The first plasmid to be designed and built using the BioBytes system was named pKan. The schematic of the construct is shown in Figure 1a and the plasmid map in Figure 1b.

Figure 1a. Kan is the kanamycin resistance gene. Ori is the origin of replication pMB1. J23100 is the 75% promoter from the Anderson collection.

Figure 1b. Kan is the kanamycin resistance gene. Ori is the origin of replication pMB1. J23100 is the 75% promoter from the Anderson collection. A- and B-scar sequences are those derived from A and B overhangs annealed to eachother. The Anchor/Term.-scar contains the I-SceI site and results from the circularization of the pKan construct. RBS is the ribosome binding sequence.

pKan Construction

pKan was designed to test the BioByte method of rapid DNA assembly on a bead platform. BA-Kan, AB-Ori, and AB-promoter was prepared and assembled by the methods discussed in the protocols section. Assembly took place with the following order:

• 1) A-Anchor bound to bead
• 2) AB-J23100 (promoter)
• 3) BA-kanamycin resistance gene
• 4) AB-pMB1 origin of replication
• 5) B-Terminator piece

We designed the Ori to be the last brick to allow for selection for full length construct.

Once the linear chain was complete, an annealed terminator was added and the contruct was cleaved from the bead using USER. The sample was heated and cooled slowly to allow proper annealing of the ends before transformation. The anneal construct was transformed into E. coli and plated on Kanamycin containing LB plates. After growing overnight, we counted 105 colonies. Ten colonies were screened by digestion with I-SceI, which was expected to linearize the plasmid. The expected length of the linearized construct was 1562bp Figure 2.

Figure 2. shows one of the gels run to screen for successful pKan transformants. 1kbp+ is the 1 Kb ladder plus from fermentas. The label "CX" is circular miniprep product from colony number "X". "LX" denotes lanes where linearized plasmids, by I-SceI digestion, have been loaded. Expected size of linearized pKan is 1562 bp, which corresponds, for instance, to the single band in lane "L1" as well as most LX lanes. In lanes with two bands (most CX lanes) correspond to different circular forms of the plasmid: the top band is most likely nicked and the bottom supercoiled.

From the I-SceI digestion lanes of this gel and the other gel (not shown) of the minipreps, we observed that 8/10 of the screened colonies are the expected length. This was confirmed by sequencing. The sequence results can be viewed:

Genbank pKan Sequence

Although our time was limited, we still wish to further validate the BioBytes assembly method by constructing longer plasmids. The next construct is planned to contain an artificial operon composed of 2 resistance marker ORFs and a red fluorescent protein ORF.