Revision as of 20:51, 8 August 2018

Phactory

Redefine Phage Production

Create DNA

One Problem of cell extract, is the stability of linear DNA. In escherichia coli, endogenous and exogenous linear DNA are differentiated by specific proteins, such as the recombinase complex RecBCD. It recognizes genomic double strand breaks via a specific sequence of 8 bases called Chi-site and prepares the DNA for homologous repair. However, foreign DNA lacks the Chi-site and is therefore degraded by RecBCD. For experiments in cell extract expensive and inefficient inhibitors are used. Since a knockout of RecBCD is lethal, we chose to modify the enzyme to enable selective depletion after cell lysis. Therefore, we came up with several depletion strategies:

Create Soup

The financial advantage of producing home-made cell extract for in vitro protein expression is virtually reversed by the fact that it is tedious and time-consuming, with huge batch to batch variations of the resulting product. Therefore, our objective is to optimize the protocol for self-made cell extract from E. coli, by simplifying time consuming steps while eliminating the central sources of variation during the preparation process. The focus of our effort is to determine the influence of cultivation conditions on extract quality and optimizing the cell lysis for speed, protein yield and nal product quality. By implementing novel ways of extract characterization, we aim to gain a deeper understanding of cell-free systems.

Create Phages

Generation of a universal platform for phage engineering: Our TX-TL system is superior to conventional bacterial based phage production in the following aspects:
- Assembly of phages in vitro
- Exchange of phage fibers in vitro
- Establishment of homogeneous phage batches
- Reduction of endotoxins within phage batches We will exemplarily engineer a non lytic fluorescent T7 phage, which targets a non primary E. coli strain.