Hi. Ali.

Thank you for your response.

Please could you suggest to me the softewares i could use for the design of the plasmid DNA/ gene construct?

Dear Uchechukwu

for express 2 different gene using a single plasmid you need a specific vector or carryng two different promoters and 2 different MCs ad happen for the pET-Duet ( http://www.merckmillipore.com/IT/it/product/pETDuet-1-DNA-Novagen,EMD_BIO-71146 ) suitable for co-expression or a single promoter but souble SD sequences to allow a bicistronic expression (as happen in Stargate sistem (see page 10 of https://www.iba-lifesciences.com/isotope/5/5-1632-001-Manual_StarGate.pdf )

Once you choose your vector, primer design happen similar to a single cloning approahc. You can find some bioinformatic tools and general guidelines about primer desing on my blog ProteoCool ( http://proteocool.blogspot.com/ )

ProteoCool n°2 (Primer Design: Restiction/Ligation ) at page 1

ProteoCool n°17 (Simple Tools for DNA sequence analysis) at page 6

good luck

ciao

Manuele

Hi Uchechukwu,

There are two basic solutions.

1. As Manuele Martinelli mentioned, you can use a plasmid that can simultaneously express multiple genes (with several MCS), such as the pACYCDuet series. However, this method is slightly more complicated when designing clones, and you need to consider the compatibility of enzyme cutting sites between your target genes.

2. You can also separately clone your target genes into two expression vectors that are compatible with each other (usually with different resistance), and then transform the two recombinant expression vectors into one competent cell for expression. The disadvantage of this method is that you can’t control the expression ratio of different target genes. If you need to obtain a protein complex assembled in a certain proportion, this method may not be suitable.

Hope it helps!

https://www.frontiersin.org/articles/10.3389/fmicb.2014.00172/full