Help:Protocols/PCR Standardization

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. 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' GTTTCTT C GAATTC GCGGCCGC  T  TCTAGA  G   [part] 3'
3' CAAAGAA G CTTAAG CGCCGGCG  A  AGATCT  C   [part] 5'
   (1)    (2)(3)    (4)      (5) (6)    (7)  (8)
  1. Extra bases designed to both
    1. permit cutting of the PCR product with EcoRI by providing extra "spacer" bases.
    2. promote addition of an A base on the opposite strand by Taq polymerase for high efficiency TA cloning if desired.
  2. Random extra spacer base
  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. XbaI 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 G AAGAAAC   3'
3' [part] A TGATCA  T  CGCCGGC GACGTC C TTCTTTG   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. Random extra spacer base
  7. Extra bases designed to both
    1. permit cutting of the PCR product with PstI by providing extra "spacer" bases.
    2. promote addition of an A base on the opposite strand by Taq polymerase for high efficiency TA cloning if desired.

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 TCG 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 TCC 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' GTTTCTT C GAATTC GCGGCCGC  T  TCTAG [ATG Remaining CDS] 3'
3' CAAAGAA G CTTAAG CGCCGGCG  A  AGATC [TAC Remaining CDS] 5'
   (1)    (2)(3)    (4)      (5) (6)    (7) (8)
  1. Extra bases designed to both
    1. permit cutting of the PCR product with EcoRI by providing extra "spacer" bases.
    2. promote addition of an A base on the opposite strand by Taq polymerase for high efficiency TA cloning if desired.
  2. Random extra spacer base
  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. XbaI 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 G AAGAAAC  3'
3' [part] ATTATT A TGATCA  T  CGCCGGC GACGTC C TTCTTTG  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. Random extra spacer base
  8. Extra bases designed to both
    1. permit cutting of the PCR product with PstI by providing extra "spacer" bases.
    2. promote addition of an A base on the opposite strand by Taq polymerase for high efficiency TA cloning if desired.

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 TCG AAT TCG CGG CCG CTT CTA G [ATG start]
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 TCC TGC AGC GGC CGC TAC TAG TA [TTA TTA double stop codon]

Cloning your PCR'd Part

The GTTTCTTC overhangs of the PCR primers suggested above are designed to encourage Taq or Taq containing enzyme mixtures to add a 3' A to the final PCR product. This is designed to make TA cloning or TOPO-TA cloning of the PCR product easier, if required.

The overhang also provides sufficient DNA overhang to allow the standard Biobrick enzymes to cut the PCR product for direct assembly into an existing Biobrick vector. Cutting with EcoRI and SpeI, EcoRI and PstI, or XbaI and PstI, while cutting the vector with the identical enzymes allow direct ligation of the PCR product into the vector. Cutting with EcoRI and PstI, followed by ligation to a Biobrick vector backbone is probably the most straightforward cloning method.

Colonies can be checked for the correct length with colony PCR using the primers VF2 and VR. Correct length clones should be sequence verified by sequencing with either or both VF2 and VR primers.

Remember to add part descriptions and sequence to the Registry of Standard Biological Parts, and to send plasmid or transformed cell samples to the Registry.

Source: http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication