The Theory of Extractory

A. Autolysis

The challenge in designing a good autolytic system is to ensure the cells lyses specifically at a particular period of time, which can be controlled by the manufacturer. Our manufacturing process here combines a heat-inducible promoter from bacteriophage lambda with the T4 phage lysis system. This system will make huge time-savings as the major bottleneck in current plasmid manufacturing process are the heat or alkaline lysis steps.

The heat-inducible component of the genetic circuit involves the pR promoter from the bacteriophage lambda and the temperature-sensitive cl repressor from the PGW7 plasmid ^[1]. The cl repressor protein is constitutively expressed and as a result it prevents expression of any genes downstream of the pR promoter. However thermo-induction in the bioreactor between 35 and 42 degrees celsius causes a conformational change in the cl repressor, which then loses its binding capability to the promoter and consequentially expression from the pR promoter is upregulated.

The xylene-inducible switch also has a similar mechanism and it involves the XylR protein (a transcriptional activator) and the Pu promoter ^[2]. Constitutive expression of the XylR protein maintains a constant level inside the cell and in the presence of chemicals like Xylene, toluene or benzene it undergoes a conformational change, which then enables it to bind to and upregulate expression from the Pu promoter.

Lysis system in the T4 bacteriophage involves 2 protein: lysozyme and holin. The lysozyme is the effector enzyme which degrades the peptidoglycan cell wall and lyses the bacterial cell open. However, in order to do that the lysozyme needs to cross the inner membrane and gain access to the periplasmic space, which is ensured by holin, a membrane spanning protein that can form channels in the membrane ^[3]. Therefore, expressing the lysozyme without the holin, only leaves the enzyme isolated in the cytoplasm and the lysis process cannot happen (Williams, et al. 2006). Only after the holin in expressed, the lysozyme can pass through the channel into the periplasmic spaces and start degrading the cell wall.

In our design we have positioned both the regulatory proteins (cl and XylR) under a constitutive promoter, the T4 lysozyme under the pR promoter and the holin under the pu promoter. Therefore thermo-induction only expresses the lysozyme, but the lysis process does not happen until xylene is added to activate the pU promoter and express holin.

B. Exonuclease

The bacteriophage T5 exonuclease is a unique DNase which is ideal for plasmid DNA manufacturing. This nuclease can digest linear single and double stranded DNA, but will leave supercoiled plasmids alone. Furthermore, this DNase can also digest irreversibly denatured plasmids which resemble supercoiled plasmids and are otherwise also resistant to digestion by restriction enzymes. The D15 exonuclease has a 5’ to 3’ activity, which is complementary to E coli exonuclease III with a 3’ to 5’ activity ^[4]. Also compared to other similar DNases like, E coli exonuclease III and T7 phage exonulcease which can only digest double-stranded DNA, D15 can digest both single and duplex DNA molecules. Experiments carried out by Sayers et al has substantially proved the absence of double-stranded endonuclease activity of the D15 nuclease and which is why this exonuclease does not affect closed circular supercoiled plasmids at all and are referred to as a ‘plasmid-safe nuclease’. Interestingly, this particular exonuclease share 54% similarity to the E Coli DNA polymerase I. Therefore the T5 exonuclease is a very useful tool in plasmid DNA manufacturing, as it reduces the complexity and length of the entire downstream process, by getting rid of ‘junk plasmids’ (nicked and linear forms), which have less therapeutic value than supercoiled plasmids ^[5]. In our BioBrick device we positioned the exonuclease downstream of the pu promoter, so that its expression would be initiated simultaneously as the holin enzyme by induction with xylene. In this way even after the cell is lysed open the exonuclease enzyme will be present in the cell lysate and will be able to to digest unwanted plasmids.

An additional gene for ribonuclease also aids further in the purification process, by getting rid of the cellular RNAs from the DNAs. Chimeric constructs of ribonuclease and T5 exonuclease gene encoded on E coli chromosomes have also been designed and tried in the industry with good success and no harmful effects so far.

C. Simplified autolysis process

Another novel autolysis process for plasmid purification has been reported by Carnes, A. E., et al ^[6]. This process utilizes new autolytic host strains expressing phase endolysin gene, λR, cytoplasmically. Chromosome engineering to over-express λR was performed using an integrated vector called pAH1444 modified to allow expression of cloned genes from the bacteriophage λPR and λPL dual promoters. This vector overproduces target proteins specifically during the 42°C induction phase of the fermentation process. The expressed endolysin remains in the cytoplasm, where it is separated from its peptidoglycan substrate in the cell wall. So that cells can be harvested by normal methods after fermentation. The current autolysis conducted in common buffers works efficiently. However, it results in complete lysis and non-specific release of all the cell content which raises the viscosity of the cell lysate and poses difficulties in downstream processing. To address this issue, it has been suggested that a buffer with pH range of 5.0 to 5.5 should be used, which results in efficient plasmid recovery, while reducing the release of genomic DNA and cell debris. Besides, an outer membrane permeabilizing agent and a cytoplasmic membrane permeabilizing agent should also be applied along with. . According to Carnes, A.E., the purity of plasmid DNA can achieve 98.3% after acidic plasmid extraction.