Synthetic Biology:BioBricks/Part fabrication

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 approach being used as well as the type of part being constructed.

Constructing a BioBrick part via PCR

Standard part fabrication

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)

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. SpeI recognition site
6. Extra G base to prevent inadvertent creation of either
   1. a GATC site (which can undergo methylation thereby inhibiting digestion by XbaI)
   2. an ATG start codon
7. 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)

• 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. NotI recognition site
4. PstI recognition site
5. 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.
6. Random extra bases

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