If we are able to engineer our FastPETase into Alteromonas macleodii, we will need a way to implement the bacterium into the environment. Direct implementation is not possible due to environmental and ecological concerns. However, implementation can still be done via bioreactors, in locations such as recycling facilities.
Here, we designed a bioreactor using CAD that would work as a theoretical implementation for our Alteromonas, either out in the open ocean or grounded on land.
Our design was checked by Professor Christian Diercks at Scripps Research, who mentioned the possibility of running as a “continuous culture (keeping the optical density of the culture constant by exchanging media using the nutrient valve and seawater intake valve, as well as the exit valve” and the addition of a turbidity meter to keep optical density constant.
He also mentioned one large benefit of the usage of Alteromonas in a bioreactor, which is the lack of need for desalination:
“As mentioned before, the fact that your bacteria can grow in sea water is important for economical reasons. In fact, this is a challenge that has long limited bio-manufacturing in microbial hosts (e.g., microbial biofuels). If the cost of desalination of water (both economically and energetically) is too high, such processes will not be adopted. in my mind the same will hold true for your proposed application. The elegance of your process relies on its ease of implementation and the use of seawater for growth certainly adds to that!” - Professor Diercks
For our project, our team chose the loop assembly protocol in the beginning of our research in order to have ease when interchanging plasmid parts during our L1 plasmid engineering phase. Loop Assembly was pioneered in our laboratory (J. Craig Venter Institute) and is a hierarchical system of plasmid assembly similar to MoClo. L0 plasmids are constructed with Gibson Assembly to contain singular basic parts (Promoters, Terminators, RBSs). L1 plasmids are built using Golden Gate- Assembly from multiple L0s, and contains a full transcriptional unit (Promoter, RBS, Gene of Interest, 3x stop or sfGFP, and terminator). L2 plasmids are constructed from L1s and contain multiple transcriptional units that often come together to make an enzymatic pathway (PETase + MHETase for example).
Our L0 plasmid map contains the gene for Fast PETase, and some extra base pairs for the overhang. The gene is labeled as the “g-block”, which is the sequence that encodes the Fast PETase enzyme. Other L0 plasmids were sourced from JCVI, and are listed below for our L1 plasmid map.
The sequencing primers for the L0 plasmid are found by getting the reverse sequence of specific parts of the g-block. Through PCR, these primers provide attachment sites for DNA polymerase, which allows us to obtain a nucleotide sequence in sequencing (Plasmidsaurus). This verifies that the correct gene is present.
Our L1 plasmid map contains the J23101 promoter, B0034 RBS, gene for Fast PETase, a 3x stop, and rrNB terminator. The L1 plasmid is constructed from five separate L0 plasmids in a Golden-Gate reaction (L0 plasmids besides the FastPETase were sourced from JCVI). The promoter encourages RNA polymerase to bind, the ribosomal binding site provides a place for ribosomes to initiate translation of mRNA, while the terminator helps to end transcription of mRNA.