Difference between revisions of "Part:BBa K4604015"
 
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piG_01b is a construct consisting of the tet promoter/repressor, the modified T7 RBS, a functional <i>bluB</i> gene and the rrnB terminator. The backbone we used for experiments is pGGAselect. The tet operator is a BioBrick from iGEM Freiburg 2022 (<a href="https://parts.igem.org/Part:BBa_K4229059">BBa_K4229059</a>) This BioBrick can be used to produce Adenosylcobalamin (AdoCbl) in <i>E. coli</i> when supplemented with cobinamide. The tetA/B promoter used in this version of the plasmid is a modified version of piG_01a/<a href="https://parts.igem.org/Part:BBa_K4604016">BBa_K4604016</a>.  
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piG_01b is consisting of the tet promoter/repressor, the modified T7 RBS, a functional <i>bluB</i> gene and the rrnB terminator. The backbone we used for experiments is pGGAselect. The tet operator is a BioBrick from iGEM Freiburg 2022 (<a href="https://parts.igem.org/Part:BBa_K4229059">BBa_K4229059</a>). Our BioBrick can be used to produce Adenosylcobalamin (AdoCbl) in <i>E. coli</i> when supplemented with cobinamide. The tetA/B promoter used in this version of the plasmid is a modified version of piG_01a (<a href="https://parts.igem.org/Part:BBa_K4604016">BBa_K4604016</a>).  
 
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AdoCbl is a bioavailable form of B12. Vitamin B12 is an essential nutrient, humans are dependent on for the production of red blood cells, the synthesis of the DNA and the function of nerves. To form the complete AdoCbl synthesis pathway in <i>E. coli</i>, it would require 28 additional genes. Since this is not realistic nor practical for an iGEM project, we decided on an alternative method. When supplemented with cobinamide, a precursor for AdoCbl, <i>E. coli</i> is capable of producing AdoCbl on their own in small amounts. With the overexpression of a naturally occurring gene of the synthesis pathway of <i>sinorhizobium meliloti 2011</i>, called <i>bluB</i> <a href="https://parts.igem.org/Part:BBa_K4604005">BBa_K4604005</a>, a greater yield can be achieved [2].         
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AdoCbl is a bioavailable form of B12, which is an essential nutrient required for the production of red blood cells, the synthesis of the DNA and the function of nerves. To form the complete AdoCbl synthesis pathway in <i>E. coli</i>, it would require 28 additional genes. Since this is not realistic nor practical for an iGEM project, we decided on an alternative method. When supplemented with cobinamide, a precursor for AdoCbl, <i>E. coli</i> is capable of producing AdoCbl on their own in small amounts. With the overexpression of a naturally occurring gene of the synthesis pathway of <i>sinorhizobium meliloti 2011</i>, called <i>bluB</i> (<a href="https://parts.igem.org/Part:BBa_K4604005">BBa_K4604005</a>), a greater yield can be achieved [1].         
 
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===Characterization===
 
===Characterization===
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<img src="figure 15 bioproduction results" width="700">
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<img src="https://static.igem.wiki/teams/4604/wiki/b12-results/prodf15.png" width="700">
 
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<span style="font-size: smaller;"><b>Figure 1: BluB enzyme expression for different inducer concentrations.</b> Detection of recombinantly expressed, his-tagged BluB enzyme with SDS-PAGE followed by Western Blot. Detection of the BluB protein was performed with an anti-his antibody. Loading control: RNA polymerase β-subunit.
 
<span style="font-size: smaller;"><b>Figure 1: BluB enzyme expression for different inducer concentrations.</b> Detection of recombinantly expressed, his-tagged BluB enzyme with SDS-PAGE followed by Western Blot. Detection of the BluB protein was performed with an anti-his antibody. Loading control: RNA polymerase β-subunit.
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We examined the expression of BluB using Western Blot. Cells containing this BioBrick were compared to ones with <a href="https://parts.igem.org/Part:BBa_K4604020">BBa_K4604020</a> (piG_07)
 
  
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<img src="figure 18 bioproduction results" width="700">
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We examined the expression of BluB using Western Blot. Cells containing this BioBrick were compared to ones with piG_07 (<a href="https://parts.igem.org/Part:BBa_K4604020">BBa_K4604020</a>). </html>
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<span style="font-size: smaller;"><b>Figure 2: BluB expression in modified <i>E. coli</i> MG1655 cells with different substrates, induced and non-induced.</b> Detection of recombinantly expressed, his-tagged BluB enzyme with SDS-PAGE followed by Western Blot. Detection of the BluB protein was performed with an anti-his antibody. Loading control: RNA polymerase β-subunit.</span>
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As seen in the graph above, induced cultures show clear bands that prove BluB expression. There are slight bands indicating BluB expression for non-induced cells containing this BioBrick. There was no bluB expression in cells carrying piG_07 (<a href="https://parts.igem.org/Part:BBa_K4604020">BBa_K4604020</a>) or pGGAselect.  
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LC-MS is a method used to determine the concentration of molecules based on their mass. With this, it was possible to detect how much Hydroxocobalamin (OHCbl) is produced (relative to dry cell mass). When AdoCbl is exposed to light, it is converted to another derivative of B12, namely OHCbl. To make the preparation of the samples and measurement easier, we worked in sunlight, thereby converting AdoCbl into OHCbl and measured the amount of this compound. We cultivated bacteria containing either a functional or a mutated version of the <i>blub</i> gene and afterwards sent them to Hannibal Lab at the University Medical Center Freiburg, to be measured via mass spectrometry.  
  
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Next a LC-MS measurement of OHCbl was performed.
 
  
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<img src="figure 19 ODER 20 lc-ms production" width="700">
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As seen in the graph above, induced cultures show clear bands that prove BluB expression. There are slight bands indicating BluB expression for non-induced cells containing this BioBrick. There was no bluB expression in cells carrying piG_07 (<a href="https://parts.igem.org/Part:BBa_K4604020">BBa_K4604020</a>) or pGGAselect. </html>
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<span style="font-size: smaller;"><b>Figure 3: OHCbl and Cbi contents in modified <i>E. coli</i> MG1655 cells from production cultures, measured by LC-MS.</b> (A) OHCbl in cell pellets (B) Cbi in cell pellets (C) OHCbl and Cbi in supernatants. LC-MS performed at Hannibal Lab, University Medical Center Uniklinik Freiburg. n=2 span>
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LC-MS measurement shows that the induced cultures of <i>E. coli</i> MG1655[piG_01b] express BluB (Figure 18). This in turn leads to the production of DMB, leading to AdoCbl synthesis when cells are supplied with Cbi. <i>E. coli</i> cells, which do not carry the BluB gene, also produce AdoCbl when treated with Cbi and DMB. These results prove that <i>E. coli</i> MG1655 cells take up the precursor Cbi to then produce AdoCbl.
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Next a LC-MS measurement of OHCbl was performed using constructs with either a functional (piG_01b) or non-functional (piG_07/<a href="https://parts.igem.org/Part:BBa_K4604020">BBa_K4604020</a>) version of <i>bluB</i>.</html>
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<img src="https://static.igem.wiki/teams/4604/wiki/parts-registry/comp-p-mc-results.png" width="700">
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<span style="font-size: smaller;"><b>Figure 2: OHCbl content measurement with LC-MS in dry cell pellet.</b> <i>E. coli MG1655</i> cultures induced with 100 ng/mL DOX were supplemented with 500nM cobinamide. Samples taken immediately before induction and after cultivation for 24 h. LC-MS performed at Hannibal Lab, University Medical Center Freiburg.</span>
  
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===References===
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LC-MS measurement shows that the induced cultures of <i>E. coli</i> containing this BioBrick produce AdoCbl as opposed to the ones containing piG_07 (<a href="https://parts.igem.org/Part:BBa_K4604020">BBa_K4604020</a>). Thereby we have both proven the expression of BluB as well as AdoCbl synthesis.</html>
  
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1] Hallberg ZF, Su Y, Kitto RZ, Hammond MC. Engineering and In Vivo Applications of Riboswitches. Annual Review of Biochemistry [Internet]. 2017 Jun 20;86(1):515–39. Available from: https://doi.org/10.1146/annurev-biochem-060815-014628
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===References===
  
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[2] Fowler CC, Brown ED, Li Y. Using a Riboswitch Sensor to Examine Coenzyme B12 Metabolism and Transport in E. coli. Chemistry & Biology [Internet]. 2010 Jul 1;17(7):756–65. Available from: https://doi.org/10.1016/j.chembiol.2010.05.025
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[1] Fowler CC, Brown ED, Li Y. Using a Riboswitch Sensor to Examine Coenzyme B12 Metabolism and Transport in E. coli. Chemistry & Biology [Internet]. 2010 Jul 1;17(7):756–65. Available from: https://doi.org/10.1016/j.chembiol.2010.05.025
  
 
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Latest revision as of 05:56, 12 October 2023


piG_01b (tetR_bluB)

piG_01b is consisting of the tet promoter/repressor, the modified T7 RBS, a functional bluB gene and the rrnB terminator. The backbone we used for experiments is pGGAselect. The tet operator is a BioBrick from iGEM Freiburg 2022 (BBa_K4229059). Our BioBrick can be used to produce Adenosylcobalamin (AdoCbl) in E. coli when supplemented with cobinamide. The tetA/B promoter used in this version of the plasmid is a modified version of piG_01a (BBa_K4604016).

Usage and Biology

AdoCbl is a bioavailable form of B12, which is an essential nutrient required for the production of red blood cells, the synthesis of the DNA and the function of nerves. To form the complete AdoCbl synthesis pathway in E. coli, it would require 28 additional genes. Since this is not realistic nor practical for an iGEM project, we decided on an alternative method. When supplemented with cobinamide, a precursor for AdoCbl, E. coli is capable of producing AdoCbl on their own in small amounts. With the overexpression of a naturally occurring gene of the synthesis pathway of sinorhizobium meliloti 2011, called bluB (BBa_K4604005), a greater yield can be achieved [1].

Characterization

Figure 1: BluB enzyme expression for different inducer concentrations. Detection of recombinantly expressed, his-tagged BluB enzyme with SDS-PAGE followed by Western Blot. Detection of the BluB protein was performed with an anti-his antibody. Loading control: RNA polymerase β-subunit.


We examined the expression of BluB using Western Blot. Cells containing this BioBrick were compared to ones with piG_07 (BBa_K4604020).

LC-MS is a method used to determine the concentration of molecules based on their mass. With this, it was possible to detect how much Hydroxocobalamin (OHCbl) is produced (relative to dry cell mass). When AdoCbl is exposed to light, it is converted to another derivative of B12, namely OHCbl. To make the preparation of the samples and measurement easier, we worked in sunlight, thereby converting AdoCbl into OHCbl and measured the amount of this compound. We cultivated bacteria containing either a functional or a mutated version of the blub gene and afterwards sent them to Hannibal Lab at the University Medical Center Freiburg, to be measured via mass spectrometry.


As seen in the graph above, induced cultures show clear bands that prove BluB expression. There are slight bands indicating BluB expression for non-induced cells containing this BioBrick. There was no bluB expression in cells carrying piG_07 (BBa_K4604020) or pGGAselect. Next a LC-MS measurement of OHCbl was performed using constructs with either a functional (piG_01b) or non-functional (piG_07/BBa_K4604020) version of bluB.

Figure 2: OHCbl content measurement with LC-MS in dry cell pellet. E. coli MG1655 cultures induced with 100 ng/mL DOX were supplemented with 500nM cobinamide. Samples taken immediately before induction and after cultivation for 24 h. LC-MS performed at Hannibal Lab, University Medical Center Freiburg.

LC-MS measurement shows that the induced cultures of E. coli containing this BioBrick produce AdoCbl as opposed to the ones containing piG_07 (BBa_K4604020). Thereby we have both proven the expression of BluB as well as AdoCbl synthesis.

References

[1] Fowler CC, Brown ED, Li Y. Using a Riboswitch Sensor to Examine Coenzyme B12 Metabolism and Transport in E. coli. Chemistry & Biology [Internet]. 2010 Jul 1;17(7):756–65. Available from: https://doi.org/10.1016/j.chembiol.2010.05.025

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 710
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1618