# A convenient five-segment cassette procedure for DNA insertions coding for novel peptides

## TL;DR

This paper introduces a cost-effective DNA cassette method for creating and testing new peptides in E. coli using short oligomers and GFP fusions.

## Contribution

A novel five-segment DNA cassette method with three nicks for efficient peptide insertion and rapid prototyping.

## Key findings

- The five-segment cassettes with 20 nt overlaps can code for 20 amino acid peptides using 40 nt oligomers.
- Cassettes with more than three nicks fail to insert into plasmid DNA successfully.
- Peptides are isolated as C-terminal fusions with GFP, enabling easy mutation and analysis of solubility and IMAC retention.

## Abstract

A DNA cassette assembly method is described which utilizes inexpensive oligomers no longer than 40 nt in length. The five-segment cassettes have 20 nt overlaps which give an effective length of 80 nt, making it possible to code for peptides up to 20 amino acids long. The cassettes have three phosphate free nicks, which can be successfully inserted into plasmid DNA and used to transform E. coli. The nicks are repaired in vivo by an unknown mechanism. Insertions are not successful for cassettes with greater than three nicks. A procedure is provided for rapid turnaround from DNA design to peptides, which are easily isolated as C-terminal fusions with GFP. The technique generally gives the expected sequence, with errors which occur about 1% of the time. Several representative DNA inserts are described which illustrate the method, as well as chemical details on the new peptides coded for. The peptides can be readily mutated to make it possible to understand how polar and aromatic residues affect GFP-fusion solubility, and how histidine residues can be strategically placed in a peptide for good IMAC retention. The method can be used to explore a large number of new designed peptides as fusion products quickly and economically.