Future Directions

Troubleshooting Orthogonal Transcription

Both the original Universal Bacterial Expression Resource (UBER) and our modified UBER have been tested in E. coli DH10B, S. oneidensis, and P. putida. Our characterization of UBER yielded conflicting results. When UBER and the mKate2 reporter were on separate plasmids and transformed into E. coli DH10B, the modified UBER drove higher expression of mKate2 than the original, unmodified UBER. However, when UBER and the mKate reporter were contained within the same plasmid, we saw that relative expression levels for the two UBERs were flipped; the original UBER drove higher expression of reporter than our modified UBER. This inconsistency in UBER-mediated reporter expression was also seen when UBER was expressed in different strains. P. putida transformed with modified UBER showed higher reporter levels than P. putida transformed with original UBER, while the opposite was true for S. oneidensis. We suspect that recombination within the UBER constructs and heterogeneity of the recombinants among clones may be responsible for the inconsistency. Future work will include investigating and eliminating the cause of this irregularity in our data.

The orthogonal transcription module of PORTAL (UBER) behaves inconsistently depending on whether or not UBER and the reporter are on the same plasmid

The orthogonal transcription module of PORTAL (UBER) behaves inconsistently across strains

Orthogonal Translation in Other Strains

The orthogonal translation component of PORTAL can be characterized in other bacterial strains. Currently, the circuit for characterizing orthogonal ribosomes has been built and characterized in E. coli, but not yet characterized in our non-model bacterial strains.

The orthogonal translation module of PORTAL has been built and needs to be characterized in non-model bacteria

Combining Orthogonal Transcription and Translation Modules

The orthogonal transcription, translation, and reporter components of PORTAL are currently on different plasmids. We plan on combining these components into one plasmid to avoid the complications of a three-plasmid system.

Orthogonal transcription and translation components of PORTAL can be combined into one plasmid for easier use.

Further Insulation of PORTAL From Host Processes

The UBER system contains RBSs with conventional Shine Dalgarno sequences, which means that UBER itself is dependent on native transcription for expression. To further insulate PORTAL from native expression machinery, the RBSs of UBER may be changed to orthogonal RBSs.

Native UBER RBSs can be replaced with orthogonal RBSs

Additional Future Directions

The PORTAL system may be tested in more strains of scientifically and industrially relevant bacteria, such as Vibrio natriegens. Additionally, because the orthogonal translation aspect of PORTAL uses host ribosomes modified only by mutation of the anti Shine Dalgarno sequence, the ribosomes and therefore the expression of the fluorescent protein reporter may differ between species. To control for this, future work includes using a reporter system whose output is indicative of transcription, such as a toggle switch or pulse generator, as the output of PORTAL. Finally, different methods of creating orthogonal transcription may be explored; for example, the T7 RNAP in UBER and the T7 promoters may be replaced by a gene for an orthogonal sigma factor and orthogonal sigma factor promoters.