Difference between revisions of "Part:BBa K2398000"

Latest revision as of 01:37, 2 November 2017

Cytochrome c for the synthesis of organosilicons

We present a codon-optimized version of the cytochrome c protein derived from Rhodotermus marinus. This protein is able to perform the conversion of organosilicons. It is a novel catalytic unit that allows utilization of silicon compounds [Arnold et al., 2016]. When in its mature form, it catalyzes a carbene insertion into silicon-hydrogen bonds [Coelho et al., 2013].

Usage and Biology

The cytochrome c is used for the catalysis of silicon compounds. This cytochrome c variant provides an easy to use tool that is accessible to everyone in the synthetic biology community and allows the user to harness the vast potential of organosilicons. This basic part exhibits a strong tendency to form silicon-carbon bonds and is a valuable addition to perform controlled organic chemistry in microorganisms. A triple mutant of this part has already been applied in the successful synthesis of organosilicons as a proof-of-concept. As a next step, this part can be implemented in the directed evolution approach of phage-assisted continuous evolution (PACE) or in the phage-related discontinuous evolution (PREDCEL) approach to improve organosilicon synthesis by cytochrome engineering.

Characterisation

For the characterization of this part, we conducted experiments for the production of organosilicons and therefore used a previously described triple mutant, already engineered by [Arnold et al., 2016]. According to the protocol, we obtained the matured cytochrome c and performed conversion experiments analyzed via GC-MS method. The following figures show the results obtained with the previously engineered enzyme.

Figure 1: Used educts are depicted at the top while produced products are depicted on the bottom.

First, we tested the enzyme on the conversion of the compound (1) with (3) to the final product of (5) (Fig. 1). The gas chromatogram shows one product peak which emerges at 9.2 min retention time. Analysis of the gas chromatogram indicates a conversion rate of >99% on the basis of the non-existence of the respective educt peak.

Figure 2: Gas chromatogram for the reaction of educt (2) and (5) to the product (4). 9.2 minutes retention time indicates product formation.

As we performed GC-MS analysis, mass spectrometry data were also available which can be seen in Fig. 3 for the product peak that emerges at 9.2 minutes. It shows the fission of this product and can clearly be identified as our desired product (5).

Figure 3: Mass chromatogram shows the breakdown of the product (4) ethyl 2-(dimethyl(phenyl)silyl)propanoate. The product itself corresponds to a mass of 236 daltons.

Furthermore, we performed experiments on the compound (1) which differs from compound (2) in its amino functional group. The product is eluted after 11.7 minutes retention time (Fig. 4) and also confirmed as the right product via mass spectrometry in Fig. 5. Worth mentioning, there is still educt (1) present which emerges at 6.9 minutes retention time, indicating incomplete conversion. Calculations showed a conversion rate of 47.5% for the product (4).

Figure 4: Gas chromatogram for the reaction of educt (1) and (3) to the product (5). 11.7 minutes retention time indicates product formation. Unconverted educts converge 6.9 and 7.2 (7.4) minutes.

Figure 5: Mass chromatogram shows the breakdown of the product (3) ethyl 2-((4-aminophenyl)dimethylsilyl)propanoate. The product itself corresponds to a mass of 251 daltons.

As these results only show activity for a previously engineered cytochrome c, we are convinced that our part is still functional despite not as effective as the mutant. This part as our best basic part is intended as a platform for future teams to evolve their own cytochrome c for their specific target molecule.

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 93
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 2: / Line 2: @@
 ===Cytochrome c for the synthesis of organosilicons===
-We present a codon-optimized version of the cytochrome c protein derived from ''Rhodotermus marinus''. This protein is able to perform the conversion of organosilicons. It is a novel catalytic unit that allows utilization of silicon compounds [Arnold et al., 2016]. When in its mature form, it catalyzes a carbene insertion into silicon-hydrogen bonds.
+We present a codon-optimized version of the cytochrome c protein derived from ''{{#tag:html|<a href="http://www.rcsb.org/pdb/explore.do?structureId=3cp5">Rhodotermus marinus</a>}}''. This protein is able to perform the conversion of organosilicons. It is a novel catalytic unit that allows utilization of silicon compounds [Arnold et al., 2016]. When in its mature form, it catalyzes a carbene insertion into silicon-hydrogen bonds [Coelho et al., 2013].
 ===Usage and Biology===
-The cytochrome c is used for the catalysis of silicon compounds
+The cytochrome c is used for the catalysis of silicon compounds. This cytochrome c variant provides an easy to use tool that is accessible to everyone in the synthetic biology community and allows the user to harness the vast potential of organosilicons. This basic part exhibits a strong tendency to form silicon-carbon bonds and is a valuable addition to perform controlled organic chemistry in microorganisms. A triple mutant of this part has already been applied in the successful synthesis of organosilicons as a proof-of-concept. As a next step, this part can be implemented in the directed evolution approach of phage-assisted continuous evolution ({{#tag:html|<a href="http://2017.igem.org/Team:Heidelberg/Pace">PACE</a>}}) or in the phage-related discontinuous evolution ({{#tag:html|<a href="http://2017.igem.org/Team:Heidelberg/Predcel">PREDCEL</a>}}) approach to improve organosilicon synthesis by cytochrome engineering.
 ===Characterisation===
-[[File:https://static.igem.org/mediawiki/2017/9/92/T--Heidelberg--GCAnilineCompound.png|frame|center|Figure 1: Determination of the MIC of beta-lactamases with varying carbenicillin concentration in liquid culture. The OD600 was determined with a sample of 100 µl in 96 well plates]]
+For the characterization of this part, we conducted experiments for the production of organosilicons and therefore used a previously described triple mutant, already engineered by [Arnold et al., 2016]. According to the {{#tag:html|<a href="http://2017.igem.org/Team:Heidelberg/Experiments">protocol</a>}}, we obtained the matured cytochrome c and performed conversion experiments analyzed via GC-MS method. The following figures show the results obtained with the previously engineered enzyme.
-[[File:https://static.igem.org/mediawiki/2017/1/16/T--Heidelberg--MSAnilineCompound.png|frame|center|Figure 1: Determination of the MIC of beta-lactamases with varying carbenicillin concentration in liquid culture. The OD600 was determined with a sample of 100 µl in 96 well plates]]
+[[File:T--Heidelberg--Compounds.png|thumb|center|Figure 1: Used educts are depicted at the top while produced products are depicted on the bottom.]]
-[[File:https://static.igem.org/mediawiki/2017/e/ef/T--Heidelberg--GCNonAninlineCompound.png|frame|center|Figure 1: Determination of the MIC of beta-lactamases with varying carbenicillin concentration in liquid culture. The OD600 was determined with a sample of 100 µl in 96 well plates]]
+First, we tested the enzyme on the conversion of the compound (1) with (3) to the final product of (5) (Fig. 1). The gas chromatogram shows one product peak which emerges at 9.2 min retention time. Analysis of the gas chromatogram indicates a conversion rate of >99% on the basis of the non-existence of the respective educt peak.
-[[File:https://static.igem.org/mediawiki/2017/2/2b/T--Heidelberg--MSNonAnilineCompound.png|frame|center|Figure 1: Determination of the MIC of beta-lactamases with varying carbenicillin concentration in liquid culture. The OD600 was determined with a sample of 100 µl in 96 well plates]]
+[[File:T--Heidelberg--GCNonAninlineCompound.png|thumb|center|Figure 2: Gas chromatogram for the reaction of educt (2) and (5) to the product (4). 9.2 minutes retention time indicates product formation.]]
+As we performed GC-MS analysis, mass spectrometry data were also available which can be seen in Fig. 3 for the product peak that emerges at 9.2 minutes. It shows the fission of this product and can clearly be identified as our desired product (5).
+[[File:T--Heidelberg--MSNonAnilineCompound.png|thumb|center|Figure 3: Mass chromatogram shows the breakdown of the product (4) <i>ethyl 2-(dimethyl(phenyl)silyl)propanoate</i>. The product itself corresponds to a mass of 236 daltons.]]
+Furthermore, we performed experiments on the compound (1) which differs from compound (2) in its amino functional group. The product is eluted after 11.7 minutes retention time (Fig. 4) and also confirmed as the right product via mass spectrometry in Fig. 5. Worth mentioning, there is still educt (1) present which emerges at 6.9 minutes retention time, indicating incomplete conversion. Calculations showed a conversion rate of 47.5% for the product (4).
+[[File:T--Heidelberg--GCAnilineCompound.png|thumb|center|Figure 4: Gas chromatogram for the reaction of educt (1) and (3) to the product (5). 11.7 minutes retention time indicates product formation. Unconverted educts converge 6.9 and 7.2 (7.4) minutes.]]
+[[File:T--Heidelberg--MSAnilineCompound.png|thumb|center|Figure 5: Mass chromatogram shows the breakdown of the product (3) <i>ethyl 2-((4-aminophenyl)dimethylsilyl)propanoate</i>. The product itself corresponds to a mass of 251 daltons.]]
+As these results only show activity for a previously engineered cytochrome c, we are convinced that our part is still functional despite not as effective as the mutant. This part as our best basic part is intended as a platform for future teams to evolve their own cytochrome c for their specific target molecule.