Team:FAFU-CHINA/Engineering
DESCRIPTION
Preface
We designed and evaluated our two generations of microbial aroma production systems in order to enable microbes to produce dynamic and attractive fragrances. Through literature review, design system, brainstorming and literature review cycle, we finally determined the operation mechanism of microbial incense production factory.
Oscillator
Gene oscillator can realize the dynamic expression of target gene. Using a gene oscillator to dynamically express the core enzymes used to produce linalool and neroli, we could achieve dynamic changes in aroma, which was our initial idea. We understand the working principle of the gene oscillator in detail, and predict the actual effect of the oscillator through modeling.
In E. coli, oscillators are not difficult to construct. However, we also noted that neroli and linalool may be toxic to E. coli, and we were concerned that continuous production of neroli and linalol by E. coli would accelerate the decline of E. coli communities. And the oscillator is a closed loop control system, even if the fragrance components can be dynamically changed, it may not be in accordance with our will to change.
Photogenetic Systems
Our goal is to achieve open-loop regulation and automatic rhythm. Photoperiod gave us the idea that we could use the dynamics of light intensity throughout the day as input to the system. And we can do that by making the substrate organisms that produce linalool and neroli respond in reverse to the light signal. Therefore, BBa_K3732000 and BBa_K3127998 were used in yeast and E.coli respectively.
Under dark conditions, YF1 phosphorylation response modulator FixJ then drives the repressor gene expression downstream of FixK2 promoter, resulting in the repressor target gene expression. Under light conditions, YF1 activity is greatly reduced, FixJ is dephosphorylated, the repressor gene cannot be expressed, and the target gene (GFP) can be activated. The plasmid carrying BBa_K3127998 was transfected into competent E.coli DH5α, and the transformants were screened in chloramphenicol resistant plate. The transformants were inoculated into 1.5 mL chloramphenicol LB medium and cultured in dark for 9 hours. A small amount of bacterial solution was dipped into the inoculum needle and strewed on the resistant plate. The transformants were cultured under light/dark conditions respectively. Two days later, the petri dishes were examined under a fluorescence stereoscopic microscope.
Single colonies with green fluorescence were selected and inoculated into 1.5 mL LB medium containing chloramphenicol and cultured in light/dark
The E. coli cultured under light condition gave out strong green fluorescence, while the E. coli cultured under dark condition gave out very weak green fluorescence. This shows that BBa_K3127998 can be induced by white light, and the induction effect meets the expectation.
We fused the DNA sequence of BBa_K3732000 with the 3 'end of the DNA sequence of the red fluorescent
protein in the plasmid to study the biological function of BBa_K3732000 in the early stage. Meanwhile,
the BBa_K3732000 element and kanMX were connected in series in the plasmid. We intend to design primers
with upstream and downstream homologous arms of the termination codon of the target gene to clone 5
'mRFP1 to kanMX 3' for homologous recombination after the element is tested. As a result, BBa_K3732000
and KanMX were added to the loci of the target gene(The characterization diagram of BBa_K3732000 is
added below).
BBa_K3732000 can couple the degradation of the target protein with the light signal. The half-life of
the target protein did not change under black conditions. CODC1 fused to the tail of the target protein
is recognized by proteasome in eukaryotes under light conditions, and the target protein degrades.
Stability of co-culture system
First, we inoculated E. coli and yeast into co-culture medium, respectively, and measured their growth curves at 35 ℃ and 180rpm. In order to test whether (A, B, and C represent E.coli, S. cerevisiae, and E. coli and S. cerevisiae, respectively. Co-culture, 1, 2 and 3 respectively represent the addition of linalool and neroli in the stable phase, without linalool and neroli in the whole process, and the addition of linalool and neroli in the logarithmic phase).
The results showed that E.coli and yeast (nutrient deficiency type) grew well in the co-culture medium, and the design of the medium was successful. On the other hand, linalool and neroli at the concentration of 1.1g/L seemed to have no obvious inhibitory effect on the growth of the strain. Instead, the microbes seemed to grow faster when linalool and neroli were added to the culture, possibly because they metabolized them as nutrients. In addition, we observed that the biomass of the control group was slightly higher than that of the experimental group with linalool and neroli at the later stage of the stationary phase, possibly because linalool and neroli could accelerate the decline of the flora. After a week of growth curve measurement, we found that there was no significant decline in the number of bacteria, OD value kept at about 1.5. The co-culture system was basically stable, and linalool and nerolol at the concentration of 1.1g/L had no obvious inhibitory effect on the growth of the strain.
Construction of odoriferous strain
With HP, we obtained IPP-enriched yeast (PS100) from the laboratory of Zhejiang University of
Technology.
The genes that can synthesize nerolol were downloaded from NCBI, codon optimization was carried out, and
the sequence was synthesized by Bioengineering.
Protoplasts (PS100) are prepared using kits, and then plasmids carrying the target genes are transferred
into (PS100). Subsequently, the inverters were screened by neomycin-resistant medium plate. Several
transformants were selected and inoculated into special medium for co-culture.
Reference
[1] Lutz AP, Renicke C, Taxis C. Controlling Protein Activity and Degradation Using Blue Light. Methods
Mol Biol. 2016;1408:67-78.
[2] Jungbluth M, Renicke C, Taxis C (2010) Targeted protein depletion in Saccharomyces cerevisiae by
activation of a bidirectional degron. BMC Syst Biol 4:176