Each part to be assembled via the BioBytes method needs to have its ends altered to either the AB or the BA form. To do this, unique plasmids were developed and named pAB and pBA. These plasmids were designed and constructed from pUC19 and contain the pMB1 high copy origin. Unique cassettes were designed containing PstI, XbaI, and primer annealing regions complementary to the ‘A’ and ‘B’ ends. The cassette was synthesized and inserted using two restriction sites (EcoRI and NsiI). This left an EcoRI site on the final plasmid as well a PstI scar site. The primer annealing regions in pAB are reverse to that in pBA so that compatible sticky ends can be produced in either plasmid. Genes can be inserted using XbaI and PstI (or an enzyme which produces a compatible sticky end). The plasmids were originally designed for a previous but now obsolete system, where the sticky ends were generated via nicking enzymes.

Figure 1.

How To Create a Byte

In order to format a part as a AB or BA form Byte, the part first needs to be cloned in to pAB or pBA, respectively. This is done using a XbaI and PstI digest of both the insert and backbone, followed by ligation to place the part inside of the AB or BA cassette.

Once the part is cloned, universal PCR primers containing deoxyuridine residues are used to amplify the part. To create an AB Byte from pAB, the universal primers pAB_F and pAB_R are used. For the creation of a BA Byte from pBA, the primers pBA_F and pBA_R are used (Figure 2).

Figure 2.

After amplification, treatment with USER^TM mix (available from New England Biolabs) creates a nucleotide gap at the position of the uracil by first excising the uracil base by Uracil DNA glycosylase and then cleaving the phosphodiester backbone at the apyrimidinic site via Endonuclease VIII. The resulting short oligonucleotides are then purified away from the PCR product to produce mature 12 base 3' overhangs of the AB or BA form Byte (Figure 2).

Figure 2.

Figure 3.

Shown are the AB and BA cassettes of pAB and pBA respectively. “ORF” is the open reading frame; the gene to be edited. “RBS” is the ribosome binding site. The primer binding sites are highlighted in blue/green and the complementary - primers are aligned to the plasmid. The bolded red ‘U’s” denote the deoxyuracils that the USER™ system acts on.