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| We used six replicates with 196 µL of the mixture and measured it for about 20 min (measurement all 30 sec) with the Tecan infinite® 200. We then paused the measurement program to add 4 µL (6 µg) of the previously extracted CDA or 4 µL of water to three samples each. Then, we immediately continued the measurement for about an hour. <br> | | We used six replicates with 196 µL of the mixture and measured it for about 20 min (measurement all 30 sec) with the Tecan infinite® 200. We then paused the measurement program to add 4 µL (6 µg) of the previously extracted CDA or 4 µL of water to three samples each. Then, we immediately continued the measurement for about an hour. <br> |
| As it can be seen in Figure 2, the absorption of cytidine at 282 nm began to continuously decrease after the addition of the cytidine deaminase, whereas the absorption remained more or less constant when only water was added. With these results, the activity of our extracted cytidine deaminase could be proven. | | As it can be seen in Figure 2, the absorption of cytidine at 282 nm began to continuously decrease after the addition of the cytidine deaminase, whereas the absorption remained more or less constant when only water was added. With these results, the activity of our extracted cytidine deaminase could be proven. |
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| <div class="figure medium"> | | <div class="figure medium"> |
− | <img class="figure image" src="https://static.igem.org/mediawiki/2017/d/d4/T--Bielefeld-CeBiTec--CytidineCDA.svg"> | + | <img class="figure image" src="https://static.igem.org/mediawiki/2017/2/22/T--Bielefeld-CeBiTec--CytidineCDA.png"> |
| <p class="figure subtitle"><b>Figure (2): Enzyme activity assay for the reaction of the cytidine deaminase with cytidine.</b>The reaction took place at room temperatue. Three biological replicates were used each. After the addition of water, the absorbance at 282 nm stayed the same whereas it decreased after the addition of the CDA. </p> | | <p class="figure subtitle"><b>Figure (2): Enzyme activity assay for the reaction of the cytidine deaminase with cytidine.</b>The reaction took place at room temperatue. Three biological replicates were used each. After the addition of water, the absorbance at 282 nm stayed the same whereas it decreased after the addition of the CDA. </p> |
| </div> | | </div> |
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− | After confirming general activity of CDA, we set up a possible reaction with xanthosine instead of cytidine, all other components being the same. However, since there was no real literature on this reaction, we first had to figure out the absorption rate at which xanthosine can be measured. This was done using a general spectrum analysis of different mixtures, three samples each: | + | <div class="article"> |
| + | After confirming general activity of CDA, we set up a possible reaction with xanthosine instead of cytidine, all other components being the same. However, since there was no real literature on this reaction, we first had to figure out the absorption rate at which xanthosine can be measured. This was done using a general spectrum analysis of different mixtures, three samples each: <br><br> |
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| <ol> | | <ol> |
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| <li>without xanthosine , with CDA</li> | | <li>without xanthosine , with CDA</li> |
| <li>with xanthosine, with CDA</li> | | <li>with xanthosine, with CDA</li> |
− | </ol> | + | </ol><br><br> |
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| These mixtures were then compared to estimate the absorption rate for xanthosine. | | These mixtures were then compared to estimate the absorption rate for xanthosine. |
| + | </article><br><br> |
| <ol type=A> | | <ol type=A> |
| <li>1+3: difference between a reaction mixture with and without CDA</li> | | <li>1+3: difference between a reaction mixture with and without CDA</li> |
| <li>1+2: difference between a reaction mixture with and without xanthosine</li> | | <li>1+2: difference between a reaction mixture with and without xanthosine</li> |
| <li>2+4: difference between no reaction and a possible reaction</li> | | <li>2+4: difference between no reaction and a possible reaction</li> |
− | </ol> | + | </ol><br><br> |
− | We hereby could figure out the absorption rate at which xanthosine can be measured (B) as well as ensure that the peak was independent from the CDA (A). Further on, we could identify the absorbance of CDA at about 254-260 nm (A and C). (Figure 1) ) | + | <article> |
− | <div class="figure seventy"> | + | We hereby could figure out the absorption rate at which xanthosine can be measured (B) as well as ensure that the peak was independent from the CDA (A). Further on, we could identify the absorbance of CDA at about 254-260 nm (A and C). (Figure 1)) |
− | <img class="figure image" src="https://static.igem.org/mediawiki/2017/0/07/T--Bielefeld-CeBiTec--xanthosine_NM.svg"> | + | </article> |
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| + | <div class="figure medium"> |
| + | <img class="figure image" src="https://static.igem.org/mediawiki/2017/9/97/T--Bielefeld-CeBiTec--xanthosine_NM.png"> |
| <p class="figure subtitle"><b>Figure (1): Results of the analysis of the absorbance of xanthosine at different nanometers. </b> <br> AAll measurements made with the Tecan infinite® 200 at room temperature. The difference between a mixture with and without xanthosine (red) can clearly be made up at about 282 nm.</p> | | <p class="figure subtitle"><b>Figure (1): Results of the analysis of the absorbance of xanthosine at different nanometers. </b> <br> AAll measurements made with the Tecan infinite® 200 at room temperature. The difference between a mixture with and without xanthosine (red) can clearly be made up at about 282 nm.</p> |
| </div> | | </div> |
− | Afterwards we set up new activity assays, using 196 µL of the reaction mixture in six of the well plate’s holes. After measuring the absorbance at 282 nm, we added 4 µL of either water or the enzyme (6 µg) to three biological replicates each, continuing the (previous) measurements for about an hour. | + | |
| + | <div class="article"> |
| + | Afterwards we set up new activity assays, using 196 µL of the reaction mixture in six of the well plate’s holes. After measuring the absorbance at 282 nm, we added 4 µL of either water or the enzyme (6 µg) to three biological replicates each, continuing the (previous) measurements for about an hour. <br><br> |
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| The reaction of the cytidine deaminase with xanthosine showed diverse results (Figure 3). Here, also a slight decrease of the xanthosine concentration could be seen, which, however, was not significant. | | The reaction of the cytidine deaminase with xanthosine showed diverse results (Figure 3). Here, also a slight decrease of the xanthosine concentration could be seen, which, however, was not significant. |
− | <div class="figure seventy"> | + | </article> |
− | <img class="figure image" src="https://static.igem.org/mediawiki/2017/8/86/T--Bielefeld-CeBiTec--XanthosineCDA.svg"> | + | |
| + | <div class="figure medium"> |
| + | <img class="figure image" src="https://static.igem.org/mediawiki/2017/d/d4/T--Bielefeld-CeBiTec--XanthosineCDA.png"> |
| <p class="figure subtitle"><b>Figure (3): Enzyme activity assay for the reaction of the cytidine deaminase with xanthosine as a substrate.</b>The reaction was set up at room temperature, using three biological replicates each. After adding CDA to the reaction mixture, a slight decrease in the absorbance at 282 nm was visible. However, as there is also a very small decrease for the addition of water, no significant difference was observed.</p> | | <p class="figure subtitle"><b>Figure (3): Enzyme activity assay for the reaction of the cytidine deaminase with xanthosine as a substrate.</b>The reaction was set up at room temperature, using three biological replicates each. After adding CDA to the reaction mixture, a slight decrease in the absorbance at 282 nm was visible. However, as there is also a very small decrease for the addition of water, no significant difference was observed.</p> |
− | </div> | + | </div> |
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| + | <div class="article"> |
| The HPLC-MicroTofQ Measurements could only make up the xanthosine and various other substances. However, there were no significant masses and peaks for guanosine or iso-guanosine. (Figure 4) | | The HPLC-MicroTofQ Measurements could only make up the xanthosine and various other substances. However, there were no significant masses and peaks for guanosine or iso-guanosine. (Figure 4) |
− | <div class="figure seventy"> | + | </div> |
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| + | <div class="figure medium"> |
| <img class="figure image" src="https://static.igem.org/mediawiki/2017/2/22/T--Bielefeld-CeBiTec--HPLC_xanthosine.png"> | | <img class="figure image" src="https://static.igem.org/mediawiki/2017/2/22/T--Bielefeld-CeBiTec--HPLC_xanthosine.png"> |
| <p class="figure subtitle"><b>Figure (4): HPLC-MicroTofQ measurement for the products of the reaction of CDA with xanthosine. </b>Measurement at 40 °C. Even if many different masses could be detected, none of these could be matched to guanosine or iso-guanosine. For these, a peak should be at about 282 g/mol.</p> | | <p class="figure subtitle"><b>Figure (4): HPLC-MicroTofQ measurement for the products of the reaction of CDA with xanthosine. </b>Measurement at 40 °C. Even if many different masses could be detected, none of these could be matched to guanosine or iso-guanosine. For these, a peak should be at about 282 g/mol.</p> |
| </div> | | </div> |
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| + | <div class="article"> |
| So, with only a slight decrease of the absorbance and no detectable products in the HPLC, it seems reliable that there is only a very small amount of xanthosine converted to isoguanosine, since the reaction is not specific to the CDA and thus rare. However, supplementary tests and experiments with different reaction mixtures would be needed to further analyze it. | | So, with only a slight decrease of the absorbance and no detectable products in the HPLC, it seems reliable that there is only a very small amount of xanthosine converted to isoguanosine, since the reaction is not specific to the CDA and thus rare. However, supplementary tests and experiments with different reaction mixtures would be needed to further analyze it. |
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| <h4> GMPS </h4> | | <h4> GMPS </h4> |
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| We set up the reaction mixture of the two isoforms of the GMPS following a protocol for the enzyme activity assay by Abbott, J., Newell, J., Lightcap, C. et al.(2006). We also regarded the original paper from 1985 that stated the absorbance at 290 nm for the given amount of XMP within the mixture. For that, we set up the following reaction mixture: | | We set up the reaction mixture of the two isoforms of the GMPS following a protocol for the enzyme activity assay by Abbott, J., Newell, J., Lightcap, C. et al.(2006). We also regarded the original paper from 1985 that stated the absorbance at 290 nm for the given amount of XMP within the mixture. For that, we set up the following reaction mixture: |
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| <ul> | | <ul> |
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| <li>0.8mM EDTA</li> | | <li>0.8mM EDTA</li> |
| <li>Filled up with ddH<sub>2</sub>O</li> | | <li>Filled up with ddH<sub>2</sub>O</li> |
− | </ul> | + | </ul><br><br> |
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− | Due to their instability, XMP and ATP were always added freshly. After the samples were set up, we measured them with the Tecan infinite® 200 reader for about 20 minutes at an absorbance of 290 nm. Afterwards, 4 µL of either water or 4 µL (6 µg) of the isoforms of the GMPS (isoform1: <a href=" http://parts.igem.org/Part:BBa_K2201060"> BBa_K220160</a> and isoform 2: <a href=" http://parts.igem.org/Part:BBa_K2201061">BBa_K220161</a>) were each added to three samples. The measurement was continued for approximately an hour. The activity assays of isoforms 1 and 2 both proved that the GMPS enzymes are working correctly, reducing the amount of XMP in the reaction mixture significantly. Therefore, the absorption at 290 nm decreased a lot after adding the enzyme to the solution of isoform 1 of GMPS, whereas the initial decrease was weaker for the codon-optimized isoform 2. However, both decreased the amount of XMP about the same within the hour in which their reaction was measured. Thus, it can be said that both, isoform 1 and isoform 2 are working as expected (See Figure 5 and Figure 6 for comparison) | + | <div class="article"> |
| + | Due to their instability, XMP and ATP were always added freshly. After the samples were set up, we measured them with the Tecan infinite® 200 reader for about 20 minutes at an absorbance of 290 nm. Afterwards, 4 µL of either water or 4 µL (6 µg) of the isoforms of the GMPS (isoform1: <a href=" http://parts.igem.org/Part:BBa_K2201060"> BBa_K220160</a> and isoform 2: <a href=" http://parts.igem.org/Part:BBa_K2201061">BBa_K220161</a>) were each added to three samples. The measurement was continued for approximately an hour. The activity assays of isoforms 1 and 2 both proved that the GMPS enzymes are working correctly, reducing the amount of XMP in the reaction mixture significantly. Therefore, the absorption at 290 nm decreased a lot after adding the enzyme to the solution of isoform 1 of GMPS, whereas the initial decrease was weaker for the codon-optimized isoform 2. However, both decreased the amount of XMP about the same within the hour in which their reaction was measured. Thus, it can be said that both, isoform 1 and isoform 2 are working as expected (See Figure 5 and Figure 6 for comparison) |
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| <div class="figure hundred"> | | <div class="figure hundred"> |
− | <img class="figure image" src="https://static.igem.org/mediawiki/2017/f/f2/T--Bielefeld-CeBiTec--GMPSGeneSyn.svg"> | + | <img class="figure image" src="https://static.igem.org/mediawiki/2017/6/66/T--Bielefeld-CeBiTec--GMPSGeneSyn.png"> |
| <p class="figure subtitle"><b>Figure (5): Enzyme activity assay of iso-form 2 of the guanosine monophosphate synthetases.</b>The reaction was set up at room temperature using three biological replicates. A significant decrease in the absorption at 290 nm can be made up after the addition of the synthetized GMPS whereas the negative control with water stays at the same absorption. </p> | | <p class="figure subtitle"><b>Figure (5): Enzyme activity assay of iso-form 2 of the guanosine monophosphate synthetases.</b>The reaction was set up at room temperature using three biological replicates. A significant decrease in the absorption at 290 nm can be made up after the addition of the synthetized GMPS whereas the negative control with water stays at the same absorption. </p> |
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| <div class="half right"> | | <div class="half right"> |
| <div class="figure hundred"> | | <div class="figure hundred"> |
− | <img class="figure image" src="https://static.igem.org/mediawiki/2017/7/7f/T--Bielefeld-CeBiTec--GMPSSelf.svg"> | + | <img class="figure image" src="https://static.igem.org/mediawiki/2017/0/0c/T--Bielefeld-CeBiTec--GMPSSelf.png"> |
| <p class="figure subtitle"><b>Figure (6):Enzyme activity assay of iso-form1 of the guanosine monophosphate synthetases.</b>Three biological replicates were used. The reaction was set up at room temperature. A significant decrease in the absorption at 290 nm can be made up after the addition of the GMPS whereas the negative control with water stays at the same absorption. </p> | | <p class="figure subtitle"><b>Figure (6):Enzyme activity assay of iso-form1 of the guanosine monophosphate synthetases.</b>Three biological replicates were used. The reaction was set up at room temperature. A significant decrease in the absorption at 290 nm can be made up after the addition of the GMPS whereas the negative control with water stays at the same absorption. </p> |
| </div> | | </div> |
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| As described earlier, it took some time to figure out the right requirements for the HPLC-MicroTofQ measurements, since iso-GMP and GMP have the exact same mass and are thus only separable by their structure. However, with the method chosen in the end, it was possible to identify analytes that seem to represent iso-GMP. Therefore, at first, the general substances within the reaction mix had to be figured out to ensure that only those representing GMP/iso-GMP will be included in the analyses. The general analysis of all substances included showed significant values for all the interesting substrates and products that should be within the reaction mix, including AMP, ADP and ATP, some remaining traces of XMP and of course GMP/iso-GMP (Figures 7 and 8). | | As described earlier, it took some time to figure out the right requirements for the HPLC-MicroTofQ measurements, since iso-GMP and GMP have the exact same mass and are thus only separable by their structure. However, with the method chosen in the end, it was possible to identify analytes that seem to represent iso-GMP. Therefore, at first, the general substances within the reaction mix had to be figured out to ensure that only those representing GMP/iso-GMP will be included in the analyses. The general analysis of all substances included showed significant values for all the interesting substrates and products that should be within the reaction mix, including AMP, ADP and ATP, some remaining traces of XMP and of course GMP/iso-GMP (Figures 7 and 8). |
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| We then compared the resulting form of GMP with a GMP-standard (10^-5 diluted solution) and the exact measurements of the HPLC. For both, isoform 2 and isoform 1 of GMPS the peaks of the substance’s flow-through found at the molecular mass of GMP and iso-GMP (approximately 363.22 g/mol, in the graph at approximately 362 g/mol because of the missing H due to the measurement method) were significantly shifted to the right compared to the standard. Thus, the form of GMP that is created with the enzyme reactions of the two isoforms of GMPS and the gene synthesis has to be another form of GMP, most likely iso-GMP. (Figure 9) | | We then compared the resulting form of GMP with a GMP-standard (10^-5 diluted solution) and the exact measurements of the HPLC. For both, isoform 2 and isoform 1 of GMPS the peaks of the substance’s flow-through found at the molecular mass of GMP and iso-GMP (approximately 363.22 g/mol, in the graph at approximately 362 g/mol because of the missing H due to the measurement method) were significantly shifted to the right compared to the standard. Thus, the form of GMP that is created with the enzyme reactions of the two isoforms of GMPS and the gene synthesis has to be another form of GMP, most likely iso-GMP. (Figure 9) |
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− | <div class="figure seventy"> | + | <div class="figure medium"> |
| <img class="figure image" src="https://static.igem.org/mediawiki/2017/c/c7/T--Bielefeld-CeBiTec--HPLC_GMPvsISOGMP.png"> | | <img class="figure image" src="https://static.igem.org/mediawiki/2017/c/c7/T--Bielefeld-CeBiTec--HPLC_GMPvsISOGMP.png"> |
| <p class="figure subtitle"><b>Figure (9): HPLC-MicroTofQ measurement comparing the GMP standard and the reaction products’ flow-through. </b> In red the product of isoform 2 of GMPS. In blue, the one found for isoform 1 of GMPS, in green the standard. Even though the standard as well as the mixtures contained compounds that have the same molecular mass, they show different behaviors on the HPLC. The ordinary GMP was significantly faster than the one generated in the enzyme reactions. Thus, the form of GMP that results from the reactions is likely to be iso-GMP. </p> | | <p class="figure subtitle"><b>Figure (9): HPLC-MicroTofQ measurement comparing the GMP standard and the reaction products’ flow-through. </b> In red the product of isoform 2 of GMPS. In blue, the one found for isoform 1 of GMPS, in green the standard. Even though the standard as well as the mixtures contained compounds that have the same molecular mass, they show different behaviors on the HPLC. The ordinary GMP was significantly faster than the one generated in the enzyme reactions. Thus, the form of GMP that results from the reactions is likely to be iso-GMP. </p> |
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| In conclusion, we did not only figure out the synthesis pathways in <i>Croton tiglium</i> but could even recreate a part of it, showing that the enzymes expressed in <i>Croton tiglium</i> are more likely to generate a different form of GMP (presumably iso-GMP). | | In conclusion, we did not only figure out the synthesis pathways in <i>Croton tiglium</i> but could even recreate a part of it, showing that the enzymes expressed in <i>Croton tiglium</i> are more likely to generate a different form of GMP (presumably iso-GMP). |
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| <b>Abbott, J., Newell, J., Lightcap, C. <i>et al.</i></b>(2006) The Effects of Removing the GAT Domain from E.coli GMP Synthetase. <b>25</b> 384<br> | | <b>Abbott, J., Newell, J., Lightcap, C. <i>et al.</i></b>(2006) The Effects of Removing the GAT Domain from E.coli GMP Synthetase. <b>25</b> 384<br> |
| <b>Cohen, R.M., Wolfenden, R.</b> (1971). Cytidine Deaminase from <i>E.coli</i> – Purificarion Properties and Inhibiton by the potential transition state analog 3,4,5,6-tetrahydrouridine. The Journal of Biological Chemistry. <b>25</b><br> | | <b>Cohen, R.M., Wolfenden, R.</b> (1971). Cytidine Deaminase from <i>E.coli</i> – Purificarion Properties and Inhibiton by the potential transition state analog 3,4,5,6-tetrahydrouridine. The Journal of Biological Chemistry. <b>25</b><br> |
| <b>Zalkin, H </b>(1971), GMP sythesis | | <b>Zalkin, H </b>(1971), GMP sythesis |
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