Difference between revisions of "Part:BBa K4370000:Experience"

Revision as of 07:25, 11 September 2022

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K4370000

We performed experiments to evaluate the activity of Streptomyces bottropensis RuBisCO in native and heterologous chassis.

S. bottropensis ATCC 25435 strain was grown during 4 days in sub-minimal liquid medium (devoid of carbon source), minimal liquid medium 0.5 % glycerol and rich liquid medium (MP5), under standard atmosphere or 3 % CO2 enriched atmosphere (in jar containing BD GasPak EZ) at 30°C under agitation. E. coli BL21DE3 strains were grown in LB rich liquid medium during 24 h at 20°C under agitation.

Cell extracts were prepared following a protocol very similar to the one proposed by the iGEM Bielefeld 2014 team (https://2014.igem.org/Team:Bielefeld-CeBiTec/Results/CO2-fixation/RuBisCO), except that cells were lysed One Shot Cell Disrupters (Constant Systems Ltd - 2.5 kbar). Our precise protocol is available here: File:T--Go-Paris-Saclay-protocol-RuBisCO.pdf

We thus performed a RuBisCO activity test on S. bottropensis ATCC 25435 and E. coli BL21DE3 strains harboring a plasmid encoding ccbL (BBa_K4370000, BBa_K4370002) and cbbS (BBa_K4370001, BBa_K4370003) under the control of tetO promoter (BBa_R0040) or a control plasmid (containing BBa_K4370010). The background in the E. coli extracts in the analysis area makes the result inconclusive (Figure 1).

However, we obtained interesting results in S. bottropensis background. First, we noticed that the final biomass reached by S. bottropensis in sub-minimal liquid medium under 3 % CO₂ enriched atmosphere was twice the final biomass reached in this medium in a standard atmosphere. Such a doubling in the biomass under enriched atmosphere was not observed with another Streptomyces grown in the same conditions (Figure 2). This result suggests that at least in one condition (absence of carbon source and under 3 % CO₂), S. bottropensis may fix CO₂ to form biomass. This observation supports the hypothesis that its CO₂ fixation module encodes a functional RuBisCO that is expressed under these conditions.

Our HPLC analysis of the signal detected in the UV (220 nm) allows to detect both the substrate (RuBP, ribulose-1,5-bisphosphate) and the product (PGA, 3-phosphoglycerate), used as standards. After around 1 min incubation at room temperature of RuBP with cell extract, the RuBP was not detected in all S. bottropensis samples (Figure 3), suggesting that it was used by some enzymes (… that may be RuBisCO!). To be sure that the RuBP was consumed by the RuBisCO, we needed to detect the product of this enzyme (PGA). Interestingly, a signal was detected at the retention time of the PGA only in the sub-minimal medium (devoid of carbon source) under standard atmosphere. Neither RuBP nor GPA was detected in the reaction using cell extract of cells grown in this medium under 3 % CO₂ enriched atmosphere. This suggests that if GPA was produced in this condition, it was used by other cellular enzymes to produce compounds not detected in our window of analysis. These enzymes could be other enzymes encoded by the CO₂ fixation module of S. bottropensis and/or its endogenous pentose phosphate pathway. We note a significant baseline in some samples which can make the results ambiguous. Thus the determination of the nature of the products by mass spectrometry could allow to confirm the identity of the product and therefore these first results.

Taken together, these results provide for the first time preliminary data in favour of the functionality of a Streptomyces RuBisCO, which will need to be confirmed by further analyses.

Figure 1: HPLC analysis of RuBisCO assay perform with RuBP (ribulose-1-5-bisphosphate) and cell extract of E. coli harboring a plasmid encoding ccbL (BBa_K4370000, BBa_K4370002) and cbbS (BBa_K4370001, BBa_K4370003) under the control of tetO promoter (BBa_R0040), or control extracts. Please note the y-scale change compared to Figure 3. The very high baseline in E. coli samples makes this test inconclusive.

Figure 2: Biomass reached after 4 days of culture of Streptomyces under standard or CO₂ enriched atmosphere. The media were inoculated with an initial biomass of env. 0.04 g.

Figure 3: HPLC analysis of RuBisCO assay perform with RuBP (ribulose-1-5-bisphosphate) and cell extract of S. bottropensis grown under various conditions

User Reviews

UNIQ79ef8c912d2fa996-partinfo-0000000B-QINU UNIQ79ef8c912d2fa996-partinfo-0000000C-QINU

@@ Line 11: / Line 11: @@
 Cell extracts were prepared following a protocol very similar to the one proposed by the iGEM Bielefeld 2014 team (https://2014.igem.org/Team:Bielefeld-CeBiTec/Results/CO2-fixation/RuBisCO), except that cells were lysed One Shot Cell Disrupters (Constant Systems Ltd - 2.5 kbar). Our precise protocol is available here: [[File: T--Go-Paris-Saclay-protocol-RuBisCO.pdf]]
-We thus performed a RuBisCO activity test on <i>S. bottropensis</i> ATCC 25435 and <i>E. coli </i> BL21DE3 strains harboring a plasmid encoding <i>ccbL</i> (<partinfo>BBa_K4370000</partinfo>, <partinfo>BBa_K4370002</partinfo>) and cbbS (<partinfo>BBa_K4370001</partinfo>, <partinfo>BBa_K4370003</partinfo>) under the control of <i>tetO</i> promoter (<partinfo>BBa_R0040</partinfo>) or a control plasmid (containing <partinfo>BBa_K4370010</partinfo>). The background in the <i>E. coli</i> extracts in the analysis area makes the result inconclusive (Figure 1).
+We thus performed a RuBisCO activity test on <i>S. bottropensis</i> ATCC 25435 and <i>E. coli </i> BL21DE3 strains harboring a plasmid encoding <i>ccbL</i> (<partinfo>BBa_K4370000</partinfo>, <partinfo>BBa_K4370002</partinfo>) and cbbS (<partinfo>BBa_K4370001</partinfo>, <partinfo>BBa_K4370003</partinfo>) under the control of <i>tetO</i> promoter (<partinfo>BBa_R0040</partinfo>) or a control plasmid (containing <partinfo>BBa_K4370010</partinfo>). The background in the <i>E. coli</i> extracts in the analysis area makes the result inconclusive (<b>Figure 1</b>).
-However, we obtained interesting results in <i>S. bottropensis</i> background. First, we noticed that the final biomass reached by <i>S. bottropensis</i> in sub-minimal liquid medium under 3 % CO<sub>2</sub> enriched atmosphere was twice the final biomass reached in this medium in a standard atmosphere. Such a doubling in the biomass under enriched atmosphere was not observed with another <i>Streptomyces</i> grown in the same conditions (Figure 2). This result suggests that at least in one condition (absence of carbon source and under 3 % CO<sub>2</sub>), <i>S. bottropensis</i> may fix CO<sub>2</sub> to form biomass. This observation supports the hypothesis that its CO<sub>2</sub> fixation module encodes a functional RuBisCO that is expressed under these conditions.
+However, we obtained interesting results in <i>S. bottropensis</i> background. First, we noticed that the final biomass reached by <i>S. bottropensis</i> in sub-minimal liquid medium under 3 % CO<sub>2</sub> enriched atmosphere was twice the final biomass reached in this medium in a standard atmosphere. Such a doubling in the biomass under enriched atmosphere was not observed with another <i>Streptomyces</i> grown in the same conditions (<b>Figure 2</b>). This result suggests that at least in one condition (absence of carbon source and under 3 % CO<sub>2</sub>), <i>S. bottropensis</i> may fix CO<sub>2</sub> to form biomass. This observation supports the hypothesis that its CO<sub>2</sub> fixation module encodes a functional RuBisCO that is expressed under these conditions.
-Our HPLC analysis of the signal detected in the UV (220 nm) allows to detect both the substrate (RuBP, ribulose-1,5-bisphosphate) and the product (PGA, 3-phosphoglycerate), used as standards. After around 1 min incubation at room temperature of RuBP with cell extract, the RuBP was not detected in all <i>S. bottropensis</i> samples, suggesting that it was used by some enzymes (… that may be RuBisCO, Figure 3). To be sure that the RuBP was consumed by the RuBisCO, we needed to detect the product (PGA). Interestingly, a signal was detected at the retention time of the PGA only in the sub-minimal medium (devoid of carbon source) under standard atmosphere. Neither RuBP nor GPA was detected in the reaction using cell extract of cells grown in this medium under 3 % CO<sub>2</sub>  enriched atmosphere. This suggests that if GPA was produced in this condition, it was used by other cellular enzymes to produce compounds not detected in our window of analysis. These enzymes could be other enzymes encoded by the CO<sub>2</sub> fixation module of <i>S. bottropensis</i> and/or the endogenous pentose pathway of the strain. We note a significant baseline in some samples which can make the results ambiguous. Thus the determination of the nature of the products by mass spectrometry could allow to confirm these first results.
+Our HPLC analysis of the signal detected in the UV (220 nm) allows to detect both the substrate (RuBP, ribulose-1,5-bisphosphate) and the product (PGA, 3-phosphoglycerate), used as standards. After around 1 min incubation at room temperature of RuBP with cell extract, the RuBP was not detected in all <i>S. bottropensis</i> samples (<b>Figure 3</b>), suggesting that it was used by some enzymes (… that may be RuBisCO!). To be sure that the RuBP was consumed by the RuBisCO, we needed to detect the product of this enzyme (PGA). Interestingly, a signal was detected at the retention time of the PGA only in the sub-minimal medium (devoid of carbon source) under standard atmosphere. Neither RuBP nor GPA was detected in the reaction using cell extract of cells grown in this medium under 3 % CO<sub>2</sub>  enriched atmosphere. This suggests that if GPA was produced in this condition, it was used by other cellular enzymes to produce compounds not detected in our window of analysis. These enzymes could be other enzymes encoded by the CO<sub>2</sub> fixation module of <i>S. bottropensis</i> and/or its endogenous pentose phosphate pathway. We note a significant baseline in some samples which can make the results ambiguous. Thus the determination of the nature of the products by mass spectrometry could allow to confirm the identity of the product and therefore these first results.
 <b>Taken together, these results provide for the first time preliminary data in favour of the functionality of a <i>Streptomyces</i> RuBisCO, which will need to be confirmed by further analyses. </b>
-[[Image: T--Go-Paris-Saclay-BBa_K4370000_1_2_3_CBB_results_1.png|800px|thumb|left|'''Figure 1:''' HPLC analysis of RuBisCO assay perform with RuBP (ribulose-1-5-bisphosphate) and cell extract of <i>E. coli </i> harboring a plasmid encoding ccbL (<partinfo>BBa_K4370000</partinfo>, <partinfo>BBa_K4370002</partinfo>) and cbbS (<partinfo>BBa_K4370001</partinfo>, <partinfo>BBa_K4370003</partinfo>) under the control of tetO promoter (<partinfo>BBa_R0040</partinfo>), or control extracts. Please note the y-scale change compared to Figure 3. The very high baseline in <i>E. coli</i> samples makes this test inconclusive.]]
+[[Image: T--Go-Paris-Saclay-BBa_K4370000_1_2_3_CBB_results_1.png|400px|thumb|left|'''Figure 1:''' HPLC analysis of RuBisCO assay perform with RuBP (ribulose-1-5-bisphosphate) and cell extract of <i>E. coli </i> harboring a plasmid encoding ccbL (<partinfo>BBa_K4370000</partinfo>, <partinfo>BBa_K4370002</partinfo>) and cbbS (<partinfo>BBa_K4370001</partinfo>, <partinfo>BBa_K4370003</partinfo>) under the control of tetO promoter (<partinfo>BBa_R0040</partinfo>), or control extracts. Please note the y-scale change compared to Figure 3. The very high baseline in <i>E. coli</i> samples makes this test inconclusive.]]
 [[Image: T--Go-Paris-Saclay-BBa_K4370000_1_2_3_CBB_results_2.png|800px|thumb|left|'''Figure 2:''' Biomass reached after 4 days of culture of <i>Streptomyces</i> under standard or CO<sub>2</sub> enriched atmosphere. The media were inoculated with an initial biomass of env. 0.04 g.]]
-[[Image: T--Go-Paris-Saclay-BBa_K4370000_1_2_3_CBB_results_3.png|800px|thumb|left|'''Figure 3:''' HPLC analysis of RuBisCO assay perform with RuBP (ribulose-1-5-bisphosphate) and cell extract of <i>S. bottropensis</i> grown under various conditions]]
+[[Image: T--Go-Paris-Saclay-BBa_K4370000_1_2_3_CBB_results_3.png|400px|thumb|left|'''Figure 3:''' HPLC analysis of RuBisCO assay perform with RuBP (ribulose-1-5-bisphosphate) and cell extract of <i>S. bottropensis</i> grown under various conditions]]
 ===User Reviews===