Plasmid Design & Construction:
Nitrogen
Level 0 plasmid
In our Level 0 plasmid builds, two different bacteria were used as a basis for two versions of nosZ: nosZ from Pseudomonas stutzeri and nosZ from Dechloromonas denitrificans. These original hosts of nosZ were chosen due to their part compatibility with “chlamy”.
Level 0 plasmid
P. stutzeri and D. denitrificans
By building two versions of nosZ, we could determine which build would work best for reducing nitrous oxide. These part sequences were found using GenBank and then codon optimized with IDT’s software for C. reinhardtii which prefers a high G, C content in its genome. Once optimized, the correct restriction sites were added to each end of the sequence to allow for cloning using Golden Gate technology. We confirmed that these plasmids were correctly built using diagnostic digests.
Level 1 plasmid
Next, the Level 1 plasmid builds were built using parts from the Chlamydomonas MoClo kit. This kit allows for the assembly of parts through the choice of many pieces that align with each other in a system of genetic constructs.
Level 1 plasmid
P. stutzeri and D. denitrificans
Each part fits together with unique sticky ends in a designated spot.We used a C. reinhardtii Psad promoter from the MoClo kit to allow for robust gene expression. Following this, the nosZ coding sequence was cloned in-frame with the gene for mCherry, a red-fluorescent protein, and this was followed by a beta-tubulin terminator. We confirmed that these plasmids were correctly built using diagnostic digests.
Phosphorous
To increase phosphate uptake from wastewater, we used the MoClo kit to manipulate the expression of two additional genes. First, we wanted to overexpress the Psr1 gene from C. reinhardtii that promotes increased uptake of phosphate. We obtained the Level 0 part from the Alison Baker Lab at the University of Leeds.
Diagnostic Digest
Diagnostic Digest
The Level 1 build started with the promoter from the Psad gene. We attached the PSR1 gene to the NanoLuc reporter gene that can be used in a luminescence reporting assay. We confirmed that this plasmid was built successfully using a diagnostic digest.
Level 0 plasmid
Additionally, we designed an artificial miRNA to silence expression of the PTC1 gene, which codes for a phosphate export protein.
Diagnostic Digest
Level 1 plasmid
For this Level 1 plasmid, we chose to use the beta-tubulin promoter, to prevent all genes from relying on the same promoter for the eventual assembly of the Level 2 plasmid. With the PTC1 gene silenced, the algae will be unable to release the phosphate it absorbs and will be forced to continue phosphate uptake. We confirmed the successful creation of this part by PCR.
We designed all L1 plasmid builds to allow us to assemble an L2 plasmid build that expresses nosZ, PSR1, and the miRNA to reduce PTC1 expression, along with a gene that allows for antibiotic selection through an additional round of Golden Gate Cloning. We call this plasmid the de-Slimer!
For a description of our use of the plasmid in Chlamydomonas reinhardtii, please see our Results page!
Also, please see our most-commonly used Experiment Protocols below.