Difference between revisions of "Part:BBa K4370012:Experience"

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Applications of BBa_K4370012

The pCRISPR-dCas9 (Tong et al., ACS Synth. Biol., 2015) encoding the sgRNA BBa_K4370012 designed to silence gpm gene in Streptomyces has been introduced by conjugation in Streptomyces ambofaciens ATCC 23877 (please refer to our STREPTObook for detailed protocols and medium composition). Exconjugants were reisolated on SFM containing selection marker (apramycin). Thereafter, spores from three independent exconjugants were collected and grown in MP5 liquid medium at 30°C under agitation. A strain containing the pCRISPR-dCas9 plasmid not encoding any sgRNA was used as a control. After washes in liquid sub-minimal medium (devoid of organic carbon source), 5 µl of bacteria (at 0.3 g of wet biomass/ml) were spotted either on rich medium (MP5) or on subminimal medium plate. After 48 h, a SNA top containing the indicative strain, Micrococcus luteus (OD_{600 nm} = 0.04), was poured onto the plates. Plates were incubated overnight incubation at 37°C. The results are presented in the Figure 1.

We noticed that clones containing the BBa_K4370012 biobrick show a slight growth defect, which is indicative of an effect of the presence of the sgRNA. This observation is consistent with the fact that the a decrease in the expression of the phosphoglycerate mutase (encoded by gpm) disrupts carbon flow in the glycolytic/gluconeogenic backbone as previously reported in E. coli (Antonovsky et al., Cell, 2016).

Morever, the production of antibiotics is decreased in the strains harboring pCRISPR-dCas9-BBa_K4370012 plasmid. Again, this observation is indicative of a disrupted metabolism. Fortunately, production in absence of organic carbon source is still significant, which is encouraging for our project.In this experiment, we observed more antibacterial activity in the poorest condition. This illustrates that antibiotic production by S. ambofaciens increases with nutritional stress. Indeed, it is generally proposed that antibiotic production is associated with resource conservation in favor of the Streptomyces colony.

One may be surprised to observe growth on a medium without an organic carbon source. We have observed this for all the Streptomyces strains we have tested in the STREPTObook. In fact, this is a first stage of growth that we suppose is probably based on intracellular carbon reserves. Indeed, after striking colonies from subminimal plates, the culture does not restart (we did this experiment). This illustrates the necessity to implement a Calvin cycle for a prolonged growth of the bacteria in the absence of organic carbon source.

In the course of this work, we designed an sgRNA based on a rationale derived from both the literature analysis and our mathematical modelling. After constructing and testing the vectors, we learned that the presence of the biobrick slightly affects the developmental cycle of Streptomyces without abolishing antibiotic production. This is encouraging in the context of an application of this biobrick to facilitate the implementation of the Calvin cycle in Streptomyces for antibiotic production after CO₂ fixation. Please refer to our STREPTObook for detailed protocols concerning conjugation, medium composition, bioassays experiments.

Figure : Bioassays performed with Streptomyces ambofaciens>/i> ATCC 23877 strains containing or not the pCRISPR-dCas9-BBa_K4370012 plasmid

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References Yaojun Tong, Pep Charusanti, Lixin Zhan, Tilmann Weber, and Sang Yup Lee, “CRISPR-Cas9 Based Engineering of Actinomycetal Genomes”, <i>ACS Synth. Biol. 2015, 4, 9, 1020–1029 https://doi.org/10.1021/acssynbio.5b00038 Antonovsky N, Gleizer S, Noor E, Zohar Y, Herz E, Barenholz U, Zelcbuch L, Amram S, Wides A, Tepper N, Davidi D, Bar-On Y, Bareia T, Wernick DG, Shani I, Malitsky S, Jona G, Bar-Even A, Milo R. "Sugar Synthesis from CO₂ in Escherichia coli." Cell<i>. 2016 Jun 30;166(1):115-25. https://doi.org/10.1016/j.cell.2016.05.064

User Reviews

UNIQb4a4737993878929-partinfo-00000002-QINU UNIQb4a4737993878929-partinfo-00000003-QINU

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 ===Applications of BBa_K4370012===
-The pCRISPR-dCas9 (Tong <i>et al., ACS Synth. Biol.</i>, 2015) encoding the sgRNA <part>BBa_K4370012</part> designed to silence <i>gpm</i> gene in <i>Streptomyces</i> has been introduced by conjugation in <i>Streptomyces ambofaciens</i> ATCC 23877 (please refer to our STREPTObook for detailed protocols and medium composition). Exconjugants were reisolated on SFM containing selection marker (apramycin). Thereafter, spores from three independent exconjugants were collected and grown in MP5 liquid medium at 30°C under agitation. A strain containing the pCRISPR-dCas9 plasmid not encoding any sgRNA was used as a control. After washes in liquid sub-minimal medium (devoid of organic carbon source), 5 µl of bacteria (at 0.3 g of wet biomass/ml) were spotted either on rich medium (MP5) or on subminimal medium plate. After 48 h, a SNA top containing the indicative strain, <i>Micrococcus luteus</i> (OD<sub>600 nm</sub> = 0.04), was poured onto the plates. Plates were incubated overnight incubation at 37°C. The results are presented in the <b>Figure 1</i>.
+The pCRISPR-dCas9 (Tong <i>et al., ACS Synth. Biol.</i>, 2015) encoding the sgRNA <partinfo>BBa_K4370012</partinfo> designed to silence <i>gpm</i> gene in <i>Streptomyces</i> has been introduced by conjugation in <i>Streptomyces ambofaciens</i> ATCC 23877 (please refer to our STREPTObook for detailed protocols and medium composition). Exconjugants were reisolated on SFM containing selection marker (apramycin). Thereafter, spores from three independent exconjugants were collected and grown in MP5 liquid medium at 30°C under agitation. A strain containing the pCRISPR-dCas9 plasmid not encoding any sgRNA was used as a control. After washes in liquid sub-minimal medium (devoid of organic carbon source), 5 µl of bacteria (at 0.3 g of wet biomass/ml) were spotted either on rich medium (MP5) or on subminimal medium plate. After 48 h, a SNA top containing the indicative strain, <i>Micrococcus luteus</i> (OD<sub>600 nm</sub> = 0.04), was poured onto the plates. Plates were incubated overnight incubation at 37°C. The results are presented in the <b>Figure 1</b>.
-We noticed that clones containing the <part>BBa_K4370012</part> biobrick show a slight growth defect, which is indicative of an effect of the presence of the sgRNA. This observation is consistent with the fact that the a decrease in the expression of the phosphoglycerate mutase (encoded by <i>gpm</i>) disrupts carbon flow in the glycolytic/gluconeogenic backbone as previously reported in <i>E. coli</i> (Antonovsky <i>et al., Cell</i>, 2016).
-Morever, the production of antibiotics is decreased in the strains harboring pCRISPR-dCas9- BBa_K4370012 vector. Again, this observation is indicative of a disrupt metabolism. Fortunately, production in a carbon-free environment is still significant, which is encouraging for our project.
+We noticed that clones containing the <partinfo>BBa_K4370012</partinfo> biobrick show a slight growth defect, which is indicative of an effect of the presence of the sgRNA. This observation is consistent with the fact that the a decrease in the expression of the phosphoglycerate mutase (encoded by <i>gpm</i>) disrupts carbon flow in the glycolytic/gluconeogenic backbone as previously reported in <i>E. coli</i> (Antonovsky <i>et al., Cell</i>, 2016).
-One may be surprised to observe growth on a medium without an organic carbon source. We have observed this for all the Streptomyces strains we have tested in the STREPTObook. In fact, this is a first stage of growth that we suppose is probably based on intracellular carbon reserves. Indeed, after striking colonies from subminimal plates, the culture does not restart. This illustrates the necessity to implement a Calvin cycle for a prolonged growth of the bacteria in the absence of organic carbon source.
+Morever, the production of antibiotics is decreased in the strains harboring pCRISPR-dCas9-BBa_K4370012 plasmid. Again, this observation is indicative of a disrupted metabolism. Fortunately, production in absence of organic carbon source is still significant, which is encouraging for our project.In this experiment, we observed more antibacterial activity in the poorest condition. This illustrates that antibiotic production by <i>S. ambofaciens</i> increases with nutritional stress. Indeed, it is generally proposed that antibiotic production is associated with resource conservation in favor of the <i>Streptomyces</i> colony.
+One may be surprised to observe growth on a medium without an organic carbon source. We have observed this for all the <i>Streptomyces</i> strains we have tested in the STREPTObook. In fact, this is a first stage of growth that we suppose is probably based on intracellular carbon reserves. Indeed, after striking colonies from subminimal plates, the culture does not restart (we did this experiment). This illustrates the necessity to implement a Calvin cycle for a prolonged growth of the bacteria in the absence of organic carbon source.
 In the course of this work, we designed an sgRNA based on a rationale derived from both the literature analysis and our mathematical modelling. After constructing and testing the vectors, we learned that the presence of the biobrick slightly affects the developmental cycle of <i>Streptomyces</i> without abolishing antibiotic production. This is encouraging in the context of an application of this biobrick to facilitate the implementation of the Calvin cycle in <i>Streptomyces</i> for antibiotic production after CO<sub>2</sub> fixation.
 Please refer to our STREPTObook for detailed protocols concerning conjugation, medium composition, bioassays experiments.