Although we could not get into laboratory and conduct experiments, we were ready to conduct experiments.
Extraction of nicotine
We will follow the experimental procedure as we showed on the extraction of nicotine page. (extraction of nicotine) We contacted cigarette manufacturers and volunteer organizations that collect cigarette butts and negotiated with them to get their cigarette waste.As a result, we were able to learn about the tobacco farmer's waste and made a commitment to provide us with the cigarette butts they picked up. we were lucky to get samples of tobacco waste as shown on figure 1. All other materials are ready at laboratory.
Plan A (E. coli as chassis)
For the overview of this plan, please check the description(Converting nicotine into 5-aminolevulinic acid) First, we will insert genes form metabolic pathway of P. putida S16 and check if they function as previously reported. We codon optimized the genes using IDT codon optimization tool. We will ask IDT to synthesis our DNA fragments including promoter(Part:BBa_J23100), RBS(BBa_B0034) and terminator(BBa_B0015). We will insert the genes to pSB1C3 vector by Gibson assembly. You can see plasmid maps of them on figure 2. After confirming the transformation of plasmid DNA to E. coli by restriction enzymes, we will add the cells to water containing substrates and check if they degrade them by thin layer chromatography. After this is checked, we will introduce plasmid containing luxI, luxR, lux promoter, and gfp.(lux-gfp-pSB1C3 on figure2) This is for checking the timing of the expression of genes under lux promoter. By assembling all together, we will get the E. coli we want.
|Name of the plasmid||Link|
|nicA2 expression device||https://benchling.com/s/seq-xsEECePdAspGs5OF7iPY|
|pnao expression device||https://benchling.com/s/seq-05GEiNs82c0DrFPus4VN|
|sapd expression device||https://benchling.com/s/seq-e0fK86luSbKmeGxjoWmt|
|spmABC expression device||https://benchling.com/s/seq-DDvHTheksQ4L1TG8oy7G|
|hspB expression device||https://benchling.com/s/seq-mKBtdNJpU0GR42sT9zgt|
PlanB(P. putida S16 as chassis)
For the overview of this plan, please check the description. (Using Pseudomonas putida S16 as a chassis) First we will make a hpo deletion mutant. This is done by introducing hpo plasmid and homologous recombination. (figure 3.) After incubating P. putida on kanamycin plasmid and checking deletion by PCR, we will transform plasmid containing lux, luxR, lux promoter, tetR, tet promoter, and gfp by triparental mating. This is for checking the timing of the expression of genes under lux system and tet promotor. Finally, changing the gfp to hpo, we will complement hpo.
|Name of the plasmid||Link|
|hpo complement device||https://benchling.com/s/seq-JcAzK3pPjkcB1geGuAO8|
Converting to 5-aminolevulinic acid.
For the overview of this, please check the description. (Converting 2,5-hydroxypyridine to 5-aminolevulinic acid) We will conduct experiments as shown on the page.