2024 AIChE Annual Meeting

Integration of Degron Tags in Plasmid Cloning for Controlled Protein Translation

Synthetic biology facilitates the engineering of biological parts within organisms to unlock new functions and purposes. One way to accomplish this is by introducing proteins not native to the organism via protein translation—a vital tool in biomanufacturing. However, expressing these proteins often proves challenging due to toxicity, which induces metabolic stress to cells and can cause cell death. Even proteins native to the organism can sometimes be toxic when overexpressed, posing significant challenges to the advancement of protein synthesis.

Addressing these issues, this study examines the integration of degradation tags to genes of interest for plasmid cloning. When proteins are expressed in E. coli, Lon protease enzymes, native to the cell, recognize and degrade the tagged proteins; this allows the cell to grow before succumbing to toxicity. As the strain grows and amplifies the plasmid number, these DNA copies can be collected for employing a cell-free system (CFS) to translate the protein of interest. CFS is an in vitro tool that utilizes transcription-translation (TXTL) machinery that enables the rapid production of functional proteins from cell extracts. The strain used in CFS is Lon protease deficient; thus, most tagged proteins remain intact.

To validate this approach, researchers selected three degradation tags shown in the literature with the highest degradation rate and energy efficiency with Lon enzymes. These tags were then attached to the end of sfGFP biosensor protein from different promoter sites: pJ23119 (strong constitutive), pJ23105 (weak constitutive), and pT7 (strong constitutive) via site-directed mutagenesis PCR cloning method. The plasmid was then transformed into DH10β (strain used for cloning), and the plate reader was used to measure the protein concentration and cell growth after 24 hours. Preliminary data indicate significant degradation of sfGFP with tags compared to untagged ones at different promoter sites, for which 43.68-53.80% degradation in pJ23105, 73.11-76.70% degradation in pADS4, and 22.83-32.80% degradation in pJ23119 was observed. Results indicated that the overall degradation between the tags did not differ significantly, but the degradation rates could vary. Therefore, a growth curve will be measured to determine the most energy-efficient tags out of the three. Furthermore, with CFS, the protein expression for tagged and untagged plasmids has all shown high sfGFP expression (~25,000 RFU). This signifies that the protein degradation seen in DH10β was indeed by Lon protease enzymes, not due to other factors like misfolding that could have played a role. The following steps are to replicate data in a controlled setting, find ways to have even more degradation in vivo, and apply the novel mechanism to express other toxic proteins. The success of this research holds immense potential in the realm of biotechnology. Such development offers numerous real-world applications, including the ability to express various biosensor proteins that cannot be translated with the current methodology.