NUS Enterprise

Synthetic Biology – Scalable, Compatible, Antibiotic-free Multi-Plasmid System

Technology #13350s

 Synthetic Biology - Tool to Modulate Multiple Gene Expression Levels in Microbes


The engineering of microbes for production of useful chemicals, such as food additives, pharmaceuticals or biofuels is the main aim of synthetic biology. In bacteria, a replicable plasmid is an excellent recombinant gene carrier since it is easily manipulated and can have multiple copies in cells. However, significant limitations restrict the utility of multiple plasmids in large scale fermentation processes:

  • the co-existence of two or more incompatible plasmids will lead to unstable copy numbers
  • plasmids may be lost during the scaling-up process especially when cells are metabolically stressed by the over-expression of genes carried by the plasmid. One approach to circumvent this is to use antibiotics but the use of antibiotics is cost prohibitive and unstable in long term growth


The invention is based on a parental plasmid which has been specifically engineered at the recognition sites of the plasmid copy number control mechanism to produce a library of compatible plasmids. Thus, an unlimited number of plasmids with various copy numbers can be generated and introduced into the same cell. In order to maintain these multiple plasmids without using antibiotics, multiple conditional essential genes (CEG) from the host genome have been grafted into these plasmids. All of the co-existing plasmids carrying the CEG are maintained during fermentation.


  • Multiple genes can be inserted into host cells with plasmids that co-exist in a cell and grow under selection pressure without the use of antibiotics
  • No incompatible plasmids and unstable copy numbers


The utility of this method is in large-scale fermentation (synthetic biology and metabolic engineering applications).


Available for licensing. Patent pending in US and Europe.


A/Prof. Too Heng Phon



Radhika Das Chakraborty

Phone: +65-66012768


ILO Ref No: 13350S