragments]]. | ||
##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 spacer bases | |||
#EcoRI recognition site | #EcoRI recognition site | ||
#NotI recognition site | #NotI recognition site | ||
Line 97: | Line 97: | ||
====Suffix==== | ====Suffix==== | ||
5' [part] TAATAA T ACTAGT A GCGGCCG CTGCAG AGAAAC | 5' [part] TAATAA T ACTAGT A GCGGCCG CTGCAG GG AGAAAC 3' | ||
3' [part] ATTATT A TGATCA T CGCCGGC GACGTC TCTTTG | 3' [part] ATTATT A TGATCA T CGCCGGC GACGTC CC TCTTTG 5' | ||
(1) (2) (3) (4) (5) (6) (7) | (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. | *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. | ||
Line 109: | Line 109: | ||
#NotI recognition site | #NotI recognition site | ||
#PstI recognition site | #PstI recognition site | ||
#Random extra spacer bases | |||
#Extra bases designed to both | #Extra bases designed to both | ||
##permit cutting of the PCR product with PstI by providing extra "spacer" bases. See [[Restriction digest#Notes | notes on cutting near the ends of linear DNA fragments]]. | ##permit cutting of the PCR product with PstI by providing extra "spacer" bases. See [[Restriction digest#Notes | notes on cutting near the ends of linear DNA fragments]]. | ||
##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]]. | ||
====Quick reference==== | ====Quick reference==== | ||
Line 120: | Line 120: | ||
includes the ATG start codon! | includes the ATG start codon! | ||
5' ---> 3' | 5' ---> 3' | ||
CCG | GTT TCT CCG AAT TCG CGG CCG CTT CTA GAT G | ||
Copy and paste the following 35 bp sequence onto the 5' end of your downstream primer for your coding sequence: | Copy and paste the following 35 bp sequence onto the 5' end of your downstream primer for your coding sequence: | ||
includes the TAATAA double stop codon! | includes the TAATAA double stop codon! | ||
5' ---> 3' | 5' ---> 3' | ||
GTT TCT CCC TGC AGC GGC CGC TAC TAG TAT TAT TA | |||
==Constructing a BioBrick part via direct synthesis== | ==Constructing a BioBrick part via direct synthesis== |
Revision as of 09:09, 12 January 2006
UNDER CONSTRUCTION: use at your own risk.
This page is intended as a how-to guide for constructing novel BioBrick parts for submission to the Registry of Standard Biological Parts.
For those already familiar with making BioBrick parts, see the quick reference guide.
For help in assembling two preexisting BioBricks parts together, see one of the following pages.
Introduction
This page serves as documentation for how to construct a BioBrick part. For a more detailed explanation of the development of the BioBricks scheme, see Tom Knight's technical report Idempotent Vector Design for Standard Assembly of BioBricks.
The exact approach used when fabricating a BioBrick part depends on the fabrication method (PCR or direct synthesis) as well as the type of part being constructed (standard part or protein coding sequence).
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' GTTTCT CC GAATTC GCGGCCGC T TCTAGA G [part] 3' 3' CAAAGA GG CTTAAG CGCCGGCG A AGATCT C [part] 5' (1) (2)(3) (4) (5) (6) (7) (8)
- Extra bases designed to both
- permit cutting of the PCR product with EcoRI by providing extra "spacer" bases. See notes on cutting near the ends of linear DNA fragments.
- 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.
- Random extra spacer bases
- EcoRI recognition site
- NotI recognition site
- Extra base to prevent inadvertent creation of EcoBI or EcoKI methylation sites which could inhibit efficient digestion by the BioBricks enzymes.
- SpeI recognition site
- Extra G base to prevent inadvertent creation of either
- a GATC site (which can undergo methylation in some strains thereby inhibiting digestion by the BioBricks enzymes.)
- an ATG start codon
- 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 GG AGAAAC 3' 3' [part] A TGATCA T CGCCGGC GACGTC CC TCTTTG 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.
- Approximately 20 bp of sequence that matches the 3' end of the part you wish to construct.
- SpeI recognition site
- Extra base to prevent inadvertent creation of EcoBI or EcoKI methylation sites which could inhibit efficient digestion by the BioBricks enzymes.
- NotI recognition site
- PstI recognition site
- Random extra spacer bases
- Extra bases designed to both
- permit cutting of the PCR product with PstI by providing extra "spacer" bases. See notes on cutting near the ends of linear DNA fragments.
- 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.
Quick reference
Once you are ready to design your primers for making a BioBrick, you can copy and paste the following sequences into your primers.
Copy and paste the following 30 bp sequence onto the 5' end of your upstream primer: 5' ---> 3' GTT TCT CCG 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' GTT TCT CCC 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.
- 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 sequential TAA stop codons.
Prefix
5' GTTTCT CC GAATTC GCGGCCGC T TCTAG [ATG Remaining CDS] 3' 3' CAAAGA GG CTTAAG CGCCGGCG A AGATC [TAC Remaining CDS] 5' (1) (2)(3) (4) (5) (6) (7) (8)
- Extra bases designed to both
- permit cutting of the PCR product with EcoRI by providing extra "spacer" bases. See notes on cutting near the ends of linear DNA fragments.
- 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.
- Random extra spacer bases
- EcoRI recognition site
- NotI recognition site
- Extra base to prevent inadvertent creation of EcoBI or EcoKI methylation sites which could inhibit efficient digestion by the BioBricks enzymes.
- SpeI recognition site
- An ATG start codon
- 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 GG AGAAAC 3' 3' [part] ATTATT A TGATCA T CGCCGGC GACGTC CC TCTTTG 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.
- Approximately 20 bp of sequence that matches the 3' end of the CDS you wish to construct (excluding the stop codon).
- Two sequential stop codons. TAA is the default stop codon used in all BioBricks coding sequences.
- SpeI recognition site
- Extra base to prevent inadvertent creation of EcoBI or EcoKI methylation sites which could inhibit efficient digestion by the BioBricks enzymes.
- NotI recognition site
- PstI recognition site
- Random extra spacer bases
- Extra bases designed to both
- permit cutting of the PCR product with PstI by providing extra "spacer" bases. See notes on cutting near the ends of linear DNA fragments.
- 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.
Quick reference
Once you are ready to design your primers for making a BioBrick, you can copy and paste the following sequences into your primers.
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' GTT TCT CCG AAT TCG CGG CCG CTT CTA GAT G
Copy and paste the following 35 bp sequence onto the 5' end of your downstream primer for your coding sequence: includes the TAATAA double stop codon! 5' ---> 3' GTT TCT CCC TGC AGC GGC CGC TAC TAG TAT TAT TA
Constructing a BioBrick part via direct synthesis
Coming soon.
References
- Idempotent Vector Design for Standard Assembly of BioBricks by Tom Knight
- 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.