http://2012.igem.org/wiki/index.php?title=Special:Contributions/Anyakornilo&feed=atom&limit=50&target=Anyakornilo&year=&month=2012.igem.org - User contributions [en]2024-03-28T20:20:32ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:Calgary/Notebook/ElectrochemTeam:Calgary/Notebook/Electrochem2012-10-27T03:50:27Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookGreen|<br />
TITLE=Electrochemistry Notebook|<br />
CONTENT = <br />
<html><br />
<h2>Week 1 (May 1-4)</h2><br />
<p>This week involved planning various experiments and gathering chemicals to be tested. Unfortunately no lab work was conducted this week.</p><br />
<h2>Week 2 (May 7-11)</h2><br />
<p>Chlorophenol red (CPR) was oxidized in a sodium phosphate solution with zinc phthalocyanine modified carbon electrodes or just straight carbon electrodes. It was found that the modified electrodes had greater responses than their unmodified counterparts. All future work with CPR will be done with these electrodes. An IPTG induced <i>lacZ</i> was transformed for the registry and the <i>uidA</i> gene was amplified and biobricked under the control of the IPTG promoter. These genes will be used to cleave specific sugars from electrochemical analytes.</p><br />
<h2>Week 3 (May 14-18)</h2><br />
<p>A standard curve was generated using CPR as the analyte in a sodium phosphate solution. This was made with the modified electrodes. After this it was found that the reference electrode being used, a silver/ silver chloride electrode, had stopped functioning. Switching to a reduction of hydrogen electrode (RHE) reference fixed this problem. A gold working electrode was also briefly tested to reduce the background noise caused by the capacitance of carbon. Further testing will be needed to choose a final electrode.</p><br />
</html>[[File:Screen Shot 2012-06-18 at 5.15.59 PM.png|thumb|500px|center|Figure 1: Standard curve for the oxidation of CPR at a carbon working electrode in 25mL pH7 0.1M PBS with Argon bubbling. The reference electrode was RHE and the counter electrode was platinum.]]<html><br />
<h2>Week 4 (May 21-25)</h2><br />
<p>This week involved testing the oxidation of CPR and para-aminophenol (PAP) in the same solution to determine if their oxidation potentials were unique. By showing that they are separate peaks it demonstrates that they can be used as two of the electrochemical components of a multiplexed biosensor. After a morning of failed tests on Thursday we finally managed to show that the peaks are unique (0.7V for PAP and 1.3V for CPR vs RHE), giving us the first step towards a final sensor.</p></html>[[File:CPR+PAP.png|thumb|500px|center|Figure 2: Electrochemical detection of CPR and PAP in the same cell. PAP has the reversible wave seen at 0.75V vs RHE, while CPR has the irreversible wave at 1.3V vs RHE. These values were recorded in 25mL pH7 0.1M PBS with Ar bubbling on a carbon working electrode with a platinum counter electrode and a RHE as the reference electrode.]]<html><br />
<h2>Week 5 (May 28- June 1)</h2><br />
<p>This week the data collected was inconsistent across multiple runs even though the electrodes used were from the same batch. It seems that each of these electrodes is unique, presenting a challenge for analyzing results when multiple electrode setups are used.</p><br />
<h2>Week 6 (June 4-8)</h2><br />
<p>This week marked the start of the construction of a mathematical model of the electrochemical system. The goal of this model is to identify the shortest time in which an electrochemical signal could be reached and what conditions would decrease this detection time. To do this the MATLAB toolbox SimBiology will be used as well as differential solvers and published rate constants. The model will include translation, transcription, RNA and protein degradation, and the actual conversion from the sugar conjugate into the electrochemical analyte.</p><br />
<h2>Week 7 (June 11-15)</h2><br />
<p>The list of final analytes was compiled this week, giving a goal of chemicals to test with and without their sugar conjugates. The genes necessary to break these compounds apart were also noted and primers were designed so that they could be amplified. More work was placed on the model as well, getting it to the point where it can go from a constant amount of plasmid DNA into the RNA transcript and then the protein product.</p><br />
<h2>Week 8 (June 18-22)</h2><br />
<p>The model has been updated to include the transport of PAPG, the sugar conjugated form of PAP, into the cell through the LacY transport protein. The diffusion of PAP across the membrane out into the solution was also included into the model. A diagramatic representation of this is shown below. The last of the chemicals needed for the electrochemical testing was ordered this week too, meaning that work can begin at an increased pace on the wetlab front when the shipment arrives.</p><br />
</html>[[File:Calgary_Model.png|thumb|500px|center|Figure 3: Diagramatic representation of the mathematical model of electrochemical detection. PAPGE and PAPE stand for PAPG and PAP external to the bacterial cell. Green circles are enzymatic reactions and red circles are degradation reactions.]]<html><br />
<h2>Week 9 (June 25-29)</h2><br />
<p>Results from the model have been obtained, hinting at a detection time of approximately 250 seconds, or just over 4 minutes. This is based on the detection of PAP at 0.06mM, which is equivalent to the 1500 &mu;L of PAP sweep shown in Figure 2. The sequencing results from the <i>lacZ</i> and <i>uidA</i> genes have arrived and the <i>lacZ</i> construct is fine while the <i>uidA</i> construct has some mutations.</p><br />
<h2>Week 10 (July 3-6)</h2><br />
<p>A standard curve was attempted for PAP, however the graphs recorded resembled a solution of intense resistivity. A possible explanation for this is the clip connecting the potentiostat to the electrode having an incomplete connection. An electrode with a stronger connection was constructed and tested to remedy this, however it presented no detection of the PAP at any concentration. Switching to a platinum working electrode showed a strong response and testing will continue with this next week.</p><br />
<h2>Week 11 (July 9-13)</h2><br />
<p>Testing continued using platinum to detect the presence of PAP in a solution of PBS. Problems were encountered with the conductivity of the testing solution, but were remedied through the preparation of a new batch of PBS. One other concern was that the electrodes were becoming coated in various chemicals from the solution and thus degrading the strength of the signal obtained. To clean the electrodes they were submerged in a solution of concentrated H<sub>2</sub>SO<sub>4</sub> for two days. On the genetics side the <i>bglX</i> gene was biobricked and sent for sequencing this week, giving us that last brick for the electrochemistry project for now.</p><br />
<h2>Week 12 (July 16-20)</h2><br />
<p>Using the cleaned platinum electrode a standard curve of PAP was attempted however no results were obtain. After consulting with multiple graduate students and professors in electrochemistry it was discovered that the reason for this could be the solubility of PAP. With this in mind the PAP system was shelved for the time being until it can be brought into solution. Moving on to the chemical PDP (para-diphenol, commonly called hydroquinone) and PNP (para-nitrophenol), two other chemicals with specific sugar conjugates to be tested. These two compounds are more soluble than PAP and went into solution immediately. A quick run with both chemicals was conducted to determine if they were detectable, and it appeared that their oxidation potentials were around 1.0V and 1.6V vs RHE and that they were easily detectable.</p><br />
<h2>Week 13 (July 23-27)</h2><br />
<p>More characterization was done on PDP, including a potentiostatic run. This type of run is where the working electrode is held at a certain potential, in this case the oxidation potential of PDP, and as more of the analyte is added the current increases proportionally. The benefit of this kind of testing is the increased speed as there is no need to sweep over potentials where the compound would not react.</p><br />
<h2>Week 14 (July 30- Aug 3)</h2><br />
<p>Using the potentiostatic method PDP was consistently detected at 0.825V and 0.85V vs RHE with no detection of the sugar conjugate PDPG (para-diphenol-B-D-glucopyranoside) at this potential. As the graphs were cleaner at 0.825V vs RHE this potential will be used for future analysis of PDP and Beta-Glucosidase activity. A graph of potentiostatic testing at 0.825V vs RHE is shown below. Testing with PNP shows that the oxidation potential is between 1.5 and 1.7V.</p></html><br />
[[File:Calgary_2012_0.825V_PDP.jpg|thumb|500px|center|Figure 4: Potentiostatic run at 0.825V vs RHE with additions of 0.4&mu;M every 60 seconds after a 5 minute current stabilization period at the beginning of the experiment. At the end of the graph PDPG was added twice and caused no increase in current.]]<html><br />
<h2>Week 15 (Aug 6-10)</h2><br />
<p>Potentiostatic runs of PDP were further characterized and runs on PNP at 1.5V and 1.6V vs RHE were started. PNPG (para-nitrophenol-B-D-glucorinide) was tested for any electrochemical reactions from 0-2V vs RHE but no reactions were observed. As this was the case no effect was observed during the potentiostatic runs with the addition of PNPG.</p><br />
<h2>Week 16 (Aug 13-17)</h2><br />
<p>This week some testing was conducted on the Biosensor prototype in parallel with the potentiostat usually used for the electrochemistry experiments. It turns out that the prototype only works with the blue carbon strip electrodes and cannot perform experiments with other electrodes. The oxidation potential for PDP was tested on these electrodes and determined to be -0.1V vs the pseudo-Ag/AgCl reference electrode on the strips. This was confirmed through potentiostatic detection at this voltage.</p><br />
<h2>Week 17 (Aug 20-24)</h2><br />
<p>PNP was tested on the blue strip electrodes and it's oxidation potential was determined to be at around +0.6V vs the pseudo-Ag/AgCl reference electrode. The potentiostat prototype was able to detect this chemical at the same oxidation potential when using the blue strip electrodes. Some software glitches were encountered but they were resolved by the end of the testing day.</p><br />
<h2>Week 18 (Aug 27-31)</h2><br />
<p>ONP (ortho-nitrophenol) was tested as a substitute for PAP. No discernible detection was noted during initial testing. More work was done to bring the prototype up to the same detection thresholds as that of the commercial potentiostat.</p><br />
<h2>Week 19 (Sept 3-7)</h2><br />
<p>The genetic circuit with <i>uidA</i> under the control of the IPTG-inducible <i>lacI</i> promoter was confirmed through sequencing this week. More work is still needed on the circuits for <i>lacZ</i> and <i>bglX</i>, as the Registry <i>lacZ</i> has a frameshift mutation, and the <i>bglX</i> gene has proven difficult to work with due to a recently discovered pstI cut site in the gene.</p><br />
<h2>Week 20 (Sept 10-14)</h2><br />
<p>Testing was done on the <i>uidA</i> circuit and a response was observed in the presence of IPTG. This is shown below in Figure 5. The experiment was repeated with and without IPTG to detect any leaky expression.</p><br />
</html><br />
[[File:Calgary2012 ECHEM PNP.png|thumb|600px|center|Figure 5: Potentiostatic detection of PNP at 1.6V vs RHE by either an IPTG induced <i>uidA</i> gene or leaky expression of an uninduced <i>uidA</i> gene.]]<br />
<html><br />
<h2>Week 21 (Sept 17-21)</h2><br />
<p>This week work on a new <i>lacZ</i> substrate was halted and testing with CPR resumed. Due to limited time before the competition it was deemed best to use a working compound rather than to explore new options. Experiments mostly focused around confirming old data.</p><br />
<h2>Week 22 (Sept 24-28)</h2><br />
<p>A lot of coding was done on the wiki this week as the wiki freeze is just around the corner. Tests were performed on a suspected <i>bglX</i> culture but no response was observed. A new circuit is being constructed under the control of the constitutive <i>tetR</i> promoter to see if a response can be generated.</p><br />
<h2>Week 23 (Oct 1-23)</h2><br />
<p>WIKI FREEZE!!! Lots of coding was done to get the wiki to the point where it was presentable to the judges. Testing was also done with <i>lacZ</i> and CPRG to show that a response is possible. The constitutive <i>bglX</i> was also tested and you can find the results on our <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">electroreporting page.</a></p><br />
</html><br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/ElectrochemTeam:Calgary/Notebook/Electrochem2012-10-27T03:37:52Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookGreen|<br />
TITLE=Electrochemistry Notebook|<br />
CONTENT = <br />
<html><br />
<h2>Week 1 (May 1-4)</h2><br />
<p>This week involved planning various experiments and gathering chemicals to be tested. Unfortunately no lab work was conducted this week.</p><br />
<h2>Week 2 (May 7-11)</h2><br />
<p>Chlorophenol red (CPR) was oxidized in a sodium phosphate solution with zinc phthalocyanine modified carbon electrodes or just straight carbon electrodes. It was found that the modified electrodes had greater responses than their unmodified counterparts. All future work with CPR will be done with these electrodes. An IPTG induced <i>lacZ</i> was transformed for the registry and the <i>uidA</i> gene was amplified and biobricked under the control of the IPTG promoter. These genes will be used to cleave specific sugars from electrochemical analytes.</p><br />
<h2>Week 3 (May 14-18)</h2><br />
<p>A standard curve was generated using CPR as the analyte in a sodium phosphate solution. This was made with the modified electrodes. After this it was found that the reference electrode being used, a silver/ silver chloride electrode, had stopped functioning. Switching to a reduction of hydrogen electrode (RHE) reference fixed this problem. A gold working electrode was also briefly tested to reduce the background noise caused by the capacitance of carbon. Further testing will be needed to choose a final electrode.</p><br />
</html>[[File:Screen Shot 2012-06-18 at 5.15.59 PM.png|thumb|500px|center|Figure 1: Standard curve for the oxidation of CPR at a carbon working electrode in 25mL pH7 0.1M PBS with Argon bubbling. The reference electrode was RHE and the counter electrode was platinum.]]<html><br />
<h2>Week 4 (May 21-25)</h2><br />
<p>This week involved testing the oxidation of CPR and para-aminophenol (PAP) in the same solution to determine if their oxidation potentials were unique. By showing that they are separate peaks it demonstrates that they can be used as two of the electrochemical components of a multiplexed biosensor. After a morning of failed tests on Thursday we finally managed to show that the peaks are unique (0.7V for PAP and 1.3V for CPR vs RHE), giving us the first step towards a final sensor.</p></html>[[File:CPR+PAP.png|thumb|500px|center|Figure 2: Electrochemical detection of CPR and PAP in the same cell. PAP has the reversible wave seen at 0.75V vs RHE, while CPR has the irreversible wave at 1.3V vs RHE. These values were recorded in 25mL pH7 0.1M PBS with Ar bubbling on a carbon working electrode with a platinum counter electrode and a RHE as the reference electrode.]]<html><br />
<h2>Week 5 (May 28- June 1)</h2><br />
<p>This week the data collected was inconsistent across multiple runs even though the electrodes used were from the same batch. It seems that each of these electrodes is unique, presenting a challenge for analyzing results when multiple electrode setups are used.</p><br />
<h2>Week 6 (June 4-8)</h2><br />
<p>This week marked the start of the construction of a mathematical model of the electrochemical system. The goal of this model is to identify the shortest time in which an electrochemical signal could be reached and what conditions would decrease this detection time. To do this the MATLAB toolbox SimBiology will be used as well as differential solvers and published rate constants. The model will include translation, transcription, RNA and protein degradation, and the actual conversion from the sugar conjugate into the electrochemical analyte.</p><br />
<h2>Week 7 (June 11-15)</h2><br />
<p>The list of final analytes was compiled this week, giving a goal of chemicals to test with and without their sugar conjugates. The genes necessary to break these compounds apart were also noted and primers were designed so that they could be amplified. More work was placed on the model as well, getting it to the point where it can go from a constant amount of plasmid DNA into the RNA transcript and then the protein product.</p><br />
<h2>Week 8 (June 18-22)</h2><br />
<p>The model has been updated to include the transport of PAPG, the sugar conjugated form of PAP, into the cell through the LacY transport protein. The diffusion of PAP across the membrane out into the solution was also included into the model. A diagramatic representation of this is shown below. The last of the chemicals needed for the electrochemical testing was ordered this week too, meaning that work can begin at an increased pace on the wetlab front when the shipment arrives.</p><br />
</html>[[File:Calgary_Model.png|thumb|500px|center|Figure 3: Diagramatic representation of the mathematical model of electrochemical detection. PAPGE and PAPE stand for PAPG and PAP external to the bacterial cell. Green circles are enzymatic reactions and red circles are degradation reactions.]]<html><br />
<h2>Week 9 (June 25-29)</h2><br />
<p>Results from the model have been obtained, hinting at a detection time of approximately 250 seconds, or just over 4 minutes. This is based on the detection of PAP at 0.06mM, which is equivalent to the 1500 &mu;L of PAP sweep shown in Figure 2. The sequencing results from the <i>lacZ</i> and <i>uidA</i> genes have arrived and the <i>lacZ</i> construct is fine while the <i>uidA</i> construct has some mutations.</p><br />
<h2>Week 10 (July 3-6)</h2><br />
<p>A standard curve was attempted for PAP, however the graphs recorded resembled a solution of intense resistivity. A possible explanation for this is the clip connecting the potentiostat to the electrode having an incomplete connection. An electrode with a stronger connection was constructed and tested to remedy this, however it presented no detection of the PAP at any concentration. Switching to a platinum working electrode showed a strong response and testing will continue with this next week.</p><br />
<h2>Week 11 (July 9-13)</h2><br />
<p>Testing continued using platinum to detect the presence of PAP in a solution of PBS. Problems were encountered with the conductivity of the testing solution, but were remedied through the preparation of a new batch of PBS. One other concern was that the electrodes were becoming coated in various chemicals from the solution and thus degrading the strength of the signal obtained. To clean the electrodes they were submerged in a solution of concentrated H<sub>2</sub>SO<sub>4</sub> for two days. On the genetics side the <i>bglX</i> gene was biobricked and sent for sequencing this week, giving us that last brick for the electrochemistry project for now.</p><br />
<h2>Week 12 (July 16-20)</h2><br />
<p>Using the cleaned platinum electrode a standard curve of PAP was attempted however no results were obtain. After consulting with multiple graduate students and professors in electrochemistry it was discovered that the reason for this could be the solubility of PAP. With this in mind the PAP system was shelved for the time being until it can be brought into solution. Moving on to the chemical PDP (para-diphenol, commonly called hydroquinone) and PNP (para-nitrophenol), two other chemicals with specific sugar conjugates to be tested. These two compounds are more soluble than PAP and went into solution immediately. A quick run with both chemicals was conducted to determine if they were detectable, and it appeared that their oxidation potentials were around 1.0V and 1.6V vs RHE and that they were easily detectable.</p><br />
<h2>Week 13 (July 23-27)</h2><br />
<p>More characterization was done on PDP, including a potentiostatic run. This type of run is where the working electrode is held at a certain potential, in this case the oxidation potential of PDP, and as more of the analyte is added the current increases proportionally. The benefit of this kind of testing is the increased speed as there is no need to sweep over potentials where the compound would not react.</p><br />
<h2>Week 14 (July 30- Aug 3)</h2><br />
<p>Using the potentiostatic method PDP was consistently detected at 0.825V and 0.85V vs RHE with no detection of the sugar conjugate PDPG (para-diphenol-B-D-glucopyranoside) at this potential. As the graphs were cleaner at 0.825V vs RHE this potential will be used for future analysis of PDP and Beta-Glucosidase activity. A graph of potentiostatic testing at 0.825V vs RHE is shown below. Testing with PNP shows that the oxidation potential is between 1.5 and 1.7V.</p></html><br />
[[File:Calgary_2012_0.825V_PDP.jpg|thumb|500px|center|Figure 4: Potentiostatic run at 0.825V vs RHE with additions of 0.4&mu;M every 60 seconds after a 5 minute current stabilization period at the beginning of the experiment. At the end of the graph PDPG was added twice and caused no increase in current.]]<html><br />
<h2>Week 15 (Aug 6-10)</h2><br />
<p>Potentiostatic runs of PDP were further characterized and runs on PNP at 1.5V and 1.6V vs RHE were started. PNPG (para-nitrophenol-B-D-glucorinide) was tested for any electrochemical reactions from 0-2V vs RHE but no reactions were observed. As this was the case no effect was observed during the potentiostatic runs with the addition of PNPG.</p><br />
<h2>Week 16 (Aug 13-17)</h2><br />
<p>This week some testing was conducted on the Biosensor prototype in parallel with the potentiostat usually used for the electrochemistry experiments. It turns out that the prototype only works with the blue carbon strip electrodes and cannot perform experiments with other electrodes. The oxidation potential for PDP was tested on these electrodes and determined to be -0.1V vs the pseudo-Ag/AgCl reference electrode on the strips. This was confirmed through potentiostatic detection at this voltage.</p><br />
<h2>Week 17 (Aug 20-24)</h2><br />
<p>PNP was tested on the blue strip electrodes and it's oxidation potential was determined to be at around +0.6V vs the pseudo-Ag/AgCl reference electrode. The potentiostat prototype was able to detect this chemical at the same oxidation potential when using the blue strip electrodes. Some software glitches were encountered but they were resolved by the end of the testing day.</p><br />
<h2>Week 18 (Aug 27-31)</h2><br />
<p>ONP (ortho-nitrophenol) was tested as a substitute for PAP. No discernible detection was noted during initial testing. More work was done to bring the prototype up to the same detection thresholds as that of the commercial potentiostat.</p><br />
<h2>Week 19 (Sept 3-7)</h2><br />
<p>The genetic circuit with <i>uidA</i> under the control of the IPTG-inducible <i>lacI</i> promoter was confirmed through sequencing this week. More work is still needed on the circuits for <i>lacZ</i> and <i>bglX</i>, as the Registry <i>lacZ</i> has a frameshift mutation, and the <i>bglX</i> gene has proven difficult to work with due to a recently discovered pstI cut site in the gene.</p><br />
<h2>Week 20 (Sept 10-14)</h2><br />
<p>Testing was done on the <i>uidA</i> circuit and a response was observed in the presence of IPTG. This is shown below in Figure 5. The experiment was repeated with and without IPTG to detect any leaky expression.</p><br />
</html><br />
[[File:Calgary2012 ECHEM PNP.png|thumb|600px|center|Figure 5: Potentiostatic detection of PNP at 1.6V vs RHE by either an IPTG induced <i>uidA</i> gene or leaky expression of an uninduced <i>uidA</i> gene.]]<br />
<html><br />
<h2>Week 21 (Sept 17-21)</h2><br />
<p>This week work on a new <i>lacZ</i> substrate was halted and testing with CPR resumed. Due to limited time before the competition it was deemed best to use a working compound rather than to explore new options. Experiments mostly focused around confirming old data.</p><br />
<h2>Week 22 (Sept 24-28)</h2><br />
<p>A lot of coding was done on the wiki this week as the wiki freeze is just around the corner. Tests were performed on a suspected <i>bglX</i> culture but no response was observed. A new circuit is being constructed under the control of the constitutive <i>tetR</i> promoter to see if a response can be generated.</p><br />
<h2>Week 23 (Oct 1-3)</h2><br />
<p>WIKI FREEZE!!! Lots of coding was done to get the wiki to the point where it was presentable to the judges. Testing was also done with <i>lacZ</i> and CPRG to show that a response is possible. The constitutive <i>bglX</i> was also tested and you can find the results on our <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">electroreporting page.</a></p><br />
</html><br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/ElectrochemTeam:Calgary/Notebook/Electrochem2012-10-27T03:36:03Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookGreen|<br />
TITLE=Electrochemistry Notebook|<br />
CONTENT = <br />
<html><br />
<h2>Week 1 (May 1-4)</h2><br />
<p>This week involved planning various experiments and gathering chemicals to be tested. Unfortunately no lab work was conducted this week.</p><br />
<h2>Week 2 (May 7-11)</h2><br />
<p>Chlorophenol red (CPR) was oxidized in a sodium phosphate solution with zinc phthalocyanine modified carbon electrodes or just straight carbon electrodes. It was found that the modified electrodes had greater responses than their unmodified counterparts. All future work with CPR will be done with these electrodes. An IPTG induced <i>lacZ</i> was transformed for the registry and the <i>uidA</i> gene was amplified and biobricked under the control of the IPTG promoter. These genes will be used to cleave specific sugars from electrochemical analytes.</p><br />
<h2>Week 3 (May 14-18)</h2><br />
<p>A standard curve was generated using CPR as the analyte in a sodium phosphate solution. This was made with the modified electrodes. After this it was found that the reference electrode being used, a silver/ silver chloride electrode, had stopped functioning. Switching to a reduction of hydrogen electrode (RHE) reference fixed this problem. A gold working electrode was also briefly tested to reduce the background noise caused by the capacitance of carbon. Further testing will be needed to choose a final electrode.</p><br />
</html>[[File:Screen Shot 2012-06-18 at 5.15.59 PM.png|thumb|500px|center|Figure 1: Standard curve for the oxidation of CPR at a carbon working electrode in 25mL pH7 0.1M PBS with Argon bubbling. The reference electrode was RHE and the counter electrode was platinum.]]<html><br />
<h2>Week 4 (May 21-25)</h2><br />
<p>This week involved testing the oxidation of CPR and para-aminophenol (PAP) in the same solution to determine if their oxidation potentials were unique. By showing that they are separate peaks it demonstrates that they can be used as two of the electrochemical components of a multiplexed biosensor. After a morning of failed tests on Thursday we finally managed to show that the peaks are unique (0.7V for PAP and 1.3V for CPR vs RHE), giving us the first step towards a final sensor.</p></html>[[File:CPR+PAP.png|thumb|500px|center|Figure 2: Electrochemical detection of CPR and PAP in the same cell. PAP has the reversible wave seen at 0.75V vs RHE, while CPR has the irreversible wave at 1.3V vs RHE. These values were recorded in 25mL pH7 0.1M PBS with Ar bubbling on a carbon working electrode with a platinum counter electrode and a RHE as the reference electrode.]]<html><br />
<h2>Week 5 (May 28- June 1)</h2><br />
<p>This week the data collected was inconsistent across multiple runs even though the electrodes used were from the same batch. It seems that each of these electrodes is unique, presenting a challenge for analyzing results when multiple electrode setups are used.</p><br />
<h2>Week 6 (June 4-8)</h2><br />
<p>This week marked the start of the construction of a mathematical model of the electrochemical system. The goal of this model is to identify the shortest time in which an electrochemical signal could be reached and what conditions would decrease this detection time. To do this the MATLAB toolbox SimBiology will be used as well as differential solvers and published rate constants. The model will include translation, transcription, RNA and protein degradation, and the actual conversion from the sugar conjugate into the electrochemical analyte.</p><br />
<h2>Week 7 (June 11-15)</h2><br />
<p>The list of final analytes was compiled this week, giving a goal of chemicals to test with and without their sugar conjugates. The genes necessary to break these compounds apart were also noted and primers were designed so that they could be amplified. More work was placed on the model as well, getting it to the point where it can go from a constant amount of plasmid DNA into the RNA transcript and then the protein product.</p><br />
<h2>Week 8 (June 18-22)</h2><br />
<p>The model has been updated to include the transport of PAPG, the sugar conjugated form of PAP, into the cell through the LacY transport protein. The diffusion of PAP across the membrane out into the solution was also included into the model. A diagramatic representation of this is shown below. The last of the chemicals needed for the electrochemical testing was ordered this week too, meaning that work can begin at an increased pace on the wetlab front when the shipment arrives.</p><br />
</html>[[File:Calgary_Model.png|thumb|500px|center|Figure 3: Diagramatic representation of the mathematical model of electrochemical detection. PAPGE and PAPE stand for PAPG and PAP external to the bacterial cell. Green circles are enzymatic reactions and red circles are degradation reactions.]]<html><br />
<h2>Week 9 (June 25-29)</h2><br />
<p>Results from the model have been obtained, hinting at a detection time of approximately 250 seconds, or just over 4 minutes. This is based on the detection of PAP at 0.06mM, which is equivalent to the 1500 &mu;L of PAP sweep shown in Figure 2. The sequencing results from the <i>lacZ</i> and <i>uidA</i> genes have arrived and the <i>lacZ</i> construct is fine while the <i>uidA</i> construct has some mutations.</p><br />
<h2>Week 10 (July 3-6)</h2><br />
<p>A standard curve was attempted for PAP, however the graphs recorded resembled a solution of intense resistivity. A possible explanation for this is the clip connecting the potentiostat to the electrode having an incomplete connection. An electrode with a stronger connection was constructed and tested to remedy this, however it presented no detection of the PAP at any concentration. Switching to a platinum working electrode showed a strong response and testing will continue with this next week.</p><br />
<h2>Week 11 (July 9-13)</h2><br />
<p>Testing continued using platinum to detect the presence of PAP in a solution of PBS. Problems were encountered with the conductivity of the testing solution, but were remedied through the preparation of a new batch of PBS. One other concern was that the electrodes were becoming coated in various chemicals from the solution and thus degrading the strength of the signal obtained. To clean the electrodes they were submerged in a solution of concentrated H<sub>2</sub>SO<sub>4</sub> for two days. On the genetics side the <i>bglX</i> gene was biobricked and sent for sequencing this week, giving us that last brick for the electrochemistry project for now.</p><br />
<h2>Week 12 (July 16-20)</h2><br />
<p>Using the cleaned platinum electrode a standard curve of PAP was attempted however no results were obtain. After consulting with multiple graduate students and professors in electrochemistry it was discovered that the reason for this could be the solubility of PAP. With this in mind the PAP system was shelved for the time being until it can be brought into solution. Moving on to the chemical PDP (para-diphenol, commonly called hydroquinone) and PNP (para-nitrophenol), two other chemicals with specific sugar conjugates to be tested. These two compounds are more soluble than PAP and went into solution immediately. A quick run with both chemicals was conducted to determine if they were detectable, and it appeared that their oxidation potentials were around 1.0V and 1.6V vs RHE and that they were easily detectable.</p><br />
<h2>Week 13 (July 23-27)</h2><br />
<p>More characterization was done on PDP, including a potentiostatic run. This type of run is where the working electrode is held at a certain potential, in this case the oxidation potential of PDP, and as more of the analyte is added the current increases proportionally. The benefit of this kind of testing is the increased speed as there is no need to sweep over potentials where the compound would not react.</p><br />
<h2>Week 14 (July 30- Aug 3)</h2><br />
<p>Using the potentiostatic method PDP was consistently detected at 0.825V and 0.85V vs RHE with no detection of the sugar conjugate PDPG (para-diphenol-B-D-glucopyranoside) at this potential. As the graphs were cleaner at 0.825V vs RHE this potential will be used for future analysis of PDP and Beta-Glucosidase activity. A graph of potentiostatic testing at 0.825V vs RHE is shown below. Testing with PNP shows that the oxidation potential is between 1.5 and 1.7V.</p></html><br />
[[File:Calgary_2012_0.825V_PDP.jpg|thumb|500px|center|Figure 4: Potentiostatic run at 0.825V vs RHE with additions of 0.4&mu;M every 60 seconds after a 5 minute current stabilization period at the beginning of the experiment. At the end of the graph PDPG was added twice and caused no increase in current.]]<html><br />
<h2>Week 15 (Aug 6-10)</h2><br />
<p>Potentiostatic runs of PDP were further characterized and runs on PNP at 1.5V and 1.6V vs RHE were started. PNPG (para-nitrophenol-B-D-glucorinide) was tested for any electrochemical reactions from 0-2V vs RHE but no reactions were observed. As this was the case no effect was observed during the potentiostatic runs with the addition of PNPG.</p><br />
<h2>Week 16 (Aug 13-17)</h2><br />
<p>This week some testing was conducted on the Biosensor prototype in parallel with the potentiostat usually used for the electrochemistry experiments. It turns out that the prototype only works with the blue carbon strip electrodes and cannot perform experiments with other electrodes. The oxidation potential for PDP was tested on these electrodes and determined to be -0.1V vs the pseudo-Ag/AgCl reference electrode on the strips. This was confirmed through potentiostatic detection at this voltage.</p><br />
<h2>Week 17 (Aug 20-24)</h2><br />
<p>PNP was tested on the blue strip electrodes and it's oxidation potential was determined to be at around +0.6V vs the pseudo-Ag/AgCl reference electrode. The potentiostat prototype was able to detect this chemical at the same oxidation potential when using the blue strip electrodes. Some software glitches were encountered but they were resolved by the end of the testing day.</p><br />
<h2>Week 18 (Aug 27-31)</h2><br />
<p>ONP (ortho-nitrophenol) was tested as a substitute for PAP. No discernible detection was noted during initial testing. More work was done to bring the prototype up to the same detection thresholds as that of the commercial potentiostat.</p><br />
<h2>Week 19 (Sept 3-7)</h2><br />
<p>The genetic circuit with <i>uidA</i> under the control of the IPTG-inducible <i>lacI</i> promoter was confirmed through sequencing this week. More work is still needed on the circuits for <i>lacZ</i> and <i>bglX</i>, as the Registry <i>lacZ</i> has a frameshift mutation, and the <i>bglX</i> gene has proven difficult to work with due to a recently discovered pstI cut site in the gene.</p><br />
<h2>Week 20 (Sept 10-14)</h2><br />
<p>Testing was done on the <i>uidA</i> circuit and a response was observed in the presence of IPTG. This is shown below in Figure 5. The experiment was repeated with and without IPTG to detect any leaky expression.</p><br />
</html><br />
[[File:Calgary2012 ECHEM PNP.png|thumb|600px|center|Figure 5: Potentiostatic detection of PNP at 1.6V vs RHE by either an IPTG induced <i>uidA</i> gene or leaky expression of an uninduced <i>uidA</i> gene.]]<br />
<html><br />
<h2>Week 21 (Sept 17-21)</h2><br />
<p>This week work on a new <i>lacZ</i> substrate was halted and testing with CPR resumed. Due to limited time before the competition it was deemed best to use a working compound rather than to explore new options. Experiments mostly focused around confirming old data.</p><br />
<h2>Week 22 (Sept 24-28)</h2><br />
<p>A lot of coding was done on the wiki this week as the wiki freeze is just around the corner. Tests were performed on a suspected <i>bglX</i> culture but no response was observed. A new circuit is being constructed under the control of the constitutive <i>tetR</i> promoter to see if a response can be generated.</p><br />
<h2>Week 23 (Oct 1-3)</h2><br />
<p>WIKI FREEZE!!! Lots of coding was done to get the wiki to the point where it was presentable to the judges. Testing was also done with <i>lacZ</i> and CPRG to show that a response is possible. This is shown below in Figure 6. The constitutive <i>bglX</i> was also tested and you can find the results on our <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">electroreporting page.</a></p><br />
</html><br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradationTeam:Calgary/Project/OSCAR/CatecholDegradation2012-10-04T03:56:23Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Decatecholization|<br />
<br />
CONTENT=<html><br />
<img src="https://static.igem.org/mediawiki/2012/1/1c/UCalgary2012_OSCAR_Catechol_Low-Res.png" style="float: right; padding: 10px;"></img><br />
<br />
<br />
<p>Catechol is a toxic compound found in tailings ponds that is a by-product of polyaromatic hydrocarbon metabolism (Vaillancourt <i>et al.</i>, 2006, Schweigert <i>et al.</i>, 2001)). The chemical properties of catechol allow it to react with biomolecules, causing cellular damage including DNA damage, enzyme inactivation and membrane uncoupling (Schweigert <i>et al.</i>, 2001). </p><br />
<p><br />
Catechol is characterized as having a benzene ring with two hydroxyl groups at the 2,3 position. It can be converted to 2-hydroxymuconic acid by the enzyme catechol 2,3-dioxygenase, encoded by the <i>xylE</i> gene on the Tol plasmid of <i>Pseudomonas putida</i> (Nakai <i>et al.</i>, 1983).</p><br />
<br />
<p><br />
Currently the registry has two BioBricks available of <i>xylE</i>. One contained <i>xylE</i> with its native ribosome-binding site (<a href=http://partsregistry.org/Part:BBa_J33204>BBa_J33204</a>), while the other part contained <i>xylE</i> under the glucose-repressible promoter <i>cstA </i>(<a href=http://partsregistry.org/Part:BBa_K118021>BBa_K118021</a>). Given that <i>E. coli</i> is grown in the presence of glucose, we designed a new construct to keep <i>xylE</i> repressed by using the <i>tetR</i> promoter (<a href= http://partsregistry.org/Part:BBa_R0040>BBa_R0040</a>).</p> <br />
<br />
</html>[[File:UCalgary2010_R0040-XylE.png|400px|thumb|Figure 1: BioBrick genetic circuit for catechol degradation showing <i>xylE</i> under the ''tetR'' promoter|center]]<html><br />
<h3></h3><br />
<br />
<p>Catechol 2,3-dioxygenase is an extradiol dioxygenase which cleaves catechol adjacent to the two hydroxyl groups. When this occurs 2-hydroxymuconate semialdehyde is produced, which is yellow in colour. This change in colour allows for visual assay to assess the activity of XylE.</p><br />
<br />
</html>[[File:UCalgary2012_Catechol_to_2-HMS.PNG|400px|thumb|Figure 2: Catechol 2,3-dioxygenase (XylE) converts catechol to 2-Hydroxymuconate semialdehyde in the presence of oxygen. Adapted from Shu <i>et al</i>., 1995.|center]]<html><br />
<br />
<p>The visual assays were performed with <i>E. coli</i> cells transformed with (<a href=http://partsregistry.org/Part:BBa_K118021>BBa_K118021</a>) as well as with <i>E. coli</i> cells transformed with the newly constructed part (<a href=http://partsregistry.org/Part:BBa_K902048 >BBa_K902048</a>) by bringing the supernatant of an overnight culture to a concentration of 0.1 M of catechol. When the part (<a href=http://partsregistry.org/Part:BBa_K118021>BBa_K118021</a>) was used, the pellet was first washed in M9-MM and centrifuged before catechol was added to the supernatant. This was necessary to avoid the glucose in the LB from repressing the cstA promoter (<a href=http://partsregistry.org/Part:BBa_K118011>BBa_K118011</a>). Catechol was added to the supernatant because the reaction takes place outside of the cell. Within minutes of the addition of catechol to the supernatant, the solution turned from the pale yellow of LB to a bright yellow. This was indicative that catechol was breaking down into 2-Hydroxymuconate semialdehyde, which was exactly what we expected! This assay was completed by following the protocol written by the 2008 Edinburgh iGEM team.</p><br />
<br />
</html>[[File:UCalgary2012_Catechol_assay.jpg|500px|thumb|Figure 3: Results of the catechol visual assay using ''xylE'' [http://partsregistry.org/Part:BBa_K118021 BBa_K118021]. Cultures were grown overnight in LB and the pellets were washed with M9-MM at various times (From left to right: 0 min, 5 min, 10 min, 15 min, and 20 min.). Cells were then spun down and catechol was added to the supernatant to 0.1 M. The amount of time didn't affect the colour change in the cultures containing the <i>xylE</i> gene. The far right tube has <i>E. coli</i> cells without the <i>xylE</i> gene as a negative control and the supernatant remained clear when the catechol was added. |center]]<html><br />
<br />
<h2> Converting Catechol into hydrocarbons? </h2><br />
<p>After verifying that we could in fact degrade catechol into 2-hydroxymuconate semialdehyde using our <i>xylE</i> construct (<a href=http://partsregistry.org/Part:BBa_J33204>BBa_J33204</a>), we wondered if we could take this any further. What if we could convert this by-product page into hydrocarbons too? As catechol is the breakdown product of a number of different degradation pathways in bacteria, this could be particularly useful.</p><br />
<br />
<p>As 2-hydroxymuconate semialdehyde can be further metabolized to pyruvate and acetaldehyde (Harayama S et al., 1987), it seemed possible that these products could be routed into the fatty acid biosynthesis pathway and converted to alkanes using the PetroBrick or the OleT enzyme. Given that the Catechol 2,3-dioxygenase reaction is extracellular, it creates a possible scenario in which cells with the <i>xylE</i> construct could be co-cultured with Petrobrick-containing cells to cooperatively metabolise catechol into hydrocarbons. </p><br />
<br />
<p> In order to test this, we followed this <a href=https://2012.igem.org/Team:Calgary/Notebook/Protocols/decatecholization>protocol</a>, where we co-cultured cells expressing our <i>xylE</i> construct with either <i>E. coli</i> cells expressing the PetroBrick, or <i>Jeotgalicoccus</i> sp. ATCC 8456 cells expressing OleT. in the presence of catechol.<br />
<br />
<br />
</html>[[File:Calgary PetrobrickCatechol.jpg|600px|thumb|centre|Figure 4: Gas chromatograph of catechol degradation assay using the PetroBrick. While there is limited differences between <i>xylE</i> incubated with and without the PetroBrick, there was one peak with a retention time of 10.5 min which was dramatically increased in the co-culture.]]<html><br />
<br />
</html>[[File:Calgary MSCatecholPetroPeak.jpg|600px|thumb|centre|Figure 5: Mass spectra of the Petrobrick/<i>xylE</i> co-culture retention peak at 10.5 min as shown in Figure 4. While the identity of this compound is currently unknown, there are changes occuring to some of the catechol breakdown products.]]<html><br />
<br />
</html>[[File:Calgary CatechololeTGC.jpg|600px|thumb|centre|Figure 6: Gas chromatograph of catechol degradation assay using <i>Jeotgalicoccus</i> sp. ATCC 8456 a species of bacteria that converts fatty acids into alkenes. This identified a similar peak change in the PetroBrick with a retention time of 10.5 min as shown in Figure 4. This provides additional support that the PetroBrick and this organism can further degrade catechol into breakdown products.]]<html><br />
<br />
</html>[[File:Calgary CatecholMSoleT.jpg|600px|thumb|centre|Figure 7: Mass spectra of <i>Jeotgalicoccus</i> sp. ATCC 8456/<i>xylE</i> co-culture retention peak at 10.5 min as shown in Figure 6. This peak is similar to the peak from the Petrobrick/<i>xylE</i> co-culture, suggesting the breakdown product for both of these cultures is modified catechol from <i>xylE</i>. The identification of this compound is ongoing.]]<html><br />
<br />
<p> Based on our GC-MS results, we were able to show the appearance of a new peak when cells expressing <i>xylE</i> and the PetroBrick were co-cultured. Although we don't know the exact identity of this peak, it is distinct form our control. Interestingly, a similar peak appeared when cells expressing our <i>xylE</i> construct were co-cultured with <i>Jeotgalicoccus</i> sp. ATCC 8456 cells. This suggests that although we don't know the exact identity of this new peak, it is likely that it may be in fact a further breakdown product of catechol. This is a very promising result, as it suggests that in addition to converting naphthenic acids into hydrocarbons, we may also be able to break down catechol, one of the other major toxic components in tailings ponds.</p><br />
<br />
<br />
<br />
<br />
<br />
</html>}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Project/DataPageTeam:Calgary/Project/DataPage2012-10-04T03:54:22Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Detect and Destroy: Data Page|<br />
CONTENT=<br />
<html><br />
<head><br />
<style><br />
<br />
a.green{<br />
color: #159900 !important;<br />
}<br />
a.blue{<br />
color: #1088CC !important;<br />
}<br />
a.orange{<br />
color: #FF7A00 !important;<br />
}<br />
a.green:visited{<br />
color: #06660B; !important;<br />
}<br />
a.blue:visited{<br />
color: #05B !important;<br />
}<br />
a.orange:visited{<br />
color: #C40 !important;<br />
}<br />
<br />
</style><br />
</head><br />
<br />
<body><br />
<div align="justify"><br />
<br />
<br />
<br />
</html>[[File:UCalgary2012_GraphicalAbstract.png|thumb|700px|center|thumb|Figure 1. The Calgary team has developed a dual system for the detection of toxic components in tailing ponds and the remediation of these compounds. Tailing ponds are large bodies of water containing waste products produced from the extraction of bitumen in the oil sands. Our biosensor involved the identification of a toxin promoter through a transposon screen. An electrochemical detector was implemented with a multiple output system allowing for the detection of multiple compounds simultaneously. This promoter/detector system was then complemented with the production of a biosensor prototype involving both a physical device and a software program for easy data analysis.<br />
<br />
Rather than just sensing toxins in the tailings ponds, a major objective was to detoxify the tailings through the reduction of toxins such as carboxylic acids (mainly naphthenic acids) and catechol, turning them into usable hydrocarbons. Purification of these hydrocarbons would contribute an added economic and industrial benefit. In order to house this system, we also aimed to design a bioreactor for our bacteria as well as optimize product output through a flux-variability based model. Finally, in order to create higher quality hydrocarbons, we explored desulfurization and denitrogenation pathways to upgrade our fuel. To do this in a safe and environmentally sound manner, we built into our design structural containment, as well as genetic control mechanisms through novel inducible ribo-killswitches.]]<html> <br />
<br />
<h2>Characterization of new parts submitted to the Registry</h2><br />
<br />
<ul><li><p>(<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902000">BBa_K902000</a>) and <a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902004">BBa_K902004, </a>): two novel hydrolase enzymes were submitted to the registry for the hydrolysis of two different sugar-conjugated electroactive compounds: PNPG and PDPG. Used in conjunction with the existing lacZ part in the registry (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_I732005">BBa_I732005</a>) which hydrolyzes CPRG, this allows for the electrochemical detection of three compounds with a single electrode. A <i>uidA</i> inducible generator (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902002">BBa_K902002,</a>) was submitted and characterized electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<br />
<li><p>(<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>),(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902023">BBa_K902023</a>) and (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902074">BBa_K902074</a>): three novel riboswitches sensitive to magnesium, molybdate, and manganese were submitted along with two associated promoters (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902009">BBa_K902009</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902073">BBa_K902073</a>) in addition to a rhamnose inducible, glucose repressible promoter (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902065">BBa_K902065</a>). One of these riboswitches (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>) was tested with GFP and a constitutive promoter using this construct, (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902021">BBa_K902021</a>), with its promoter and GFP using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902017">BBa_K902017</a>) and with its promoter and the <i>S7</i> kill gene using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902018">BBa_K902018</a>). This data can be found on our killswitch <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a> page. </li></p><br />
<br />
<li><p>Genes for denitrogenation and desulfurization were biobricked and submitted. A novel oxidoreductase part (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058">BBa_K902058</a>) was also submitted and characterized for use in the desulfurization project. This data can be found on our upgrading <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page.</p></li></ul>. <br />
<br />
<h2>Further characterization of parts already present within the registry </h2><br />
<br />
<ul><li><p>The IPTG inducible lacI regulated promoter (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010">BBa_R0010</a>) was tested electrochemically to demonstrate its leakiness when not used in conjunction with strong expression of regulatory elements. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<li><p>(<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>), the PetroBrick, submitted by the Washington team in 2011, was characterized for a novel function: the conversion of naphthenic acids and 2-hydroxymuconate- a catechol break-down product from from the xylE gene (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J33204">BBa_J33204</a>) into hydrocarbons and potential value added products. This data can be found on both the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> page and the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. We feel that these new and meaningful applications of this part present a distinct improvement on its usefulness for other teams.</li><br />
<br />
<li><p> The output of (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) was also optimized thorugh a program we developed in MATLAB for the optimization of metabolic pathways in synthetic biology metabolic networks. The program allows you to build an artificial synthetic biology network in <i>E. coli</i> and predicts substrates that should be fed to the organism to increase production of the compound. This was characterized and validated in the wetlab with the Petrobrick. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a> page.</li><br />
<br />
<li><p> An existing <i>xylE</i> gene in the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_J33204">BBa_J33204</a>) was constructed with a constitutive promoter instead of the glucose-repressible part available within the registry. This allows for increased output in media containing glucose, making it more suitable for a variety of applications such as our own. We validated the functionality of this part which can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. In addition, we documented a novel application for this part, by using it in conjunction with Washington's PetroBrick (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) to degrade catechol into a further break-down product. </p></li><br />
<br />
</ul><br />
<br />
<br />
<h2>Additional Work and Characterization </h2><br />
<br />
<ul><br />
<li><p>Developed and tested both hardware and software for a biosensor using an electrochemical sensor. The software is available on our wiki as are the results fom the hardware. These are on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a> page</p></li><br />
<br />
<li><p> Characterized one of our constitutively expressed transposon clones to test the lacZ gene electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</p></li><br />
<li><p> Submitted novel parts involved in decarboxylation and validated the functionality of an additional enzyme (oleT), capable of converting fatty acids into alkenes by itself. This was done in its host organism. This data can be found in our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> section, however this gene has not yet been submitted due to problems cloning it.</p></li><br />
<br />
<li><p>Designed and prototyped a physical bioreactor for which we obtained both qualitative and quantitative data for its functionality. This is outlined on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a> page. </p></li><br />
<br />
<li><p>Characterized the biodegradation of carbazole and various sulfur-containing compounds resembling naphthenic acids in the organisms from which we got our genes. This data can be found on our (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a>) and (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a>)</p></li><br />
<br />
<br />
<br />
<br />
<li><p>Resubmitted (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K206009">BBa_K26009</a>) an inconsistent registry composite part that we had to construct from basic parts, resubmitting as the sequence-verified (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902016">BBa_K902016</a>)</p></li><br />
<br />
</body><br />
<br />
</html><br />
<br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/carbazoleTeam:Calgary/Notebook/Protocols/carbazole2012-10-04T03:45:26Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=Carbazole Degradation Time-course Assay|<br />
CONTENT=<html><br />
<h2>Media Preparation</h2><br />
<p><b>B-N Medium for Culturing <i>Pseudomonas sp.</i> LD2</b></p><br />
<p>The B-N (basic with no nitrogen) medium with trace metals is specifically designed for <i>Pseudomonas sp.</i> LD2 growth when analyzing biodegradation of a nitrogen-containing compound.</p><br />
<p>Weigh out appropriate amounts of all compounds except FeSO<font style="text-transform: lowercase;">4</font>.7H<font style="text-transform: lowercase;">2</font>O and dissolve, with stirring, in 1L of milliQ H<font style="text-transform: lowercase;">2</font>O. </p><br />
<br />
<br />
<center><br />
<table border="0"><br />
<br />
<tr><br />
<td> K<font style="text-transform: lowercase;">2</font>HPO<font style="text-transform: lowercase;">4</font>:</td><br />
<td>0.5g</td><br />
</tr><br />
<br />
<tr><br />
<td> Na<font style="text-transform: lowercase;">2</font>SO<font style="text-transform: lowercase;">4</font>:</td><br />
<td>2.0g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>MgSO<font style="text-transform: lowercase;">4</font> 7H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>0.2g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>FeSO<font style="text-transform: lowercase;">4</font> 7H<font style="text-transform: lowercase;">2</font>O </td><br />
<td>Trace</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> Trace metals:</td><br />
<td>1.0mL</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>Up to 1.0L</td><br />
</tr><br />
<br />
</table><br />
</center><br />
<br />
<p>When all the salts are dissolved, add FeSO<font style="text-transform: lowercase;">4</font>.7H<font style="text-transform: lowercase;">2</font>O and trace metals and stir for at least 5min to ensure that all compounds are dissolved. <br />
<p>The trace metal solution is prepared as follows:</p><br />
<br />
<center><br />
<table border="0"><br />
<br />
<tr><br />
<td>CaCl<font style="text-transform: lowercase;">2</font> 2H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>3.7g</td><br />
</tr><br />
<br />
<tr><br />
<td>H<font style="text-transform: lowercase;">3</font>BO<font style="text-transform: lowercase;">3</font>:</td><br />
<td>2.5g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> MnCl<font style="text-transform: lowercase;">2</font>:</td><br />
<td>0.87g</td><br />
</tr><br />
<br />
<tr><br />
<td> FeCl<font style="text-transform: lowercase;">3</font>:</td><br />
<td>0.44g </td><br />
</tr><br />
<br />
<tr><br />
<td>ZnCl<font style="text-transform: lowercase;">3</font>:</td><br />
<td>0.44g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> Na<font style="text-transform: lowercase;">2</font >MoO<font style="text-transform: lowercase;">2</font > 2H<font style="text-transform: lowercase;">2</font >O:</td><br />
<td>0.29g</td><br />
</tr><br />
<br />
<tr><br />
<td>CoCl<font style="text-transform: lowercase;">2</font >:</td><br />
<td>0.01g </td><br />
</tr><br />
<br />
<tr><br />
<td>CuCl<font style="text-transform: lowercase;">2</font >:</td><br />
<td>0.0001g </td><br />
</tr><br />
<br />
<tr><br />
<td> H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>Up to 1.0L</td><br />
</tr><br />
<br />
</table><br />
</center><br />
<br />
<p>Dispense appropriate amounts of B-N media into glass culture flasks and autoclave.</p><br />
<br />
<h2>Carbazole Degradation Time-course Experiment</h2><br />
<p>This is a time course experiment designed to monitor carbazole loss by collecting bacterial culture samples over time, followed by <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oextraction"> organic extraction</a> and <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Carbazole_GC-MS_Analysis">GC-MS</a> analysis.</p><br />
<ol><br />
<li>Grow up <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/onculture">overnight cultures</a> of <i>Pseudomonas sp.</i> LD2.</li><br />
<li>Make B-N media, as described above, and add 50mL to each 250mL Erlenmeyer flask and autoclave.</li><br />
<li>Prepare 2 Erlenmeyer flasks as blanks (B-N media only) for day 0 and day 14 as negative controls.</li><br />
<li>Add 15 mg carbazole in its crystalline form, 2.5mg glucose (glucose can be dissolved in a small amount of water and filter sterilized), and 100uL of cells to one set of flasks. This serves as the positive control (2 samples at each time point).</li><br />
<li>Add 15 mg carbazole in its crystalline form and 100uL of cells to another set of flasks. This serves as the experimental sample (2 samples at each time point).</li><br />
<li>Place stopper on the flasks and incubate in the shaker at 28oC.</li><br />
<li>At the appropriate time points (day 0, day 1, day 4, day 7, day 11, day 14), take out the flasks from the shaker and add enough acid to bring the pH of the solutions to ~2.</li><br />
<li>Each sample is extracted following the organic extractions protocol and analysed using GC-MS.</li><br />
</ol><br />
<br />
</html>}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/SponsorsTeam:Calgary/Sponsors2012-10-04T03:38:33Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/MainHeader|<html><img src="https://static.igem.org/mediawiki/2012/c/c5/UCalgary2012_HeaderLogoOrange.png"></img></html>}}<br />
{{Team:Calgary/BasicPage|sponsors|<br />
SECTION=Sponsors|<br />
SIDELIST=<br />
<html><br />
<ul><br />
</ul><br />
</html>|<br />
<br />
<br />
TITLE=Sponsors|<br />
CONTENT=<br />
<html><br />
<p>The University of Calgary's 2012 iGEM Team would like to thank all of our generous sponsors! </p><br />
<head><br />
<style><br />
#bodycontainer img{<br />
float: right;<br />
margin: 15px 15px 5px 15px;<br />
padding-top: 15px;<br />
display: block;<br />
}<br />
h2{<br />
margin-top: 20px;<br />
}<br />
<br />
</style><br />
</head><br />
<br />
<body><br />
<img style="display: block; margin: auto; padding: 0;float: none;" src="https://static.igem.org/mediawiki/2012/6/65/UCalgary2012_FRED_and_OSCAR_Sponsors.png"></img><br />
<a name="uofc"></a><br />
<a href="http://www.ucalgary.ca"><img style="width: 190px; padding: 25px 50px 0px 50px;" src="https://static.igem.org/mediawiki/2011/b/b7/UCalgary_Logo_Small.png"></img></a><br />
<a href="http://www.ucalgary.ca/bhsc"><img style="clear:both; margin-top: 0px;" src="https://static.igem.org/mediawiki/2011/c/c7/UCalgary_BHSc_Logo.png"></img></a><br />
<h2>University of Calgary and the O'Brien Centre for the BHSc</h2><br />
<p>We graciously thank the Department of Cell Biology and Anatomy, the Department of Electrical and Computer Engineering, the Centre for Bioengineering Research and Education and the Department of Biological Sciences for all their support. We also wish to thank the Faculty of Medicine for their additional support as well. Additionally, we wish to thank the office of the Vice President of Research for their generous support.</p> <br />
<p>The O'Brien Centre for the Bachelor of Health Sciences is a part of the University of Calgary, located in the Foothills campus. It is responsible for providing iGEM team with lab space as well as funding. The O’Brien Centre was founded in part by a generous $5-million donation by David and Gail O’ Brien, after whom the Centre was named. David O'Brien has been a member of the University's Board of Governors, while Gail O'Brien has chaired the Faculty of Medicine's Dean's Advisory Council. The O’Brien Centre is the hub of top-notch research, interdisciplinary research, and education. It is responsible for providing approximately $9-million/year for researchers. The 2012 iGEM team thanks O’Brien Centre for its generous contribution of the O’Brien Centre Summer Studentship as well as lab space this year.</p><br />
<br />
<a href="http://www.albertatechfutures.ca/"><img src="https://static.igem.org/mediawiki/2011/3/3f/UCalgary2011_AITF_Logo_Small.png"></img></a><br />
<br />
<h2>Alberta Innovates Technology Futures</h2><br />
<p>Alberta Innovates Technology Futures facilitates the growth of technical industries by supporting quality research in Alberta's particular fields of expertise. These include nanotechnology, information communication technology, and genomics. AITF works in joint effort with entrepreneurs and researchers to ensure that Alberta’s technologies can be sustained at a business level through commercialization and growth. This is accomplished by ensuring that useful products, protocols and services are delivered to meet global demands.</p><br />
<br />
<a href="http://www.osli.ca/"><img style="width:200px;" src="https://static.igem.org/mediawiki/2012/2/27/Ucalgary2012_Full_OSLI_Swirl.png"></a><br />
<a href="http://www.osrin.ualberta.ca/"><img style="clear: both; width: 200px;" src="https://static.igem.org/mediawiki/2011/9/9a/UCalgary_OSRIN_Logo.png"></img></a><br />
<h2>OSLI/OSRIN</h2><br />
<p>The Oil Sands Leadership Initiative (OSLI) and the Oil Sands Research and Information Network (OSRIN) are graciously supporting iGEM this year. OSLI is a collaborative network between Nexen Inc., ConocoPhillips Canada, Statoil Canada, Suncor Energy Inc., and Total E&P Canada. OSLI strives to improve the reputation of the oil sands and lead the industry to responsible development of Alberta's bitument resources.</p><br />
<p>OSRIN is an independent organization within the School of Energy and Environment at the University of Alberta. OSRIN compiles, interprets, analyzes, and communicates data to and from oil sands mining and the area surrounding them. The organization strives for a future where Alberta can economically benefit from oil sands development without harming the environment.</p><br />
<br/><br/><br/><br />
<a href="http://www.vwr.com/"><img style="margin-bottom: 5px; height: 90px;" src="https://static.igem.org/mediawiki/2011/6/65/UCalgary_VWR_Logo.png"></img></a><br />
<br />
<h2>VWR</h2><br />
<p>VWR International is one of the world leaders in the distribution of laboratory materials. There are currently over 250 000 customers in North America and Europe, which is quite large for a biotechnology distributor. They are based in West Chester, Pennsylvania and specialize in the supply of chemicals, equipment, instruments, lab apparel, and lab furniture. They have many offices based worldwide from Northern Ireland to Switzerland and even one based in Singapore. The unique guarantee that they offer is the shipping of ordered material within 24 hours, due to large quantities of stock and many distribution offices in various countries. They have been a significant sponsor of our iGEM team in the past and we appreciate their contribution to our team this year.</p><br />
<br />
<br />
<a href="http://www.neb.com/nebecomm/default.asp"><img style="width: 220px;" src="https://static.igem.org/mediawiki/2011/d/d6/UCalgary_NEB_Logo.png"></img></a><br />
<br />
<h2>New England Biolabs</h2><br />
<p>For over 30 years, New England Biolabs has led the industry in the discovery and production of enzymes for molecular biology applications. At NEB, enzyme production is linked to basic research in the cloning and overexpression of restriction/modification systems. Their focus on providing the largest selection of recombinant enzymes has resulted in lower dollar-per-unit costs and improved purity and consistency of product. Presently, NEB supplies more than 240 restriction enzymes, over 160 of which are available in recombinant form, as well as numerous recombinant DNA and protein modifying enzymes.</p><br />
<a href="http://www.sarstedt.com/php/main.php"><img style="height:50px;" src="https://static.igem.org/mediawiki/2011/e/ee/UCalgary_Sarstedt_Logo.png"></img></a><br />
<br />
<h2>Sarstedt</h2><br />
<p>The Sarstedt Group develops, manufactures and sells equipment and consumables in the field of medicine and research. Ever since it was set up in 1961 the company has continued to grow to the point where it now employs a workforce of 2,500. They produce consumables and analytical equipment for medical diagnostics and consumables for use in medical patient care as well as for research laboratories and also for environmental analytical work. The Sarstedt group manufactures their own products at their own production sites as well as develops concepts and finished products in their own R&D centre.</p><br />
<a href="http://www.eurofins.com/en.aspx"><img style="width: 250px;" src="https://static.igem.org/mediawiki/2012/a/a4/UCalgary2012_Logo_Eurofins.png"></img></a><br />
<h2>Eurofins MWG Operon</h2><br />
<p>Eurofins MWG Operon, founded in 1990 and member of the Eurofins Group, is an international provider of genomic services established around the core business lines next generation sequencing, custom DNA sequencing, oligonucleotides, siRNA and gene synthesis. The company's main mission is focussed on customer convenience and high quality services in industrial scale for the life science industries and academic research institutions around the world.</p><br />
<a href="http://www.sparkscience.ca/"><img style="width: 230px;" src="https://static.igem.org/mediawiki/2012/6/6f/UCalgary2012_Logo_Telus_Spark.png"></img></a><br />
<br />
<h2>Telus Spark</h2><br />
<p>The culmination of over a decade of planning, design and construction to bring Calgary’s new Science Centre to life will light a spark of wonder and excitement in Southern Alberta. Facilitators will fuel curiosity. Exhibits and programs will ignite a sense of wonder and excitement. Conferences and events will act as a catalyst for innovation and new ideas. TELUS Spark is a place for people of all ages and abilities to let go and embrace the desire to explore and discover science, technology and art in a way that their normal day-to-day life doesn’t allow for.</p><br />
<a href="http://www.idtdna.com/site"><img src="https://static.igem.org/mediawiki/2012/1/15/UCalgary2012_Logo_IDT.png"></img></a><br />
<br />
<h2>IDT (Integrated DNA Technologies)</h2><br />
<p>Integrated DNA Technologies (IDT) is a leader in manufacturing and developing products for the research and diagnostic life science market. IDT serves the areas of academic research, biotechnology, and pharmaceutical development. Founded by Dr. Joseph Walder in 1987, IDT’s development has been guided by an uncompromising approach to quality, a belief in the value of good service, and a determination to minimize consumer costs. IDT now has over 500 employees and ships over 20,000 oligos per day. <br />
<br />
Serving over 70,000 life sciences researchers, IDT is widely recognized as the industry leader in custom oligonucleotides. </p><br />
<a href="http://genomealberta.ca/"><img src="https://static.igem.org/mediawiki/2012/6/68/UCalgary2012_Logo_Genome_Alberta.png"></img></a><br />
<br />
<h2>Genome Alberta</h2><br />
<p>In partnership with Genome Canada Industry Canada and the Province of Alberta, Genome Alberta was established in 2005. They are a publicly funded non-profit corporation that initiates, funds, and manages genomics research and partnerships. <br />
Genome Alberta strives to be the leading source of information and administration related to genomics, proteomics, bioinformatics and bioethics research in Alberta. Genome Alberta is dedicated to informing students, researchers, research organizations, our partners, and the public regarding opportunities and challenges in genomics and proteomics, and in encouraging the development of a Life Sciences research industry in Alberta. <br />
</p><br />
<br />
<br />
<a href="http://www.teamlab.com/"><img style="width: 130px;" src="https://static.igem.org/mediawiki/2012/b/be/UCalgary2012_Logo_TeamLab.png"></img></a><br />
<br />
<h2>Team Lab</h2><br />
<p>TeamLab is a multifunctional online service for business collaboration, document and project management. It has been developed by Ascensio System SIA, a fast-growing company that offers IT-solutions for personal and corporate use.<br />
TeamLab was founded on the idea of making social networking and project management efficient. It combines a wide range of features that assist a company team to work as one organism at solving common tasks and achieving results.<br />
</p><br />
<br />
</body><br />
<br />
</html><br />
<br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/catalaseTeam:Calgary/Notebook/Protocols/catalase2012-10-04T03:03:45Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=Catalase Assay|<br />
CONTENT=<html><br />
<br />
<ol><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/onculture">Grow cultures</a> of <i>E. coli J04500-katG+LAA</i> and <i>J04500</i> overnight at 37*C in LB with appropriate antibiotics.</li><br />
<li>The following evening, innoculate 20 uL into 3 mL LB with varying concentrations of hydrogen peroxide (0 mM, 1 mM, 5 mM, 10 mM) and grow overnight.</li><br />
<li>The following morning, observe and record culture turbidity.</li><br />
</ol><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
</html>}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/potentiostaticTeam:Calgary/Notebook/Protocols/potentiostatic2012-10-04T03:01:35Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
<br />
TITLE=Reporter Expression Detection|<br />
CONTENT = <html><br />
<br />
<p>Similar to when creating a standard curve potentiostatically the working electrode must be held at a specific oxidation potential for the detection of that analyte. Testing for this requires a culture of cells that will be able to report through a hydrolase enzyme. To perform the detection a three electrode setup will be needed.</p><br />
<br />
<ol><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/onculture">Grow cells overnight</a> in 3mL of LB.</li><br />
<li>Pellet cells at 3750rpm for 10 minutes.</li><br />
<li>Remove the supernatant.</li><br />
<li>Resuspend in 1mL of 0.1M pH7 PBS.</li><br />
<li>Add to 25mL 0.1M pH7 PBS in an electrochemical cell.</li><br />
<li>Add the solution to be tested for activation of the reporter gene.</li><br />
<li>Add the electrodes and close the cell.</li><br />
<li>Insert a needle and bubble nitrogen or argon gas into the solution for 5 minutes.</li><br />
<li>Hold the working electrode at the oxidation potential for the analyte product.</li><br />
<li>Wait 5 minutes for current stabilization.</li><br />
<li>Add the sugar-analyte substrate.</li><br />
<li>Observe any current changes over a time period.</li><br />
<li>Relate to the concentration of the analyte produced using a standard curve.</li><br />
</ol><br />
<br />
<br />
<br />
</html><br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/potentiostaticTeam:Calgary/Notebook/Protocols/potentiostatic2012-10-04T03:00:51Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
<br />
TITLE=Reporter Expression Detection|<br />
CONTENT = <html><br />
<br />
<p>Similar to when creating a standard curve potentiostatically the working electrode must be held at a specific oxidation potential for the detection of that analyte. Testing for this requires a culture of cells that will be able to report through a hydrolase enzyme. To perform the detection a three electrode setup will be needed.</p><br />
<br />
<ol><br />
<li>Grow cells <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/onculture">overnight</a> in 3mL of LB.</li><br />
<li>Pellet cells at 3750rpm for 10 minutes.</li><br />
<li>Remove the supernatant.</li><br />
<li>Resuspend in 1mL of 0.1M pH7 PBS.</li><br />
<li>Add to 25mL 0.1M pH7 PBS in an electrochemical cell.</li><br />
<li>Add the solution to be tested for activation of the reporter gene.</li><br />
<li>Add the electrodes and close the cell.</li><br />
<li>Insert a needle and bubble nitrogen or argon gas into the solution for 5 minutes.</li><br />
<li>Hold the working electrode at the oxidation potential for the analyte product.</li><br />
<li>Wait 5 minutes for current stabilization.</li><br />
<li>Add the sugar-analyte substrate.</li><br />
<li>Observe any current changes over a time period.</li><br />
<li>Relate to the concentration of the analyte produced using a standard curve.</li><br />
</ol><br />
<br />
<br />
<br />
</html><br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/transformationTeam:Calgary/Notebook/Protocols/transformation2012-10-04T02:58:26Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
<br />
TITLE= Bacterial Transformation|<br />
CONTENT = <html><br />
<br />
<ol><br />
<li> Thaw 100 μL of competent cells (per transformation) on ice just before they are needed</li><br />
<li> Add DNA (max 20μl) thawed cells and mix by flicking the side of the tube. Leave on ice for 30 minutes</li><br />
<li> Heat shock 5 minutes at 37 degrees Celsius</li><br />
<li> Place on ice for 5 minutes </li><br />
<li> Add 250ul SOC medium to each tube</li><br />
<li> Incubate for 30 to 60 minutes with shaking at 37&deg;C. (Note that for Kanamycin containing plasmids always use one hour)</li><br />
<li> Spin down to remove all supernatant except approximately 100 μL</li><br />
<li> Plate approximately 30 μL on each of two antibiotic plates </li><br />
<li> <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/onculture">Grow overnight</a> at 37&deg;C </li><br />
</ol><br />
<p> For this protocol we used a couple of controls<br />
<ul><br />
<li> <b> Positive Control </b> - pBluescript in TOP10 cells on ampicillin plates </li><br />
<li> <b> Negative Control </b> - TOP10 cells grown on ampcillin plates </li><br />
</ul><br />
</p><br />
<br />
<br></br><br></br><br></br><br />
<br />
</html>}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/decatecholizationTeam:Calgary/Notebook/Protocols/decatecholization2012-10-04T02:54:51Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=Catechol Assay in <i>E. coli</i> Cells|<br />
CONTENT=<html><br />
<br />
<p>This assay is used to verify that catechol 2,3-dioxygenase (XylE) is converting catechol to the yellow compound 2-hydroxymuconic semialdehyde (2-HMS). For this procedure we used to the newly constructed XylE part. This part contains the TetR promoter the XylE gene and its native rbs site:</p><br />
<ol><br />
<li>Grow up 2 ml <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/onculture">overnight cultures</a> in LB.</li><br />
<li>Spin the cultures down and keep the supernatant.</li><br />
<li>Bring the supernatant to a concentration of 0.1 M of Catechol by using a 1M catechol stock solution.</li><br />
<li>The colour of the supernatant should change to bright yellow very quickly (in about 30 seconds).</li><br />
</ol><br />
<br />
<h2>GC-MS Catechol Assay</h2><br />
In order to identify if the PetroBrick or <i>Micrococcus</i> was capable of further reducing catechol products, GC-MS was used. <br />
<ol><br />
<li>Grow overnight cultures of the PetroBrick in LB at 37<sup>o</sup>C and <i>xylE</i> construct, or <i>Microccus</i> in yeast tryptone broth at 26<sup>o</sup>C.<br />
<li>Subculture into the micrococcus or PetroBrick broth respectively and set up cultures with and without <i>xylE</i> as well as the appropriate control. <br />
<li>Note: Prior to mixing, <i>xylE</i> and PetroBrick cultures were spun down at 4000 RPM for 5 min at room temperature and washed with 2x volume LB, spun down again, and then resuspended to the original volume to remove antibiotics which they were initially cultured in.<br />
<li>Add 10 mM catechol to each tube, mix well, and cover in tin foil. <br />
<li>Culture the cells at the appropriate temperature for 72 hours.<br />
<li>Sonicate samples for 1 min using a Misonix Cell Disrupter set to Dial 11. <br />
<li>Add 5 mL of ethyl acetate, shake well, and spin down at 4000 RPM for 5 min. at 4<sup>o</sup>C. <br />
<li> Add 1mL of the ethyl acetate to a tube and reduce the volume to 0.3mL through heating.<br />
<li> Samples were dried using sodium sulfate if any water layer was present and modified using trimethyl sylyl reagent.<br />
<li> Samples were ran onto the GC-MS (<a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Carbazole_GC-MS_Analysis">see protocol</a>)<br />
<br><br><br><br><br><br><br><br><br><br />
<br />
</html>}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/carbazoleTeam:Calgary/Notebook/Protocols/carbazole2012-10-04T02:51:26Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=Carbazole Degradation Time-course Assay|<br />
CONTENT=<html><br />
<h2>Media Preparation</h2><br />
<p><b>B-N Medium for Culturing <i>Pseudomonas sp.</i> LD2</b></p><br />
<p>The B-N (basic with no nitrogen) medium with trace metals is specifically designed for <i>Pseudomonas sp.</i> LD2 growth when analyzing biodegradation of a nitrogen-containing compound.</p><br />
<p>Weigh out appropriate amounts of all compounds except FeSO<font style="text-transform: lowercase;">4</font>.7H<font style="text-transform: lowercase;">2</font>O and dissolve, with stirring, in 1L of milliQ H<font style="text-transform: lowercase;">2</font>O. </p><br />
<br />
<br />
<center><br />
<table border="0"><br />
<br />
<tr><br />
<td> K<font style="text-transform: lowercase;">2</font>HPO<font style="text-transform: lowercase;">4</font>:</td><br />
<td>0.5g</td><br />
</tr><br />
<br />
<tr><br />
<td> Na<font style="text-transform: lowercase;">2</font>SO<font style="text-transform: lowercase;">4</font>:</td><br />
<td>2.0g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>MgSO<font style="text-transform: lowercase;">4</font> 7H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>0.2g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>FeSO<font style="text-transform: lowercase;">4</font> 7H<font style="text-transform: lowercase;">2</font>O </td><br />
<td>Trace</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> Trace metals:</td><br />
<td>1.0mL</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>Up to 1.0L</td><br />
</tr><br />
<br />
</table><br />
</center><br />
<br />
<p>When all the salts are dissolved, add FeSO<font style="text-transform: lowercase;">4</font>.7H<font style="text-transform: lowercase;">2</font>O and trace metals and stir for at least 5min to ensure that all compounds are dissolved. <br />
<p>The trace metal solution is prepared as follows:</p><br />
<br />
<center><br />
<table border="0"><br />
<br />
<tr><br />
<td>CaCl<font style="text-transform: lowercase;">2</font> 2H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>3.7g</td><br />
</tr><br />
<br />
<tr><br />
<td>H<font style="text-transform: lowercase;">3</font>BO<font style="text-transform: lowercase;">3</font>:</td><br />
<td>2.5g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> MnCl<font style="text-transform: lowercase;">2</font>:</td><br />
<td>0.87g</td><br />
</tr><br />
<br />
<tr><br />
<td> FeCl<font style="text-transform: lowercase;">3</font>:</td><br />
<td>0.44g </td><br />
</tr><br />
<br />
<tr><br />
<td>ZnCl<font style="text-transform: lowercase;">3</font>:</td><br />
<td>0.44g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> Na<font style="text-transform: lowercase;">2</font >MoO<font style="text-transform: lowercase;">2</font > 2H<font style="text-transform: lowercase;">2</font >O:</td><br />
<td>0.29g</td><br />
</tr><br />
<br />
<tr><br />
<td>CoCl<font style="text-transform: lowercase;">2</font >:</td><br />
<td>0.01g </td><br />
</tr><br />
<br />
<tr><br />
<td>CuCl<font style="text-transform: lowercase;">2</font >:</td><br />
<td>0.0001g </td><br />
</tr><br />
<br />
<tr><br />
<td> H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>Up to 1.0L</td><br />
</tr><br />
<br />
</table><br />
</center><br />
<br />
<p>Dispense appropriate amounts of B-N media into glass culture flasks and autoclave.</p><br />
<br />
<h2>Carbazole Degradation Time-course Experiment</h2><br />
<p>This is a time course experiment designed to monitor carbazole loss by collecting bacterial culture samples over time, followed by <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oextraction"> organic extraction</a> and <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Carbazole_GC-MS_Analysis">GC-MS</a> analysis.</p><br />
<ol><br />
<li>Grow up <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/onculture">overnight cultures</a> of <i>Pseudomonas sp.</i> LD2.</li><br />
<li>Make B-N media, as described above, and add 50mL to each 250mL Erlenmeyer flask and autoclave.</li><br />
<li>Prepare 2 Erlenmeyer flasks as blanks (B-N media only) for day 0 and day 14 as negative controls.</li><br />
<li>Add 15 mg carbazole in its crystalline form, 2.5mg glucose (glucose can be dissolved in a small amount of water and filter sterilized), and 100uL of cells to one set of flasks. This serves as the positive control (2 samples at each time point).</li><br />
<li>Add 15 mg carbazole in its crystalline form and 100uL of cells to another set of flasks. This serves as the experimental sample (2 samples at each time point).</li><br />
<li>Place stopper on the flasks and incubate in the shaker at 28oC.</li><br />
<li>At the appropriate time points (day 0, day 1, day 4, day 7, day 11, day 14), take out the flasks from the shaker and add enough acid to bring the pH of the solutions to ~2.</li><br />
<li>Each sample is extracted following the organic extractions protocol and analyzed using GC-MS.</li><br />
</ol><br />
<br />
</html>}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/nucleaseassayTeam:Calgary/Notebook/Protocols/nucleaseassay2012-10-04T02:48:29Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=Nuclease assay|<br />
CONTENT=<html><br />
<ol><br />
<li>E. coli genome was prepared using MP bio kit and the DNA was quantified using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/picogreen">picogreen assay.</a><br />
<li>2µg of DNA was put into each test condition.<br />
<li>Appropriate buffer(s) were added for each enzyme/enzyme combination in the tubes.<br />
<li>10 units of enzyme was added into appropriate conditions.<br />
<li>At each timepoint aliquots were taken out of the reaction tubes and run on a 1% gel for 40 minutes and imaged.<br />
</ol><br />
<br><br><br><br><br><br><br><br><br><br><br><br />
</html>}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/nucleaseassayTeam:Calgary/Notebook/Protocols/nucleaseassay2012-10-04T02:48:06Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=Nuclease assay|<br />
CONTENT=<html><br />
<ol><br />
<li>E. coli genome was prepared using MP bio kit and the DNA was quantified using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/picogreen">picogreen assay.</a><br />
<li>2µg of DNA was put into each test condition.<br />
<li>Appropriate buffer(s) were added for each enzyme/enzyme combination in the tubes.<br />
<li>10 units of enzyme was added into appropriate conditions<br />
<li>At each timepoint aliquots were taken out of the reaction tubes and run on a 1% gel for 40 minutes and imaged.<br />
</ol><br />
<br><br><br><br><br><br><br><br><br><br><br><br />
</html>}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/carbazoleTeam:Calgary/Notebook/Protocols/carbazole2012-10-04T02:44:54Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=Carbazole Degradation Time-course Assay|<br />
CONTENT=<html><br />
<h2>Media Preparation</h2><br />
<p><b>B-N Medium for Culturing <i>Pseudomonas sp.</i> LD2</b></p><br />
<p>The B-N (basic with no nitrogen) medium with trace metals is specifically designed for <i>Pseudomonas sp.</i> LD2 growth when analyzing biodegradation of a nitrogen-containing compound.</p><br />
<p>Weigh out appropriate amounts of all compounds except FeSO<font style="text-transform: lowercase;">4</font>.7H<font style="text-transform: lowercase;">2</font>O and dissolve, with stirring, in 1L of milliQ H<font style="text-transform: lowercase;">2</font>O. </p><br />
<br />
<br />
<center><br />
<table border="0"><br />
<br />
<tr><br />
<td> K<font style="text-transform: lowercase;">2</font>HPO<font style="text-transform: lowercase;">4</font>:</td><br />
<td>0.5g</td><br />
</tr><br />
<br />
<tr><br />
<td> Na<font style="text-transform: lowercase;">2</font>SO<font style="text-transform: lowercase;">4</font>:</td><br />
<td>2.0g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>MgSO<font style="text-transform: lowercase;">4</font> 7H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>0.2g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>FeSO<font style="text-transform: lowercase;">4</font> 7H<font style="text-transform: lowercase;">2</font>O </td><br />
<td>Trace</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> Trace metals:</td><br />
<td>1.0mL</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>Up to 1.0L</td><br />
</tr><br />
<br />
</table><br />
</center><br />
<br />
<p>When all the salts are dissolved, add FeSO<font style="text-transform: lowercase;">4</font>.7H<font style="text-transform: lowercase;">2</font>O and trace metals and stir for at least 5min to ensure that all compounds are dissolved. <br />
<p>The trace metal solution is prepared as follows:</p><br />
<br />
<center><br />
<table border="0"><br />
<br />
<tr><br />
<td>CaCl<font style="text-transform: lowercase;">2</font> 2H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>3.7g</td><br />
</tr><br />
<br />
<tr><br />
<td>H<font style="text-transform: lowercase;">3</font>BO<font style="text-transform: lowercase;">3</font>:</td><br />
<td>2.5g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> MnCl<font style="text-transform: lowercase;">2</font>:</td><br />
<td>0.87g</td><br />
</tr><br />
<br />
<tr><br />
<td> FeCl<font style="text-transform: lowercase;">3</font>:</td><br />
<td>0.44g </td><br />
</tr><br />
<br />
<tr><br />
<td>ZnCl<font style="text-transform: lowercase;">3</font>:</td><br />
<td>0.44g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> Na<font style="text-transform: lowercase;">2</font >MoO<font style="text-transform: lowercase;">2</font > 2H<font style="text-transform: lowercase;">2</font >O:</td><br />
<td>0.29g</td><br />
</tr><br />
<br />
<tr><br />
<td>CoCl<font style="text-transform: lowercase;">2</font >:</td><br />
<td>0.01g </td><br />
</tr><br />
<br />
<tr><br />
<td>CuCl<font style="text-transform: lowercase;">2</font >:</td><br />
<td>0.0001g </td><br />
</tr><br />
<br />
<tr><br />
<td> H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>Up to 1.0L</td><br />
</tr><br />
<br />
</table><br />
</center><br />
<br />
<p>Dispense appropriate amounts of B-N media into glass culture flasks and autoclave.</p><br />
<br />
<h2>Carbazole Degradation Time-course Experiment</h2><br />
<p>This is a time course experiment designed to monitor carbazole loss by collecting bacterial culture samples over time, followed by <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oextraction"> organic extraction</a> and <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Carbazole_GC-MS_Analysis">GC-MS</a> analysis.</p><br />
<ol><br />
<li>Grow up overnight cultures of <i>Pseudomonas sp.</i> LD2.</li><br />
<li>Make B-N media, as described above, and add 50mL to each 250mL Erlenmeyer flask and autoclave.</li><br />
<li>Prepare 2 Erlenmeyer flasks as blanks (B-N media only) for day 0 and day 14 as negative controls.</li><br />
<li>Add 15 mg carbazole in its crystalline form, 2.5mg glucose (glucose can be dissolved in a small amount of water and filter sterilized), and 100uL of cells to one set of flasks. This serves as the positive control (2 samples at each time point).</li><br />
<li>Add 15 mg carbazole in its crystalline form and 100uL of cells to another set of flasks. This serves as the experimental sample (2 samples at each time point).</li><br />
<li>Place stopper on the flasks and incubate in the shaker at 28oC.</li><br />
<li>At the appropriate time points (day 0, day 1, day 4, day 7, day 11, day 14), take out the flasks from the shaker and add enough acid to bring the pH of the solutions to ~2.</li><br />
<li>Each sample is extracted following the organic extractions protocol and analyzed using GC-MS.</li><br />
</ol><br />
<br />
</html>}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/carbazoleTeam:Calgary/Notebook/Protocols/carbazole2012-10-04T02:44:30Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=Carbazole Degradation Time-course Assay|<br />
CONTENT=<html><br />
<h2>Media Preparation</h2><br />
<p><b>B-N Medium for Culturing <i>Pseudomonas sp.</i> LD2</b></p><br />
<p>The B-N (basic with no nitrogen) medium with trace metals is specifically designed for <i>Pseudomonas</i> LD2 growth when analyzing biodegradation of a nitrogen-containing compound.</p><br />
<p>Weigh out appropriate amounts of all compounds except FeSO<font style="text-transform: lowercase;">4</font>.7H<font style="text-transform: lowercase;">2</font>O and dissolve, with stirring, in 1L of milliQ H<font style="text-transform: lowercase;">2</font>O. </p><br />
<br />
<br />
<center><br />
<table border="0"><br />
<br />
<tr><br />
<td> K<font style="text-transform: lowercase;">2</font>HPO<font style="text-transform: lowercase;">4</font>:</td><br />
<td>0.5g</td><br />
</tr><br />
<br />
<tr><br />
<td> Na<font style="text-transform: lowercase;">2</font>SO<font style="text-transform: lowercase;">4</font>:</td><br />
<td>2.0g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>MgSO<font style="text-transform: lowercase;">4</font> 7H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>0.2g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>FeSO<font style="text-transform: lowercase;">4</font> 7H<font style="text-transform: lowercase;">2</font>O </td><br />
<td>Trace</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> Trace metals:</td><br />
<td>1.0mL</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>Up to 1.0L</td><br />
</tr><br />
<br />
</table><br />
</center><br />
<br />
<p>When all the salts are dissolved, add FeSO<font style="text-transform: lowercase;">4</font>.7H<font style="text-transform: lowercase;">2</font>O and trace metals and stir for at least 5min to ensure that all compounds are dissolved. <br />
<p>The trace metal solution is prepared as follows:</p><br />
<br />
<center><br />
<table border="0"><br />
<br />
<tr><br />
<td>CaCl<font style="text-transform: lowercase;">2</font> 2H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>3.7g</td><br />
</tr><br />
<br />
<tr><br />
<td>H<font style="text-transform: lowercase;">3</font>BO<font style="text-transform: lowercase;">3</font>:</td><br />
<td>2.5g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> MnCl<font style="text-transform: lowercase;">2</font>:</td><br />
<td>0.87g</td><br />
</tr><br />
<br />
<tr><br />
<td> FeCl<font style="text-transform: lowercase;">3</font>:</td><br />
<td>0.44g </td><br />
</tr><br />
<br />
<tr><br />
<td>ZnCl<font style="text-transform: lowercase;">3</font>:</td><br />
<td>0.44g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> Na<font style="text-transform: lowercase;">2</font >MoO<font style="text-transform: lowercase;">2</font > 2H<font style="text-transform: lowercase;">2</font >O:</td><br />
<td>0.29g</td><br />
</tr><br />
<br />
<tr><br />
<td>CoCl<font style="text-transform: lowercase;">2</font >:</td><br />
<td>0.01g </td><br />
</tr><br />
<br />
<tr><br />
<td>CuCl<font style="text-transform: lowercase;">2</font >:</td><br />
<td>0.0001g </td><br />
</tr><br />
<br />
<tr><br />
<td> H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>Up to 1.0L</td><br />
</tr><br />
<br />
</table><br />
</center><br />
<br />
<p>Dispense appropriate amounts of B-N media into glass culture flasks and autoclave.</p><br />
<br />
<h2>Carbazole Degradation Time-course Experiment</h2><br />
<p>This is a time course experiment designed to monitor carbazole loss by collecting bacterial culture samples over time, followed by <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oextraction"> organic extraction</a> and <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Carbazole_GC-MS_Analysis">GC-MS</a> analysis.</p><br />
<ol><br />
<li>Grow up overnight cultures of <i>Pseudomonas sp.</i> LD2.</li><br />
<li>Make B-N media, as described above, and add 50mL to each 250mL Erlenmeyer flask and autoclave.</li><br />
<li>Prepare 2 Erlenmeyer flasks as blanks (B-N media only) for day 0 and day 14 as negative controls.</li><br />
<li>Add 15 mg carbazole in its crystalline form, 2.5mg glucose (glucose can be dissolved in a small amount of water and filter sterilized), and 100uL of cells to one set of flasks. This serves as the positive control (2 samples at each time point).</li><br />
<li>Add 15 mg carbazole in its crystalline form and 100uL of cells to another set of flasks. This serves as the experimental sample (2 samples at each time point).</li><br />
<li>Place stopper on the flasks and incubate in the shaker at 28oC.</li><br />
<li>At the appropriate time points (day 0, day 1, day 4, day 7, day 11, day 14), take out the flasks from the shaker and add enough acid to bring the pH of the solutions to ~2.</li><br />
<li>Each sample is extracted following the organic extractions protocol and analyzed using GC-MS.</li><br />
</ol><br />
<br />
</html>}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/DenitrogenationTeam:Calgary/Notebook/Denitrogenation2012-10-04T02:41:10Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Denitrogenation Journal|<br />
CONTENT=<br />
<html><br />
<br />
<h2>Week 1 (May 1-4)</h2><br />
<p>The following section covers wetlab aspect of our overall project focusing on microbial conversion of naphthenic acids into economically-valuable hydrocarbons. The approach taken in this endeavour will be from four strategic starting points - ring cleavage, decarboxylation, denitrogenation, and desulfurization. Overall, the 'hydrocarbons' aspect of the project is a critical one to our overall design and construction of a bioreactor capable of not only detecting but also converting naphthenic acids and other toxins to useful or at least innocuous compounds. </p><br />
<br />
<h2>Week 2-3 (May 7-18)</h2><br />
<p>In the first two weeks of iGEM our group has focused on reviewing literature regarding the bioremediation of nitrogen groups attached to naphthenic acids. The most prevalent N heterocycle is carbazole, representing 75% of total nitrogen by mass. The upper pathway of carbazole biodegradation is catalyzed by the enzymes coded for by the <i>car</i> operon, <i>CarA</i> (<i>CarAaAcAd</i>), <i>CarB</i> (<i>CarBaBb</i>), and <i>CarC</i>. These enzymes convert carbazole to anthralinic acid. The lower pathway is catalyzed by the enzymes of the <i>ant</i> operon, <i>antA, B, and C</i>, yielding catechol while releasing CO2 and NH3. The <i>car</i> and <i>ant</i> operons are both regulated by the P<sup>ant</sup> promoter which is induced by the protein, antR. <i>carAa</i> also has its own promoter which is not induced by <i>antR</i> (Diaz & Garcia, 2010). We have also investigated an alternative pathway using <i>CarA</i> combined with an amidase (<i>amdA</i>) that selectively cleaves NH2 from an intermediate of the car pathway (Xu et al, 2006). This could bypass much of the car/ant pathway and is possibly more efficient.</p><br />
<p>We have decided to use <i>Pseudomonas resinovorans</i> and <i>Rhodococcus erythropolis</i> to derive these genes from. <i>carABC</i> and <i>antABC</i> from<i> P. resinovorans</i> has been shown to have a wide range of nitrogen containing substrate specificity. <i>R. erythropolis</i> contains the <i>amdA</i> gene that we wish to use, and some evidence suggests that it may also be able to degrade sulfur rings through its <i>carABC</i> pathway.</p><br />
<br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p>This week we reviewed the primers listed in the database and also designed some new ones. Primers for <i>carAa</i>,<i> carAc</i>, and <i>carAd</i> were designed individually, while primers for <i>CarBaBbC</i> and <i>antABC</i> were designed to encompass multiple genes in a sequence. These primers were designed to be used on <i>Pseudomonas putida</i> which we decided to use as our gene source since it was available to us as opposed to ordering <i>Pseudomonas resinovorans</i>. We also designed a primer for the<i> AmdA</i> gene from <i>Rhodococcus erthyroplois</i>. In addition to ordering these primers we also ordered the nitrogen containing compounds that we will need to test these enzymes on. We decided on using carbazole to make sure the enzymes can perform their natural function as well as pyrrolidine to test them on a similar ring structure. We also ordered cyclohexamine in order to independently test the function of the alternative <i>AmdA</i> pathway. Finally, we decided to eventually order 4-Piperidine butyric acid hydrochloride to test how the enzymes will work on nitrogen containing naphthenic acids. However, we decided since it is a very expensive compound we would wait to make sure the enzyme's work on more simple compounds before ordering it.</p><br />
<p>We also started our work in the wet lab by plating colonies and making an overnight culture of -80 freezer stock <i>Psuedomonas putida</i> on Thursday. We also resuspended the primers for <i>carAa</i>, <i>carAc</i>, <i>carAd</i>,<i> antA</i>, <i>antB</i>, and <i>ant C</i> that were already in the database. We were able to use the colonies that grew on the streak plates to start a colony PCR to attempt to isolate each of these genes on Friday.</p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2><br />
<p>On Monday and Tuesday we used the database primers to attempt to isolate <i>carAa</i>, <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i>antB</i>, and <i>antC</i> from the <i>Psuedomonas putida</i> we plated last week. <i>carAd</i>, <i>antB</i>, and <i>antC</i> were all put in the gradient PCR machine to account for the wide range of their primers’ melting points, while the others were done via regular PCR. Unfortunately, no positive results were obtained from these reactions. The PCR on <i>carAc</i> and <i>antA</i> was repeated on Wednesday, still not giving positive results. Finally, we attempted to use a salt concentration gradient on the PCR reaction for <i>carAc</i> and <i>antA</i>, using concentrations that ranged from 1.0 microlitres/tube to 2.0 microlitres/tube in increments of 0.2 microlitres/tube. This helped as <i>carAc</i> showed bands in samples that had concentrations of 1.2 and 1.4 microlitres/tube. <i> antA</i> also showed bands, however they were not the correct size, indicating contamination and/or non-specific annealing of the primer. The positive control also showed bands of the correct size. Earlier in the week we also made overnight cultures of 3 environmental strains (28, 29, 30) of <i>Psuedomonas putida</i> from -80 glycerol stock. On Friday we performed a genomic prep on these cultures, and plan on attempting to amplify genes from the isolated DNA next week. </p><br />
<br />
</html>[[File:CarAa and carAc Ucalgary12.png|500px|thumb|Gel of PCR using database primers for <i>carAa</i> and <i>carAc</i> on <i>Pseudomonas putida</i>|center]]<html><br />
<br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> We started off this week by determining the DNA concentration of our genomic prep samples from last week using the nanodrop. DNA concentration for all three<i> P. putida</i> strains was at least 1000 ng/microlitre, well above what was needed for PCR. 1/2 and 1/3 dilutions were prepared for all three strains so as not to have an excess of template DNA in PCR reactions. PCR was performed on all 3 strains using primers for <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i> antB</i>, and <i>antC</i> using 6 replicates per gene. The only successful amplification appeared to be <i>antB</i> and <i>carAc</i>, both from strain 28 (with weaker bands in strain 29). We then performed another PCR, just on those two genes with an increased amount of Taq polymerase to hopefully get enough amplified DNA to move forward with. This resulted in strong bands for both at the expected size. We then performed PCR purification using the Qiaquick kit and obtained samples containing 33.5 ng/microlitre of <i>antB</i> DNA and 129 ng/microlitre of <i>carAc </i>DNA. These concentrations were both sufficient to begin a restriction digest and ligation of these parts into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. Next week we hope to verify the results of the restriction digest, continue to amplify <i>carAc</i> and <i>antB</i> from strain 28, and hopefully submit a biobrick for sequencing.</p><br />
<br />
</html>[[File:AntB and carAc Ucalgary.png|500px|thumb|Gel of PCR ran on genomic prep of <i>Pseudomonas putida</i> environmental strain 28 using primers for <i>carAc</i> and <i>antB</i>. |center]]<html><br />
<br />
<br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p>The purified PCR products for <i>carAc</i> and <i>antB</i> were digested with restriction enzymes EcoRI+SpeI, EcoRI+PstI, and XbaI+PstI. However, only <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmids that had been digested with EcoRI+SpeI and EcoRI+PstI were available to attempt ligation. Gel results were inconclusive on the restriction digest product as the plasmid size appeared much larger than expected. However, transformation was still attempted on the ligation products for both genes (both using EcoRI+SpeI ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid). The transformation products were plated onto chloroamphenicol (chlor) plates to select for colonies that had the chloro resistance gene on the <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid. Also this week another round of PCR was performed for the <i> car</i> and <i> ant</i> genes, however all, but <i>carAc</i> showed bands in the negative control lane, possibly indicating contamination or the formation of primer dimers. The <i>carAc</i> bands were fairly weak and a PCR purification resulted in very low DNA concentration, insufficient to move onto restriction digest. Next week's plan hinges heavily on the result of the transformation from this Friday. </p><br />
<br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>The results of our transformation on <i>carAc</i> and <i>antB</i> from last week showed mostly red colonies indicating that the ligation was unsuccessful and that the PSB1C3 plasmid simply closed on itself with no insert. However, there were three colonies for <i>carAc</i> that were white (cut with both X+P and E+P). Colony PCR was performed on these three colonies using biobrick primer sets to verify that the part had been inserted in these colonies, but unfortunately all PCR results were negative indicating an unsuccessful ligation.</p><br />
<p>We also performed a miniprep on <i>Pseudomonas putida</i> strain #28 this week using a home-made protocol rather than the Qiagen kit. Miniprep A had a DNA concentration of 1539.8 ng/μL and Miniprep B had DNA concentration of 1001.2 ng/μL possibly indicating that there may have been a lot of genomic DNA contamination. However miniprep A product was still used as a PCR template for a reaction attempting to isolate <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i>antB</i>, and <i>antC</i>. Special conditions for this PCR included replacing 60 μL of water with 60 μL of betaine heated to 37 degrees Celsius and running a Mg gradient ranging from 0.5 μL - 2.5 μL per tube for each gene. Of these only <i>antB</i> had bands of the right size in 3 of the lanes (Mg concentrations of 1.5, 2, and 2.5 μL). However, all of the <i>antB</i> bands (including the negative control) contained a contamination band at around 100 bases, probably due to the formation of primer dimers. This forced us to do a gel extraction rather than a PCR purification. This gel extraction did not work as the nanodrop results indicated high amounts of agarose contamination. Another PCR was performed using the same conditions, but using miniprep B instead as there was no more miniprep A product remaining. Unfortunately there were no bands for <i>antB</i>, only the 100 base pair contamination bands. </p><br />
<br />
</html>[[File:AntA and antC Ucalgary.png|500px|thumb|Gel of PCR ran using miniprep product A of <i>Pseudomonas putida</i> as template DNA in an attempt to isolate <i>antA</i> and <i>antC</i> genes.|center]]<br />
[[File:CarAc and carAd Ucalgary.png|500px|thumb|Gel of PCR ran using miniprep product A of <i>Pseudomonas putida</i> as template in an attempt to isolate <i>carAc</i> and <i>carAd</i> genes.|center]]<br />
[[File:Minipret A antB and carAc Ucalgary.png|500px|thumb|Gel of PCR ran using miniprep product A of <i>Pseudomonas putida</i> showing bands 3-5 of the correct size corresponding to antB genes and colony PCR ran using biobrick primers attempting to determine if <i>carAc</i> genes were successfully transformed into PSB1C3 plasmid.|center]] <html><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>This week we modified our PCR protocol to adjust for the homemade Taq polymerase we were using. Template DNA was boiled for 10 minutes on its own to replace the initialization step. This is because this polymerase could not tolerate the 95 degree heat during the regular initialization step. Another modification we made was to boil our primers for 5 minutes prior to adding them to the master mix. This was done to discourage the formation of primer-dimers. Finally we also attempted a touch-down PCR which started at an annealing temperature greater than the Tm of our primers and gradually worked its way down to below the Tm of the primers. This was done to increase the specificity of our reaction and also eliminate the formation of primer-dimers. Unfortunately these techniques still saw the formation of dimers, preventing us from performing a PCR purification. Again, the gel purification procedure did not lead to successful purification of our target gene. Due to the problems we have had so far our goal for next week is to perform an assay experiment to try and determine which of the <i>Pseudomonas putida</i> strains we have access to can actually degrade carbazole and therefore contain the pCAR1 plasmid. </p><br />
<br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The goal for this week was to grow different strains of<i> Pseudomonas</i> in media containing B-N media + glucose with only carbazole as a source of nitrogen. In theory only those strains which contained the pCAR1 plasmid would be able to use carbazole as a nitrogen source and thrive in this media. The strains we tested from glycerol stock included <i>P. putida</i> LD1, <i>P.putida</i> environmental strains 28-30, and <i>P. fluorescens</i> PF5. Each of these strains had two cultures grown; one with carbazole and one without it. <i>E. coli</i> grown with carbazole was used as a negative control as it does not contain the pCAR plasmid. At timepoints of 22, 46, and 117 hours of growth samples were taken to do an OD600 reading to measure bacterial growth and had an ammonia assay performed on it. The detection of ammonia acted as a proxy for detecting carbazole degradation as it is an end-product of the pathway. With this in mind, an additional control strain of LD1 was grown in media containing glucose and ammonia. This is to detect the possibility that <i>Pseudomonas</i> can use ammonia as a source of nitrogen after it has degraded carbazole which would be problematic as far as using ammonia levels to determine carbazole degradation. So far we have been having difficulties reading the results of the ammonia assay with a plate reader and may resort to testing our samples using a different assay protocol next week. The OD600 readings were initially very high at 22 hours and then dropped at 46 hours. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>This week we took our last readings from the experiment that we started last week. The OD600 readings taken at 117 hours showed minimal growth over the weekend for all strains except for environmental strain #29 (with carbazole) which increased from 0.086 to 0.313. No other strain showed an increase of 0.100 or greater. This shows that only strain #29 was able to thrive in the carbazole media, possibly indicating that it contains the pCAR plasmid enabling it to use carbazole as a nitrogen source. To verify this we used a new ammonia assay protocol to measure the production of ammonia in the different cultures and theoretically how much carbazole was degraded. Unlike the previous assay we were able to produce a standard curve with a consistent slope based on samples with known amounts of ammonia. However, the only samples that showed any elevation of ammonia levels above the blank were the LD1 samples that were cultured with ammonia chloride to control for the possibility of pseudomonas using ammonia as a nitrogen source. The ammonia levels in this sample were relatively constant which would indicate that the LD1 was not using ammonia as a nitrogen source. Unfortunately the fact that none of the test samples had elevated ammonia levels did not give us any evidence that these strains contain the pCAR plasmid. </p><br />
<br />
</html>[[File: Standard_curve_for_ammonia_assay_UCalgary2012_denitrification_group.png|500px|thumb|Standard curve for ammonia assay using OD635 absorbance.|center|]]<html><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>We received a new strain of <i>Pseudomonas</i> LD2, this week that had previously been used to study the pCAR1 plasmid. It was grown up in PCA media and had its colonies used for colony PCR. We also received our new primers this week, further increasing the chances of successful PCR. Using the new strain and the new primers <i>carAa</i>, <i>carAc</i>, and <i>carAd</i> all showed at least some bands of close to the correct size. After PCR purification we obtained DNA concentrations of 21.3 ng/μL for <i>carAa</i>, 41.9 ng/ μL for <i>carAc</i>, and 32.8 ng/μL for<i> carAd</i>. These products were then restriction digested using EcoRI + PstI sites. </p><br />
<br />
</html>[[File:07.20.2012 CarAa and CarAc.jpg|500px|thumb|Gel of colony PCR from <i>Pseudomonas sp. LD2</i> for <i>carAa</i> and <i>carAc</i> genes. <i>carAa</i> expected size is 1155 bp and <i>carAc</i> is 323 bp.|center]]<br />
[[File:07.20.2012 CarAd colony PCR from LD2.jpg|500px|thumb|Gel of colony PCR from <i>Pseudomonas sp. LD2</i> for <i>carAd</i> gene. <i>carAd</i> expected size is 989 bp.|center]] <html><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The restriction digested <i>car</i> genes were ligated into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> cells. White colonies grew for<i> carAa</i> and <i>carAc</i> suggesting that ligation and transformation were successful, however no colonies grew for <i>carAd</i> suggesting that the transformation did not work. Colony PCR was performed to confirm the presence of these genes within the vectors, however only <i>carAc</i> was positive. The colonies containing the <i>carAc</i> plasmid were then mini prepped and restriction digested to isolate the plasmid and send for sequencing. Further attempts at transforming<i> carAa</i> and <i>carAd</i> were not successful. Another goal was to perform colony PCR on the <i>P. putida</i> to isolate the<i> antABC</i> genes using both the primers for the entire operon and those for the individual genes. No attempt showed any bands, although salt and temperature gradients were used as well as different types of polymerase. LD2 cultures were grown up to be plasmid prepped and genomic prepped in order to have a higher concentration of template DNA in the PCR master mix and more PCR will be attempted on these genes next week. Mutagenesis primers were also designed this week to remove the illegal restriction cut sites in the<i> carAd</i> and <i>antB</i> genes (NotI and 2xEcoRI respectively). Only 1 mutagenesis primer was designed for <i>antB</i> as one of its EcoRI sites was very close to the beginning of the gene and could be removed using a new primer. During this process it was also discovered that the original ant primers were designed for the wrong genes and therefore do not match the intended sequence. Although the new <i>antABC</i> primers were designed using the correct gene the PCR may not be working due to the length of the operon so the design of new individual primers may be necessary. Finally, we also aimed to grow up the freeze dried culture of <i>Rhodococcus erythropolis</i> (source of the<i> AmdA</i> gene) that we received from DSMZ. The reactivation media was produced and autoclaved to begin the process next week.</p><br />
<br />
</html>[[File:07.23.2012.Restriction Digest of Car genes.jpg|500px|thumb|Gel of restriction digest of car genes. A refers to <i>carAa</i>, C to <i>carAc</i>, and D to <i>carAd</i>. Ladder shows that after purification and digestion DNA is still the correct size.|center]]<br />
[[File:CarAc biobrick confirmation PCR.jpg|500px|thumb|Gel of confirmation PCR done on competent Top 10 <i>E. coli</i> cells in which a PSB1C3 vector containing the <i>carAc</i> gene had been inserted. Red X marks the 500 bp mark on the weak ladder showing the product to be the expected size (323 bp gene + 2*100 bp biobrick sites on each end).|center]] <html><br />
<br />
<h2>Week 14 (July 30 - August 3)</h2><br />
<p>We received the sequencing results this week for <i>carAc</i> which came back as a 100% match to the expected nucleotide sequence. We also successfully ligated both<i> carAa</i> and<i> carAd</i> into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into top 10 <i>E. coli</i> cells. Colony PCR verified the presence of the biobrick plasmid in these colonies. These were both miniprepped, digested and sent for sequencing by the end of the week. Using a salt gradient, addition of DMSO, and the KAPA buffer specifically designed for GC-rich amplicons we were able to amplify the ant operon from a genomic prep of LD2. However, all attempts at ligating and transforming this sequence were met with the unfortunate presence of red colonies (indicating failed insertion into the plasmid). Next week we will recut a <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> vector with Xba and Pst to attempt to mitigate this problem. We also reactivated the <i>R. erythropolis</i> following the DSMZ protocol for reviving freeze-dried cultures that can be found <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/dmszfreezedry">here</a> using DSMZ media #1 and grew it on plates made from DSMZ media #220 at 28 degrees. Colonies grew very quickly and were used for colony PCR and to make overnight cultures for downstream applications. The colony PCR using the amidase primers was unsuccessful, so a genomic prep was performed on the overnight culture. Unfortunately we lacked some of the required equipment and the genomic prep did not work, but with a few procedural modifications and the right equipment we are confident it will work next week. A glycerol stock of the<i> R. erythropoplis</i> was also made from another overnight culture. We also started construction on the <i>carAc </i>part this week by inserting a ribosome binding site (B0034) in front of the gene. Finally, since the<i> ant</i> operon was successfully amplified by PCR we designed mutagenesis primers for the 2nd EcoRI site in the operon rather than making new individual primers. We also designed a mutagenesis primer to remove the SpeI site in <i>AmdA</i>. Now that we have conclusively proven that the LD2 strain contains the pCAR1 plasmid we aim to demonstrate its ability to degrade carbazole and other nitrogen containing heterocycles using an assay experiment similar to the one performed in Week 10.</p><br />
<br />
</html>[[File:07.30.2012. CarAa, CarAd.jpg|500px|thumb|<i>carAa</i> and <i>carAd</i> amplified from genomic prep of <i>Pseudomonas sp. LD2</i>. Amplicons are larger than expected, potentially the gel ran diagonally. The 6th lane for each gene was a negative control.|center]]<br />
[[File:07.31.2012.Ant operon.jpg|500px|thumb|<i>ant</i> operon amplified from genomic prep of <i>Pseudomonas sp. LD2</i> at the expected size of 3000 bp.|center]]<br />
[[File:08.01.2012 CarAdBB.jpg|500px|thumb|Lanes 2,4,5, and 11 contain <i>carAd</i> genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent top 10 <i>E.Coli</i>.|center]]<br />
[[File:08.01.2012 CarAaBB.jpg|500px|thumb|Lane 9 contains <i>carAa</i> genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent top 10 <i>E.Coli</i>. Although larger than expected, the positive control RFP generator in lane 10 is also larger than its expected size (~1200 bp including biobrick bookends) indicating that the gel may have a slant in it.|center]]<html><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p>This week we received the sequencing results for <i>carAa</i> and <i>carAd</i>. <i>carAd</i> was a match, however <i>carAa</i> did not match at all and had misplaced biobrick restriction enzyme sites. Due to this we decided to restart the biobricking process for <i>carAa</i> all the way back to PCR from the plasmid. By the end of the week we had progressed to the point of transforming it into competent <i>E. coli</i> cells, however a thick lawn of bacteria had grown overnight making it impossible to pick colonies. A streak plate was made from this plate to attempt to get less dense growth. Since the sequencing came back positive for <i>carAd</i> the next step was to perform site directed mutagenesis on its NotI restriction enzyme site using the primers that were designed previously (for site-directed mutagenesis optimization, please refer to Desulfurization Journal Week 14). The PCR was successful, showing very bright bands at 3000 bp, and the PCR product was digested with DpnI to remove parental DNA. Transformation of this product will indicate whether the mutagenesis was successful. We were also able to successfully perform a genomic prep of <i>Rhodococcus erythropolis</i> and amplify the <i>AmdA</i> gene this week. It was ligated into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> by Friday, and a confirmation colony PCR was run on Friday evening to verify that the gene was inserted. These results will be available by Monday. After many attempts the <i>ant</i> operon was finally ligated and transformed as well this week. Confirmation PCR showed bands of about 3000 bp which is consistent with the size of the operon. By Friday the successful colonies had been grown up and miniprepped, ready to send for sequencing early next week. The <i>carAc</i>/B0034 construct was also successfully transformed this week and verified by running a PCR using the B0034 forward primer. It was also miniprepped and is ready to send for sequencing next week. </p><br />
<p>In addition to this work constructing biobricks we also started an experiment to demonstrate the function of the pCAR plasmid in LD2. Strains of LD2 were grown up in LB and then washed and placed in B-N media + glucose and various experimental additions. These additions included carbazole, 4-PBAH (a nitrogen containing naphthenic acid), ammonia chloride, and a negative control with no nitrogen source. Strains of <i>E. coli</i> were also grown under the same conditions to compare growth and ammonia production of LD2 to a strain that does not contain the pCAR plasmid. These results should act as a preview of what we expect our final biobrick containing <i>E. col</i>i species to produce as well as allowing us to establish LD2 as a positive control for this experiment. </p><br />
<br />
<br />
</html>[[File:08.07.2012 amdA pcr from genomic prep.jpg|500px|thumb|<i>AmdA</i> amplified from the genomic prep of <i>R. Erythropolis</i> shows bands just greater than 1500 base pairs in lanes 2,4-6,9, and 12-13. Lane 14 was a negative control. The expected size of <i>AmdA</i> is 1566 base pairs.|center]]<br />
[[File:08.09.2012 confirmation pcr of ant operon.jpg|500px|thumb|The two labelled lanes show amplification at 3000 base pairs from a confirmation colony PCR using the biobrick primers. The expected size of the <i>ant</i> operon with the biobrick bookends is about 3100 base pairs.|center]]<br />
[[File:08.10.2012 carad notI mutagenesis.jpg|500px|thumb|Strong amplification at just above 3000 base pairs from the <i>carAd</i> PCR using mutagenic primers. Since PSB1C3 is 2070 base pairs and <i>carAd</i> is 989 base pairs the amplification should run at about 3059 base pairs. 3 different amounts of template DNA were tested in lanes 2-7 as indicated and a positive control showed weaker amplification in lanes 8-9 at about 4000 base pairs.|center]]<br />
<html><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>The sequencing for the <i>carAc</i>/<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> construct came back with only <i>carAc</i> and no evidence of a <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> site preceding it. The confirmation PCR that was run last week using the <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> forward primer seemed to indicate that it was inserted, however since <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> is only 12 base pairs the primer may not have been specific enough. The construction on this part was restarted this week. <i>carAa</i> colony PCR on last week's transformation showed two positive colonies, however upon culturing, miniprepping, and digesting these colonies the plasmid appeared to be too large (3000 base pairs as opposed to the expected 2000). This may be due to a problem of a mislabeled plasmid tube that has been experienced by other team members, causing the gene to be inserted into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a> rather than <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. To test this, a streak plate was made from these colonies and grown on an ampicillin resistant plate. Mutagenized <i>carAd</i> was successfully transformed into Top 10 <i>E. coli</i> cells, cultured, and miniprepped. The miniprep product was digested with NotI to test whether the NotI site was successfully removed. These results were good, as the plasmid ran at 2000 base pairs and the insert at just under 1000 base pairs in the mutagenized <i>carAd</i> whereas the non-mutagenized <i>carAd</i> had its insert cut into 2 bands. This will be sent for sequencing next week to confirm the mutation. Unfortunately the sequencing results came back negative for the <i>ant</i> operon meaning we will need to restart genomic PCR on it next week. Multiple transformations and confirmation PCRs were run on <i>AmdA</i> this week, however none displayed a band size of 1700-1800 base pairs as expected. We have continued to try different digested plasmids as other team members also had difficulties this week with ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, possibly indicating a problem with our Antarctic Phosphatase stock.</p><br />
<p>The other news from this week was the final results from our carbazole degradation experiment attempting to demonstrate LD2's ability to degrade carbazole and hopefully other nitrogen containing compounds. Unfortunately, these results were very difficult to make sense of as the LD2 that were cultured with carbazole grew, but did not show elevated ammonia levels which would be indicative of carbazole degradation. One of our controls was LD2 cultured with ammonium chloride to test whether LD2 was capable of using ammonia as a nitrogen source. This would pose a problem for us as we were planning to use an increase in ammonia levels as a proxy for carbazole degradation rather than measuring carbazole degradation directly using GC-MS. However, the LD2 showed significantly less ammonia after 44 hours when compared to 26 hours indicating that it may be degraded, either by the LD2 or by an unaccounted for abiotic process. This has lead us to decide to use GC-MS and directly measure carbazole levels in our sample the next time we do this experiment.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<p>The <i>carAa</i> was re-streaked onto ampicillin plates and grew, probably showing that it was ligated into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a>, not <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, which explained the irregularity of our confirmation digest last week. It was sent for sequencing, but in case it was incorrect we also started a new <i>carAa</i> pipeline by PCRing it from the <i>Pseudomonas</i> genome again and redigesting and ligating. It was transformed into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> over the weekend. <i>carAc</i> did not see a lot of progress this week as we spent much of the time attempting to construct a RBS (B0034) in front of it, but were unsuccessful. <i>carAd</i> was also subjected to this construction with B0034 and we were able to send it for sequencing by the end of the week. Several new constructs were also started for <i>carAd</i> in case the sequencing is negative. The <i>ant</i> operon was successfully transformed and confirmed by colony PCR this week, after which it was grown up and sent for sequencing. We also began to attempt mutagenesis on one of the EcoRI sites in the operon, but had no amplification of the plasmid. We also re-digested some PCR purified product to start another ligation in case sequencing failed. After many attempts we were also able to finally successfully transform an <i>AmdA</i>/PSB1C3 plasmid into Top 10 cells this week. After a miniprep they showed bands of about 1800 base pairs on the confirmation digest gel, so they were also sent for sequencing this week and had mutagenesis started on the illegal SpeI site. Unfortunately, the sequencing sent early in the week came back with ambiguous results. The forward reaction for<i> antABC</i> was a match, but its reverse reaction matched to <i>carAa</i>. Both <i>carAa</i> reactions matched nothing. Since these two genes are over 20 000 base pairs apart on the template plasmid it is virtually impossible that they were actually combined into one plasmid (PCR extension time would not have been long enough and even if it was the product would have run much above the 3000 base pair mark). This is possibly a mistake on Eurofins end, so we are not 100% sure if we have 1 good gene, both of them, or neither. Also the sequence for mutagenized <i>carAd</i> showed only about the first 100 base pairs, which were correct however not long enough meaning it was a poor reaction and inconclusive. Finally, we also re-started our <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/carbazole">carbazole degradation assay</a> using the GC-MS to quantitatively measure the loss of carbazole at biological sciences. This process will take 2 weeks to demonstrate LD2's ability to degrade carbazole. We are forcing it to use carbazole as a carbon and a nitrogen source in one sample and as only a nitrogen source in another sample (this sample has had glucose added to it).</p><br />
<br />
</html>[[File:AmdA colonyPCR calgary12.JPG|500px|thumb|Amplification is seen in lane 7 of this PCR of top 10 colonies into which AmdA/PSB1C3 had been transformed. This indicates that <i>AmdA</i> has been successfully inserted into a biobrick vector.|center]]<br />
[[File:NewcarAagenomePCR calgary12.jpg|500px|thumb|<i>carAa</i> amplified from pCAR1.2 plasmid of <i>Pseudomonas putida</i>.|center]] <html><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<p>The new stock of <i>carAa</i> that we had PCRed from LD2 was sent for sequencing this week and came back as a match, so we have begun constructing a RBS site and a TetR promoter (<a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>) in front of it. At this point all the <i>car</i> genes have been successfully amplified, inserted into a biobrick plasmid, and sequence verified. We also continued to attempt to construct <i>carAc</i> with an RBS site (<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>) in front of it, but had no success. On a better note, we confirmed this week that<i> carAd</i> was mutated properly and we were also able to insert an RBS site in front of it. The next step for this gene will be to attempt a plasmid switch as it is currently in <a href="http://partsregistry.org/Part:pSB1A3">pSB1A3</a> and needs to be in <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> for submission to the parts registry. After sending a new batch of the ant operon for sequencing we got the same confusing results as we did last week. We therefore hypothesized that our primers were annealing to a homologous region that is similar to the<i> ant</i> genes and very close to <i>carAa</i>. This means that we need to restart PCR on this gene as all of our current product is incorrect. The sequencing results for <i>AmdA</i> that came back this week were also somewhat confusing as they did not quite match the sequence we expected. However, they were a 100% match for the <i>AmdA</i> gene in a different strain of <i>Rhodococcus erythropolis</i>. This means that DSMZ probably sent us the wrong strain of bacteria and that we were very lucky that the primers we designed based off of the expected template actually worked to amplify a slightly different gene in another strain. The good news is that this new sequence contains no illegal cut sites, which explains why mutagenesis was not working (there was nothing to mutate) and allows us to immediately begin construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> in front of <i>AmdA</i> this week. </p><br />
<br />
</html>[[File:Caraa colonyPCR calgary12.JPG|500px|thumb|Colony PCR amplification from top 10 <i>E. coli</i> cells with a PSB1C3/<i>carAa</i> construct transformed into them. Lanes 1-3, 5, and 7 were sent for sequencing with lane 5 coming back positive.|center]]<html><br />
<br />
<h2>Week 19 (September 3- September 7)</h2><br />
<p><i>carAd</i>/<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> was successfully plasmid switched into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, making it ready for submission. Construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> and <i>carAa</i> may have been successful as the digest of the miniprep looked promising. It will be sent for sequencing next week. Unfortunately there was no progress made on the constructions of <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> or <i>carAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. These will be redigested and religated for next week. We could not amplify the <i>ant</i> operon despite trying many variations of PCR using high fidelity KAPA polymerase. This is probably due to the multiple, large regions of homology on the operon that the reverse primer binds to making amplification difficult. Because the function of this gene can theoretically be replaced by <i>AmdA</i>, we may scale back efforts dedicated to it, as the chances of successful amplification appear slim. </p><br />
<br />
<h2>Weeks 20-22 (September 10 - September 30)</h2><br />
<p>Constructs that we continued to work on this week included <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, <i>carAa</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, and <i>carAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. Some sequencing was sent, but no positive results were obtained. A new approach to biobrick the <i>ant</i> operon was implemented this week. A new forward primer was designed that would anneal about 1500 base pairs upstream of the <i>ant</i> operon, avoiding the region of homology that is repeated at various points on the plasmid. Once this region is successfully amplified we could use this amplified DNA as a template for another PCR reaction which would use the normal <i>ant</i> primers. Since the template would no longer include regions of homology these primers should be free to work properly and amplify the <i>ant</i> operon. The PCR using the new primers was started this week, and although amplification at 4500 base pairs was observed as expected, there was also a lot of non-specific amplification. This means that to purify the amplified DNA that we want and avoid the other products a dreaded gel extraction had to be performed which, as usual, did not work. At this point it was decided that further attempts to amplify the <i>ant</i> operon would be futile and our efforts would be better served working on constructing the parts we do have. Unfortunately no progress was made in these weeks on construction. We have also obtained GC-MS readings from the carbazole degradation experiment that showed that <i>Pseudomonas</i> LD2 can indeed degrade carbazole, however only under low glucose conditions.</p><br />
<br />
</html>[[File:LD2 degradation september data calgary12.jpg|500px|thumb|Carbazole loss over 13 days when cultured with <i>Pseudomonas sp. LD2</i> with and without the addition of glucose.|center]]<br />
<br />
<br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/ModelvalidationTeam:Calgary/Notebook/Protocols/Modelvalidation2012-10-04T02:34:09Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=Modelling Validation Experiments|<br />
CONTENT=<html><br />
<br />
<p> After running the model for the pathway in question, the PetroBrick (<b><a href="http://partsregistry.org/wiki/index.php?title=BBa_K590025">BBa_K590025</a></b>), overnight cultures were made adding different combinations of metabolites found to increase product output from the model. </p><br />
<br />
<ol><br />
<li>PetroBrick culture was grown in 3x 20mL cultures from isolated plate colonies supplemented with 50 ug/mL chloramphenicol. These cultures were grown overnight at 37<sup>o</sup>C with shaking.<br />
<li>Culture tubes had 5 mL of minimal <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/m9media">M9 media</a> with 1% (w/v) filter sterilized glucose, and 50 ug/mL chloramphenicol added to each tube.<br />
<li>All chemicals added were at a concentration of 50 mM except for AMP (100 mg/L) and L-aspartate (100mg/L). Stocks were dissolved in water at concentrations ~100x what was being added (provided that the compound was soluble) to ensure that changes in volume would not account for differences seen.<br />
<li>Cells were allowed to grow for 72 hours at 37<sup>o</sup>C with shaking.<br />
<li>Cultures then had OD<sub>600</sub> readings taken to normalize against. <br />
<li>Cultures were then sonicated with a Misonix Cell Disruptor at Dial 11 (output of 18 watts) for 1 min.<br />
<li>1 mL of ethyl acetate was added to each tube shaken vigorously and spun down at 4000 RPM for 5 min. at 4<sup>o</sup>C<br />
<li>250 uL of the ethyl acetate layer was ran onto the GC-MS (<a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Carbazole_GC-MS_Analysis">see protocol</a>).<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
</html>}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/mgtacircuitTeam:Calgary/Notebook/Protocols/mgtacircuit2012-10-04T02:19:15Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=Characterization of the mgtA regulation with S7 killgene|<br />
CONTENT=<html><br />
<ol><br />
<li>Cells with the appropriate circuits were grown in LB overnight with 10mM MgCl<font style="text-transform: lowercase;">2</font>.</li><br />
<li>The next morning the cells were spun down and washed with <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/m9media">M9 minimal media</a> and appropriate amounts of MgCl<font style="text-transform: lowercase;">2</font>.</li><br />
<li>Cells were then aliquoted into 96 well plates in triplicates for each concentration and OD was measured at 600nm for every 4 hour for 8 hours.</li><br />
<li>Additionally, the cells were plated on the appropriate antibiotic in dilutions of 1:1000 and 1:10000 for measuring CFU.</li><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
</ol><br />
</html>}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/mgcircuitTeam:Calgary/Notebook/Protocols/mgcircuit2012-10-04T02:16:23Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=Characterization of mgtA regulation with GFP|<br />
CONTENT=<html><br />
<ol><br />
<li>Cells containing the appropriate circuits were grown in LB overnight with 10mM MgCl<font style="text-transform: lowercase;">2</font>.</li><br />
<ul><li>mgtAp-rb-K082003</li><br />
<li>mgtAp-rb</li><br />
<li>R0040-mgtArb-K082003</li><br />
<li>R0040-B0034-K082003</li></ul><br />
<li>The next morning the cells were spun down and washed with <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/m9media">M9 minimal media</a> and appropriate amounts of MgCl<br />
<ul><li> 0mM MgCl<sub>2</sub></li><br />
<li> 0.01mM MgCl<sub>2</sub></li><br />
<li> 0.1mM MgCl<sub>2</sub></li><br />
<li> 1mM MgCl<sub>2</sub></li><br />
<li> 10mM MgCl<sub>2</sub></li></ul> <font style="text-transform: lowercase;"></font></li><br />
<br />
<li>Cells were then aliquoted into 96 well plates in triplicates for each concentration and florescence was measured every hour for 24 hours at 475nm excitation and 515nm emission.</li><br />
</ol><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
</html>}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/mgcircuitTeam:Calgary/Notebook/Protocols/mgcircuit2012-10-04T02:14:27Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=Characterization of mgtA regulation with GFP|<br />
CONTENT=<html><br />
<ol><br />
<li>Cells containing the appropriate circuits were grown in LB overnight with 10mM MgCl<font style="text-transform: lowercase;">2</font>.</li><br />
<ul><li>mgtAp-rb-K082003</li><br />
<li>mgtAp-rb</li><br />
<li>R0040-mgtArb-K082003</li><br />
<li>R0040-B0034-K082003</li></ul><br />
<li>The next morning the cells were spun down and washed with <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/m9media">M9 minimal media</a> and appropriate amounts of MgCl<br />
<ul><li> 0mM MgCl<sub>2</sub></li><br />
<li> 0.01mM MgCl<sub>2</sub></li><br />
<li> 0.1mM MgCl<sub>2</sub></li><br />
<li> 1mM MgCl<sub>2</sub></li><br />
<li> 10mM MgCl<sub>2</sub></li></ul> <font style="text-transform: lowercase;">2</font>.</li><br />
<br />
<li>Cells were then aliquoted into 96 well plates in triplicates for each concentration and florescence was measured every hour for 24 hours at 475nm excitation and 515nm emission.</li><br />
</ol><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
</html>}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/decatecholizationTeam:Calgary/Notebook/Protocols/decatecholization2012-10-04T02:10:21Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=Catechol Assay in <i>E. coli</i> Cells|<br />
CONTENT=<html><br />
<br />
<p>This assay is used to verify that catechol 2,3-dioxygenase (XylE) is converting catechol to the yellow compound 2-hydroxymuconic semialdehyde (2-HMS). For this procedure we used to the newly constructed XylE part. This part contains the TetR promoter the XylE gene and its native rbs site:</p><br />
<ol><br />
<li>Grow up 2 ml overnight cultures in LB.</li><br />
<li>Spin the cultures down and keep the supernatant.</li><br />
<li>Bring the supernatant to a concentration of 0.1 M of Catechol by using a 1M catechol stock solution.</li><br />
<li>The colour of the supernatant should change to bright yellow very quickly (in about 30 seconds).</li><br />
</ol><br />
<br />
<h2>GC-MS Catechol Assay</h2><br />
In order to identify if the PetroBrick or <i>Micrococcus</i> was capable of further reducing catechol products, GC-MS was used. <br />
<ol><br />
<li>Grow overnight cultures of the PetroBrick in LB at 37<sup>o</sup>C and <i>xylE</i> construct, or <i>Microccus</i> in yeast tryptone broth at 26<sup>o</sup>C.<br />
<li>Subculture into the micrococcus or PetroBrick broth respectively and set up cultures with and without <i>xylE</i> as well as the appropriate control. <br />
<li>Note: Prior to mixing, <i>xylE</i> and PetroBrick cultures were spun down at 4000 RPM for 5 min at room temperature and washed with 2x volume LB, spun down again, and then resuspended to the original volume to remove antibiotics which they were initially cultured in.<br />
<li>Add 10 mM catechol to each tube, mix well, and cover in tin foil. <br />
<li>Culture the cells at the appropriate temperature for 72 hours.<br />
<li>Sonicate samples for 1 min using a Misonix Cell Disrupter set to Dial 11. <br />
<li>Add 5 mL of ethyl acetate, shake well, and spin down at 4000 RPM for 5 min. at 4<sup>o</sup>C. <br />
<li> Add 1mL of the ethyl acetate to a tube and reduce the volume to 0.3mL through heating.<br />
<li> Samples were dried using sodium sulfate if any water layer was present and modified using trimethyl sylyl reagent.<br />
<li> Samples were ran onto the GC-MS (<a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Carbazole_GC-MS_Analysis">see protocol</a>)<br />
<br><br><br><br><br><br><br><br><br><br />
<br />
</html>}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/carbazoleTeam:Calgary/Notebook/Protocols/carbazole2012-10-04T02:04:51Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=Carbazole Degradation Time-course Assay|<br />
CONTENT=<html><br />
<h2>Media Preparation</h2><br />
<p><b>B-N Medium for Culturing <i>Pseudomonas</i> LD2</b></p><br />
<p>The B-N (basic with no nitrogen) medium with trace metals is specifically designed for <i>Pseudomonas</i> LD2 growth when analyzing biodegradation of a nitrogen-containing compound.</p><br />
<p>Weigh out appropriate amounts of all compounds except FeSO<font style="text-transform: lowercase;">4</font>.7H<font style="text-transform: lowercase;">2</font>O and dissolve, with stirring, in 1L of milliQ H<font style="text-transform: lowercase;">2</font>O. </p><br />
<br />
<br />
<center><br />
<table border="0"><br />
<br />
<tr><br />
<td> K<font style="text-transform: lowercase;">2</font>HPO<font style="text-transform: lowercase;">4</font>:</td><br />
<td>0.5g</td><br />
</tr><br />
<br />
<tr><br />
<td> Na<font style="text-transform: lowercase;">2</font>SO<font style="text-transform: lowercase;">4</font>:</td><br />
<td>2.0g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>MgSO<font style="text-transform: lowercase;">4</font> 7H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>0.2g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>FeSO<font style="text-transform: lowercase;">4</font> 7H<font style="text-transform: lowercase;">2</font>O </td><br />
<td>Trace</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> Trace metals:</td><br />
<td>1.0mL</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>Up to 1.0L</td><br />
</tr><br />
<br />
</table><br />
</center><br />
<br />
<p>When all the salts are dissolved, add FeSO<font style="text-transform: lowercase;">4</font>.7H<font style="text-transform: lowercase;">2</font>O and trace metals and stir for at least 5min to ensure that all compounds are dissolved. <br />
<p>The trace metal solution is prepared as follows:</p><br />
<br />
<center><br />
<table border="0"><br />
<br />
<tr><br />
<td>CaCl<font style="text-transform: lowercase;">2</font> 2H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>3.7g</td><br />
</tr><br />
<br />
<tr><br />
<td>H<font style="text-transform: lowercase;">3</font>BO<font style="text-transform: lowercase;">3</font>:</td><br />
<td>2.5g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> MnCl<font style="text-transform: lowercase;">2</font>:</td><br />
<td>0.87g</td><br />
</tr><br />
<br />
<tr><br />
<td> FeCl<font style="text-transform: lowercase;">3</font>:</td><br />
<td>0.44g </td><br />
</tr><br />
<br />
<tr><br />
<td>ZnCl<font style="text-transform: lowercase;">3</font>:</td><br />
<td>0.44g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> Na<font style="text-transform: lowercase;">2</font >MoO<font style="text-transform: lowercase;">2</font > 2H<font style="text-transform: lowercase;">2</font >O:</td><br />
<td>0.29g</td><br />
</tr><br />
<br />
<tr><br />
<td>CoCl<font style="text-transform: lowercase;">2</font >:</td><br />
<td>0.01g </td><br />
</tr><br />
<br />
<tr><br />
<td>CuCl<font style="text-transform: lowercase;">2</font >:</td><br />
<td>0.0001g </td><br />
</tr><br />
<br />
<tr><br />
<td> H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>Up to 1.0L</td><br />
</tr><br />
<br />
</table><br />
</center><br />
<br />
<p>Dispense appropriate amounts of B-N media into glass culture flasks and autoclave.</p><br />
<br />
<h2>Carbazole Degradation Time-course Experiment</h2><br />
<p>This is a time course experiment designed to monitor carbazole loss by collecting bacterial culture samples over time, followed by <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oextraction"> organic extraction</a> and <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Carbazole_GC-MS_Analysis">GC-MS</a> analysis.</p><br />
<ol><br />
<li>Grow up overnight cultures of <i>Pseudomonas</i> LD2.</li><br />
<li>Make B-N media, as described above, and add 50mL to each 250mL Erlenmeyer flask and autoclave.</li><br />
<li>Prepare 2 Erlenmeyer flasks as blanks (B-N media only) for day 0 and day 14 as negative controls.</li><br />
<li>Add 15 mg carbazole in its crystalline form, 2.5mg glucose (glucose can be dissolved in a small amount of water and filter sterilized), and 100uL of cells to one set of flasks. This serves as the positive control (2 samples at each time point).</li><br />
<li>Add 15 mg carbazole in its crystalline form and 100uL of cells to another set of flasks. This serves as the experimental sample (2 samples at each time point).</li><br />
<li>Place stopper on the flasks and incubate in the shaker at 28oC.</li><br />
<li>At the appropriate time points (day 0, day 1, day 4, day 7, day 11, day 14), take out the flasks from the shaker and add enough acid to bring the pH of the solutions to ~2.</li><br />
<li>Each sample is extracted following the organic extractions protocol and analyzed using GC-MS.</li><br />
</ol><br />
<br />
</html>}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/carbazoleTeam:Calgary/Notebook/Protocols/carbazole2012-10-04T01:54:36Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=Carbazole Degradation Time-course Assay|<br />
CONTENT=<html><br />
<h2>Media Preparation</h2><br />
<p><b>B-N Medium for Culturing <i>Pseudomonas</i> LD2</b></p><br />
<p>The B-N (basic with no nitrogen) medium with trace metals is specifically designed for <i>Pseudomonas</i> LD2 growth when analyzing biodegradation of a nitrogen-containing compound.</p><br />
<p>Weigh out appropriate amounts of all compounds except FeSO<font style="text-transform: lowercase;">4</font>.7H<font style="text-transform: lowercase;">2</font>O and dissolve, with stirring, in 1L of milliQ H<font style="text-transform: lowercase;">2</font>O. </p><br />
<br />
<br />
<center><br />
<table border="0"><br />
<br />
<tr><br />
<td> K<font style="text-transform: lowercase;">2</font>HPO<font style="text-transform: lowercase;">4</font>:</td><br />
<td>0.5g</td><br />
</tr><br />
<br />
<tr><br />
<td> Na<font style="text-transform: lowercase;">2</font>SO<font style="text-transform: lowercase;">4</font>:</td><br />
<td>2.0g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>MgSO<font style="text-transform: lowercase;">4</font> 7H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>0.2g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>FeSO<font style="text-transform: lowercase;">4</font> 7H<font style="text-transform: lowercase;">2</font>O </td><br />
<td>Trace</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> Trace metals:</td><br />
<td>1.0mL</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>Up to 1.0L</td><br />
</tr><br />
<br />
</table><br />
</center><br />
<br />
<p>When all the salts are dissolved, add FeSO<font style="text-transform: lowercase;">4</font>.7H<font style="text-transform: lowercase;">2</font>O and trace metals and stir for at least 5min to ensure that all compounds are dissolved. <br />
<p>The trace metal solution is prepared as follows:</p><br />
<br />
<center><br />
<table border="0"><br />
<br />
<tr><br />
<td>CaCl<font style="text-transform: lowercase;">2</font> 2H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>3.7g</td><br />
</tr><br />
<br />
<tr><br />
<td>H<font style="text-transform: lowercase;">3</font>BO<font style="text-transform: lowercase;">3</font>:</td><br />
<td>2.5g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> MnCl<font style="text-transform: lowercase;">2</font>:</td><br />
<td>0.87g</td><br />
</tr><br />
<br />
<tr><br />
<td> FeCl<font style="text-transform: lowercase;">3</font>:</td><br />
<td>0.44g </td><br />
</tr><br />
<br />
<tr><br />
<td>ZnCl<font style="text-transform: lowercase;">3</font>:</td><br />
<td>0.44g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> Na<font style="text-transform: lowercase;">2</font >MoO<font style="text-transform: lowercase;">2</font > 2H<font style="text-transform: lowercase;">2</font >O:</td><br />
<td>0.29g</td><br />
</tr><br />
<br />
<tr><br />
<td>CoCl<font style="text-transform: lowercase;">2</font >:</td><br />
<td>0.01g </td><br />
</tr><br />
<br />
<tr><br />
<td>CuCl<font style="text-transform: lowercase;">2</font >:</td><br />
<td>0.0001g </td><br />
</tr><br />
<br />
<tr><br />
<td> H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>Up to 1.0L</td><br />
</tr><br />
<br />
</table><br />
</center><br />
<br />
<p>Dispense appropriate amounts of B-N media into glass culture flasks and autoclave.</p><br />
<br />
<h2>Carbazole Degradation Time-course Experiment</h2><br />
<p>This is a time course experiment designed to monitor carbazole loss by collecting bacterial culture samples over time, followed by <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oextraction"> organic extraction</a> and GC-MS analysis.</p><br />
<ol><br />
<li>Grow up overnight cultures of <i>Pseudomonas</i> LD2.</li><br />
<li>Make B-N media in 250 mL Erlenmeyer flasks. Add 50mL to each Erlenmeyer flask and autoclave.</li><br />
<li>Prepare 2 Erlenmeyer flasks as blanks (B-N media only) at day 0 and day 14 as negative controls.</li><br />
<li>Add 15 mg carbazole in its crystalline form, 2.5mg glucose (glucose can be dissolved in a small amount of water and filter sterilized), and 100uL of cells to one set of flasks. This serves as the positive control (2 samples at each time point).</li><br />
<li>Add 15 mg carbazole in its crystalline form and 100uL of cells to another set of flasks. This serves as the experimental sample (2 samples at each time point).</li><br />
<li>Place stopper on the flasks and incubate in the shaker at 28oC.</li><br />
<li>At the appropriate time points, take out the flasks from the shaker and add enough acid to bring the pH of the solutions to ~2.</li><br />
<li>Each sample is extracted, and analyzed using a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Carbazole_GC-MS_Analysis">GC-MS</a>.</li><br />
</ol><br />
<br />
</html>}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/decatecholizationTeam:Calgary/Notebook/Protocols/decatecholization2012-10-04T01:51:27Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=Catechol Assay in <i>E. coli</i> Cells|<br />
CONTENT=<html><br />
<br />
<p>This assay is used to verify that catechol 2,3-dioxygenase (XylE) is converting catechol to the yellow compound 2-hydroxymuconic semialdehyde (2-HMS). For this procedure we used to the newly constructed XylE part. This part contains the TetR promoter the XylE gene and its native rbs site:</p><br />
<ol><br />
<li>Grow up 2 ml overnight cultures in LB.</li><br />
<li>Spin the cultures down and keep the supernatant.</li><br />
<li>Bring the supernatant to a concentration of 0.1 M of Catechol by using a 1M catechol stock solution.</li><br />
<li>The colour of the supernatant should change to bright yellow very quickly (in about 30 seconds).</li><br />
</ol><br />
<br />
<h2>GC/MS Catechol Assay</h2><br />
In order to identify if the PetroBrick or <i>Micrococcus</i> was capable of further reducing catechol products, GC/MS was used. <br />
<ol><br />
<li>Grow overnight cultures of the PetroBrick in LB at 37<sup>o</sup>C and <i>xylE</i> construct, or <i>Microccus</i> in yeast tryptone broth at 26<sup>o</sup>C.<br />
<li>Subculture into the micrococcus or PetroBrick broth respectively and set up cultures with and without <i>xylE</i> as well as the appropriate control. <br />
<li>Note: Prior to mixing, <i>xylE</i> and PetroBrick cultures were spun down at 4000 RPM for 5 min at room temperature and washed with 2x volume LB, spun down again, and then resuspended to the original volume to remove antibiotics which they were initially cultured in.<br />
<li>Add 10 mM catechol to each tube, mix well, and cover in tin foil. <br />
<li>Culture the cells at the appropriate temperature for 72 hours.<br />
<li>Sonicate samples for 1 min using a Misonix Cell Disrupter set to Dial 11. <br />
<li>Add 5 mL of ethyl acetate, shake well, and spin down at 4000 RPM for 5 min. at 4<sup>o</sup>C. <br />
<li> Add 1mL of the ethyl acetate to a tube and reduce the volume to 0.3mL through heating.<br />
<li> Samples were dried using sodium sulfate if any water layer was present and modified using trimethyl sylyl reagent.<br />
<li> Samples were ran onto the GC-MS (<a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Carbazole_GC-MS_Analysis">see protocol</a>)<br />
<br><br><br><br><br><br><br><br><br><br />
<br />
</html>}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/desulfurTeam:Calgary/Notebook/Protocols/desulfur2012-10-04T01:47:29Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
<br />
TITLE=Desulfurization Assay Protocol|<br />
CONTENT = <html><br />
<br />
<br />
<h2>Media preparation</h2><br />
<p>A modified M9 media is used for the desulfurization assay. The medium needs to be sulfur-free, as the presence of scarce amounts of sulfur represses the promoter controlling desulfurization genes expression. All the salts containing sulfate in the original recipe are replaced by equimolar amounts of chloride salts (e.g. MgCl <font style="text-transform: lowercase;">2</font> instead of MgSO <font style="text-transform: lowercase;">4</font>). Prepare the media as follows:</p>.<br />
<ol><br />
<li>Dissolve the following salts in about 500mL of ddH<font style="text-transform: lowercase;">2</font>O:<br />
<ul><br />
<li>12.8 grams Na<font style="text-transform: lowercase;">2</font>HPO<font style="text-transform: lowercase;">4</font></li><br />
<li>3.0 grams KH<font style="text-transform: lowercase;">2</font>PO<font style="text-transform: lowercase;">4</font></li><br />
<li>0.5 grams NaCl</li><br />
<li>1.0 gram NH<font style="text-transform: lowercase;">4</font>Cl</li><br />
</ul><br />
<br />
<li>Bring the media to approximately 950 ml with ddH2O and pH to 7.4 with NaOH.</li><br />
<li>Autoclave for 20 minutes on slow exhaust. Store at 4°C when finished.</li><br />
<li> The following salts were dissolved in 50mL of water and cold filtered with a sterilized 0.22 micron filter: </li><br />
<ul><br />
<li>10.8 grams glucose </li><br />
<li>0.19g of MgCl<font style="text-transform: lowercase;">2</font></li><br />
<li>0.0152 grams CaCl<font style="text-transform: lowercase;">2</font>.2H<font style="text-transform: lowercase;">2</font>O</li><br />
<li>0.0100 grams Thiamine</li><br />
<li>0.007g of FeCl<font style="text-transform: lowercase;">2</font>.4H<font style="text-transform: lowercase;">2</font>O</li><br />
</ul><br />
</ol><br />
<p>To maintain the plasmid in the bacterial strain, 0.5g of kanamycin was added to the media. However, the sulfate from kanamycin sulfate (commercially available form) needs to be removed. Equimolar amounts of BaCl<font style="text-transform: lowercase;">2</font> is added to a kanamycin sulfate solution (in distilled water) to precipitate and remove the sulfate in the form of BaSO<font style="text-transform: lowercase;">4</font>. The solution is then centrifuged to remove the precipitate from the solution. The solution is filtered through filter paper to remove all the precipitate. Finally, the solution was cold filtered into the stock media by a sterilized 0.22 micron filter.</p><br />
<br />
<h2>Experiment setup</h2><br />
<p>The following eight sulfur containing compounds are tested in this assay:</p><br />
<ul><br />
<li>Dibenzothiophene (DBT) </li><br />
<li>Cyclopentanethiol</li><br />
<li>Tetrahydro-4H-Thiopyran-4-one</li><br />
<li>Benzo[b] thiophene-2-carboxaldehyde</li><br />
<li>Sulfolane</li><br />
<li>Thiophene</li><br />
<li>Tetrahydrothiophene-1-oxide</li><br />
<li>Tetrahydrothiophene</li><br />
</ul><br />
<p>For each compound, five liquid cultures are prepared. Each of them contains 0.125 mM of the test compound. Four of them are inoculated with <i>Rhodococcus</i>. cells (prepared as described below), the growth of the cells (by OD readings) are monitored and the degradation of the organic compound is measured by <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Carbazole_GC-MS_Analysis">GC-MS</a> after 24hours, 48 hours, 72 hours, and 6 days. The fifth tube is left sterile (as a negative control) to make sure the degradation of the organic compound is by the bacteria and not due to other random factors (e.g. light, heat or natural breakdown of a volatile compound). A control containing only the M9 with no inoculant is used to make sure the prepared media is sterile. </p><br />
<br />
<h5><i>Cell preparation</i></h5><br />
<p>Rhodococcus cells are inoculated in liquid BHI media and incubated at 30°C. After two days, the cells are washed three times with M9 media to eliminate all the sulfur containing compounds. The washed cells are diluted in M9 to an OD600 of 0.287 and 1µL of the diluted culture is added to each of the samples. </p><br />
<br />
<p>The culture were incubated at a 30°C shaker. The samples were taken out at a series of time points (as indicated), frozen at -80°C freezer until they can be analysed using the <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Carbazole_GC-MS_Analysis">GC-MS</a>. </p><br />
<br />
</html><br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/desulfurTeam:Calgary/Notebook/Protocols/desulfur2012-10-04T01:43:23Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
<br />
TITLE=Desulfurization Assay Protocol|<br />
CONTENT = <html><br />
<br />
<br />
<h2>Media preparation</h2><br />
<p>A modified M9 media is used for the desulfurization assay. The medium needs to be sulfur-free, as the presence of scarce amounts of sulfur represses the promoter controlling desulfurization genes expression. All the salts containing sulfate in the original recipe are replaced by equimolar amounts of chloride salts (e.g. MgCl <font style="text-transform: lowercase;">2</font> instead of MgSO <font style="text-transform: lowercase;">4</font>). Prepare the media as follows:</p>.<br />
<ol><br />
<li>Dissolve the following salts in about 500mL of ddH<font style="text-transform: lowercase;">2</font>O:<br />
<ul><br />
<li>12.8 grams Na<font style="text-transform: lowercase;">2</font>HPO<font style="text-transform: lowercase;">4</font></li><br />
<li>3.0 grams KH<font style="text-transform: lowercase;">2</font>PO<font style="text-transform: lowercase;">4</font></li><br />
<li>0.5 grams NaCl</li><br />
<li>1.0 gram NH<font style="text-transform: lowercase;">4</font>Cl</li><br />
</ul><br />
<br />
<li>Bring the media to approximately 950 ml with ddH2O and pH to 7.4 with NaOH.</li><br />
<li>Autoclave for 20 minutes on slow exhaust. Store at 4°C when finished.</li><br />
<li> The following salts were dissolved in 50mL of water and cold filtered with a sterilized 0.22 micron filter: </li><br />
<ul><br />
<li>10.8 grams glucose </li><br />
<li>0.19g of MgCl<font style="text-transform: lowercase;">2</font></li><br />
<li>0.0152 grams CaCl<font style="text-transform: lowercase;">2</font>.2H<font style="text-transform: lowercase;">2</font>O</li><br />
<li>0.0100 grams Thiamine</li><br />
<li>0.007g of FeCl<font style="text-transform: lowercase;">2</font>.4H<font style="text-transform: lowercase;">2</font>O</li><br />
</ul><br />
</ol><br />
<p>To maintain the plasmid in the bacterial strain, 0.5g of kanamycin was added to the media. However, the sulfate from kanamycin sulfate (commercially available form) needs to be removed. Equimolar amounts of BaCl<font style="text-transform: lowercase;">2</font> is added to a kanamycin sulfate solution (in distilled water) to precipitate and remove the sulfate in the form of BaSO<font style="text-transform: lowercase;">4</font>. The solution is then centrifuged to remove the precipitate from the solution. The solution is filtered through filter paper to remove all the precipitate. Finally, the solution was cold filtered into the stock media by a sterilized 0.22 micron filter.</p><br />
<br />
<h2>Experiment setup</h2><br />
<p>The following eight sulfur containing compounds are tested in this assay:</p><br />
<ul><br />
<li>Dibenzothiophene (DBT) </li><br />
<li>Cyclopentanethiol</li><br />
<li>Tetrahydro-4H-Thiopyran-4-one</li><br />
<li>Benzo[b] thiophene-2-carboxaldehyde</li><br />
<li>Sulfolane</li><br />
<li>Thiophene</li><br />
<li>Tetrahydrothiophene-1-oxide</li><br />
<li>Tetrahydrothiophene</li><br />
</ul><br />
<p>For each compound, five liquid cultures are prepared. Each of them contains 0.125 mM of the test compound. Four of them are inoculated with <i>Rhodococcus</i>. cells (prepared as described below), the growth of the cells (by OD readings) are monitored and the degradation of the organic compound is measured by GCMS after 24hours, 48 hours, 72 hours, and 6 days. The fifth tube is left sterile (as a negative control) to make sure the degradation of the organic compound is by the bacteria and not due to other random factors (e.g. light, heat or natural breakdown of a volatile compound). A control containing only the M9 with no inoculant is used to make sure the prepared media is sterile. </p><br />
<br />
<h5><i>Cell preparation</i></h5><br />
<p>Rhodococcus cells are inoculated in liquid BHI media and incubated at 30°C. After two days, the cells are washed three times with M9 media to eliminate all the sulfur containing compounds. The washed cells are diluted in M9 to an OD600 of 0.287 and 1µL of the diluted culture is added to each of the samples. </p><br />
<br />
<p>The culture were incubated at a 30°C shaker. The samples were taken out at a series of time points (as indicated), frozen at -80°C freezer until they can be analysed using the <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Carbazole_GC-MS_Analysis">GC-MS</a>. </p><br />
<br />
</html><br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/desulfurTeam:Calgary/Notebook/Protocols/desulfur2012-10-04T01:39:03Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
<br />
TITLE=Desulfurization Assay Protocol|<br />
CONTENT = <html><br />
<br />
<br />
<h2>Media preparation</h2><br />
<p>A modified M9 media is used for the desulfurization assay. The medium needs to be sulfur-free, as the presence of scarce amounts of sulfur represses the promoter controlling desulfurization genes expression. All the salts containing sulfate in the original recipe are replaced by equimolar amounts of chloride salts (e.g. MgCl <font style="text-transform: lowercase;">2</font> instead of MgSO <font style="text-transform: lowercase;">4</font>). Prepare the media as follows:</p>.<br />
<ol><br />
<li>Dissolve the following salts in about 500mL of ddH<font style="text-transform: lowercase;">2</font>O:<br />
<ul><br />
<li>12.8 grams Na<font style="text-transform: lowercase;">2</font>HPO<font style="text-transform: lowercase;">4</font></li><br />
<li>3.0 grams KH<font style="text-transform: lowercase;">2</font>PO<font style="text-transform: lowercase;">4</font></li><br />
<li>0.5 grams NaCl</li><br />
<li>1.0 gram NH<font style="text-transform: lowercase;">4</font>Cl</li><br />
</ul><br />
<br />
<li>Bring the media to approximately 950 ml with ddH2O and pH to 7.4 with NaOH.</li><br />
<li>Autoclave for 20 minutes on slow exhaust. Store at 4°C when finished.</li><br />
<li> The following salts were dissolved in 50mL of water and cold filtered with a sterilized 0.22 micron filter: </li><br />
<ul><br />
<li>10.8 grams glucose </li><br />
<li>0.19g of MgCl<font style="text-transform: lowercase;">2</font></li><br />
<li>0.0152 grams CaCl<font style="text-transform: lowercase;">2</font>.2H<font style="text-transform: lowercase;">2</font>O</li><br />
<li>0.0100 grams Thiamine</li><br />
<li>0.007g of FeCl<font style="text-transform: lowercase;">2</font>.4H<font style="text-transform: lowercase;">2</font>O</li><br />
</ul><br />
</ol><br />
<p>To maintain the plasmid in the bacterial strain, 0.5g of kanamycin was added to the media. However, the sulfate from kanamycin sulfate (commercially available form) needs to be removed. Equimolar amounts of BaCl<font style="text-transform: lowercase;">2</font> is added to a kanamycin sulfate solution (in distilled water) to precipitate and remove the sulfate in the form of BaSO<font style="text-transform: lowercase;">4</font>. The solution is then centrifuged to remove the precipitate from the solution. The solution is filtered through filter paper to remove all the precipitate. Finally, the solution was cold filtered into the stock media by a sterilized 0.22 micron filter.</p><br />
<br />
<h2>Experiment setup</h2><br />
<p>The following eight sulfur containing compounds are tested in this assay:</p><br />
<ul><br />
<li>Dibenzothiophene (DBT) </li><br />
<li>Cyclopentanethiol</li><br />
<li>Tetrahydro-4H-Thiopyran-4-one</li><br />
<li>Benzo[b] thiophene-2-carboxaldehyde</li><br />
<li>Sulfolane</li><br />
<li>Thiophene</li><br />
<li>Tetrahydrothiophene-1-oxide</li><br />
<li>Tetrahydrothiophene</li><br />
</ul><br />
<p>For each compound, five liquid cultures are prepared. Each of them contains 0.125 mM of the test compound. Four of them are inoculated with <i>Rhodococcus</i>. cells (prepared as described below), the growth of the cells (by OD readings) are monitored and the degradation of the organic compound is measured by GCMS after 24hours, 48 hours, 72 hours, and 6 days. The fifth tube is left sterile (as a negative control) to make sure the degradation of the organic compound is by the bacteria and not due to other random factors (e.g. light, heat or natural breakdown of a volatile compound). A control containing only the M9 with no inoculant is used to make sure the prepared media is sterile. </p><br />
<br />
<h5><i>Cell preparation</i></h5><br />
<p>Rhodococcus cells are inoculated in liquid BHI media and incubated at 30°C. After two days, the cells are washed three times with M9 media to eliminate all the sulfur containing compounds. The washed cells are diluted in M9 to an OD600 of 0.287 and 1µL of the diluted culture is added to each of the samples. </p><br />
<br />
<p>The culture were incubated at a 30°C shaker. The samples were taken out at a series of time points (as indicated), frozen at -80°C freezer until they can be analyzed using the GCMS. </p><br />
<br />
</html><br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/plasmidminiprepTeam:Calgary/Notebook/Protocols/plasmidminiprep2012-10-04T01:29:23Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
<br />
TITLE= Plasmid Purification from <i>E. coli</i>|<br />
CONTENT = <html><br />
<br />
<p>We used two plasmid miniprep protocols. The first protocol is taken from the GenElute Miniprep Plasmid Preparation Kits distributed by Sigma Aldrich. We modified the elution portion of the procedure by using double distilled water to elute rather than using TE buffer. We also skipped the step with the optional wash solution. Instead, the step with the addition of Wash Solution in the Column Tube was done twice.</p><br />
<br />
<br />
<ol><br />
<li> Make overnight cultures from LB agar plate growth (The protocol for the making of overnight cultures can be found as a separate protocol).</li><br />
<li> After allowing approximately 16 hours of growth, pellet the cells using a centrifuge for 20 minutes at a speed of 4000 rpm at 4°C.</li><br />
<li> Discard the supernatant, while being careful not to discard any of the pellet.</li><br />
<li> Resuspend the pellet in 200 µL of Resuspension Solution (with RNase A added) provided from the kit. In case of <i>Rhodococcus</i> plasmid purification, 20 &micro;L of lysozyme with a concentration of 20mg/mL was added and the tube was incubated at 37&deg;C for 30 minutes.</li><br />
<li> Transfer the solution from a Falcon tube to a 1.5 µL microcentrifuge tube.</li><br />
<li> Add 200 & µL of Lysis Solution and invert gently to mix. Allow the mixture to clear for less than 5 minutes.</li><br />
<li> Add 350 & µL of Neutralization Solution and invert the tube 4-6 times to mix.</li><br />
<li> Pellet the microcentrifuge tubes at 14000 rpm using a microcentrifuge for 10 minutes. The resulting solution is the lysate.</li><br />
<li> Add 500 µL of the Column Preparation Solution to a binding column inside a collection tube. Centrifuge this tube for 1 minute at 14 000 rpm and discard the liquid underneath the binding tube.</li><br />
<li> Transfer the lysate into the binding column, being careful not to transfer any solid. Discard the microcentrifuge tube with the solid.</li><br />
<li> Centrifuge the collection tube at 14 000 rpm for 1 minute. Discard whatever liquid flowed through the binding column into the collection tube.</li><br />
<li> Add 750 µL of Wash Solution with concentrated ethanol added to the column and spin at 14 000 rpm for 1 minute. Discard the liquid that flowed through into the collection tube.</li><br />
<li> Repeat Step 12 a second time with the same quantity of Wash Solution.</li><br />
<li> Centrifuge the tube for 1 minute at 14 000 rpm to dry the column.</li><br />
<li> Transfer the column to a new 1.5 mL microcentrifuge tube.</li><br />
<li> Add 50 µL of double distilled water to the column and spin for 1 minute at 14 000 rpm.</li><br />
<li> Use a spectrophotometer to measure the concentration and the purity of your plasmid.</li><br />
<br />
</ol><br />
<br><br><br />
<br />
<p>The second protocol uses in-house reagents and ethanol precipitation of plasmid DNA. The required buffer solutions are:</p><br />
<ul><li> P1 : 50 mM TrisHCl (pH 8.0), 10 mM EDTA, 100 µg/ml RNAse A (store at 4°C)</li><br />
<li> P2 : 200 mM NaOH, 1% SDS</li><br />
<li> P3 : 3 M KAc (pH 5.5) (store at 4°C)</li><br />
</ul><br />
<ol><br />
<li> Grow 2.5 mL O/N culture and use 2 mL for below, keeping 0.5 mL for glycerol stock.</li><br />
<li> Pellet culture into 2 mL microfuge tube.</li><br />
<li> Aspire supernatant; Repeat if necessary.</li><br />
<li> Resuspend pellet in 300 µL P1 (Keep on ice).</li><br />
<li> *Perform next quickly (<1 min). * Add 300 µL P2 → Invert → Add 300 µL P3 (Keep on ice).</li><br />
<li> Centrifuge 14000 rpm for 10 min @ RT.</li><br />
<li> Aliquot the supernatant into 1.5 mL microfuge tube (~600-800 µL).</li><br />
<li> Add 650 µL isopropanol (RT) → Invert → Incubate for 10 min at room temperature.</li><br />
<li> Centrifuge 14000 rpm for 10 min @ 4°C → Aspirate.</li><br />
<li> Wash pellet with 70% cold EtOH (~500 µL).</li><br />
<li> Centrifuge 14000 rpm for 5 min @ 4°C → Aspirate.</li><br />
<li> Air-dry pellet (place tubes upside down or SpeedVac for 10 min).</li><br />
<li> Resuspend (by flicking) in 1⨉TE Buffer (30 µL).</li><br />
</ol><br />
<p>*Can leave at RT to facilitate dissolving of plasmid into TE Buffer</p><br />
<p>*Run 3-4 µL on gel to check quality AND/OR Measure concentration (A260/A280)</p><br />
<br />
<br />
<br />
<br />
<br />
</html><br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/plasmidminiprepTeam:Calgary/Notebook/Protocols/plasmidminiprep2012-10-04T01:28:52Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
<br />
TITLE= Plasmid Purification from <i>E. coli</i>|<br />
CONTENT = <html><br />
<br />
<p>We used two plasmid miniprep protocols. The first protocol is taken from the GenElute Miniprep Plasmid Preparation Kits distributed by Sigma Aldrich. We modified the elution portion of the procedure by using double distilled water to elute rather than using TE buffer. We also skipped the step with the optional wash solution. Instead, the step with the addition of Wash Solution in the Column Tube was done twice.</p><br />
<br />
<br />
<ol><br />
<li> Make overnight cultures from LB agar plate growth (The protocol for the making of overnight cultures can be found as a separate protocol).</li><br />
<li> After allowing approximately 16 hours of growth, pellet the cells using a centrifuge for 20 minutes at a speed of 4000 rpm at 4°C.</li><br />
<li> Discard the supernatant, while being careful not to discard any of the pellet.</li><br />
<li> Resuspend the pellet in 200 µL of Resuspension Solution (with RNase A added) provided from the kit. In case of <i>Rhodococcus</i> plasmid purification, 20 &micro;L of lysozyme with a concentration of 20mg/mL was added and the tube was incubated at 37&deg;C for 30 minutes.</li><br />
<li> Transfer the solution from a Falcon tube to a 1.5 µL microcentrifuge tube.</li><br />
<li> Add 200 & µL of Lysis Solution and invert gently to mix. Allow the mixture to clear for less than 5 minutes.</li><br />
<li> Add 350 & µL of Neutralization Solution and invert the tube 4-6 times to mix.</li><br />
<li> Pellet the microcentrifuge tubes at 14000 rpm using a microcentrifuge for 10 minutes. The resulting solution is the lysate.</li><br />
<li> Add 500 µL of the Column Preparation Solution to a binding column inside a collection tube. Centrifuge this tube for 1 minute at 14 000 rpm and discard the liquid underneath the binding tube.</li><br />
<li> Transfer the lysate into the binding column, being careful not to transfer any solid. Discard the microcentrifuge tube with the solid.</li><br />
<li> Centrifuge the collection tube at 14 000 rpm for 1 minute. DIscard whatever liquid flowed through the binding column into the collection tube.</li><br />
<li> Add 750 µL of Wash Solution with concentrated ethanol added to the column and spin at 14 000 rpm for 1 minute. Discard the liquid that flowed through into the collection tube.</li><br />
<li> Repeat Step 12 a second time with the same quantity of Wash Solution.</li><br />
<li> Centrifuge the tube for 1 minute at 14 000 rpm to dry the column.</li><br />
<li> Transfer the column to a new 1.5 mL microcentrifuge tube.</li><br />
<li> Add 50 µL of double distilled water to the column and spin for 1 minute at 14 000 rpm.</li><br />
<li> Use a spectrophotometer to measure the concentration and the purity of your plasmid.</li><br />
<br />
</ol><br />
<br><br><br />
<br />
<p>The second protocol uses in-house reagents and ethanol precipitation of plasmid DNA. The required buffer solutions are:</p><br />
<ul><li> P1 : 50 mM TrisHCl (pH 8.0), 10 mM EDTA, 100 µg/ml RNAse A (store at 4°C)</li><br />
<li> P2 : 200 mM NaOH, 1% SDS</li><br />
<li> P3 : 3 M KAc (pH 5.5) (store at 4°C)</li><br />
</ul><br />
<ol><br />
<li> Grow 2.5 mL O/N culture and use 2 mL for below, keeping 0.5 mL for glycerol stock.</li><br />
<li> Pellet culture into 2 mL microfuge tube.</li><br />
<li> Aspire supernatant; Repeat if necessary.</li><br />
<li> Resuspend pellet in 300 µL P1 (Keep on ice).</li><br />
<li> *Perform next quickly (<1 min). * Add 300 µL P2 → Invert → Add 300 µL P3 (Keep on ice).</li><br />
<li> Centrifuge 14000 rpm for 10 min @ RT.</li><br />
<li> Aliquot the supernatant into 1.5 mL microfuge tube (~600-800 µL).</li><br />
<li> Add 650 µL isopropanol (RT) → Invert → Incubate for 10 min at room temperature.</li><br />
<li> Centrifuge 14000 rpm for 10 min @ 4°C → Aspirate.</li><br />
<li> Wash pellet with 70% cold EtOH (~500 µL).</li><br />
<li> Centrifuge 14000 rpm for 5 min @ 4°C → Aspirate.</li><br />
<li> Air-dry pellet (place tubes upside down or SpeedVac for 10 min).</li><br />
<li> Resuspend (by flicking) in 1⨉TE Buffer (30 µL).</li><br />
</ol><br />
<p>*Can leave at RT to facilitate dissolving of plasmid into TE Buffer</p><br />
<p>*Run 3-4 µL on gel to check quality AND/OR Measure concentration (A260/A280)</p><br />
<br />
<br />
<br />
<br />
<br />
</html><br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/constructionTeam:Calgary/Notebook/Protocols/construction2012-10-04T01:16:17Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=Restriction Digest, Antarctic Phosphotase, and Ligation|<br />
CONTENT=<html><br />
<br />
<p> Determine the order of the two parts you will be putting together; the one in front will be referred to as the insert, while the one behind will be referred to as the vector. Both the vector and the insert need to have their own separate tube, at least in the beginning. </p><br />
<p><em> Restriction Digest Protocol</em></p><br />
<p> In the Insert Tube...<br />
<ul><br />
<li>600 ng of DNA (To figure out the volume, the calculation is 600 / concentration of plasmid. This gives you volume in μL).</li><br />
<li>Water, so that the volume of both DNA and water in the tube is 35 μL total</li><br />
<li>4 μL of React 1 Buffer </li><br />
<li>0.5 μL of EcoRI </li><br />
<li>0.5 μL of SpeI</li><br />
</ul><br />
</p><br />
<p> In the vector Tube...<br />
<ul><br />
<li>250ng of DNA (To figure out the volume, the calculation is 250 / concentration of plasmid. This gives you volume in μL).</li><br />
<li>Add water, so that the volume of both DNA and water in the tube is 35 μL total</li><br />
<li>4 μL of React 2 Buffer</li><br />
<li>0.5 μL of EcoRI</li><br />
<li>0.5 μL of XbaI</li><br />
</ul><br />
</p><br />
<p> Put both tubes into the 37°C water bath for one hour. After, place them into the 65°C heating block for 10 minutes. This deactivates any enzymes in the tube (which is ok, because by now they’ve done all they need to).<br />
Take the insert out, and put it in a -20°C freezer. </p><br />
<p><em> Antarctic Phosphatase Protocol</em></p><br />
<p> To the vector tube, add 5 μL of 10x Antarctic Phosphatase Buffer, 4 μL of water, and 1 μL of Antarctic Phosphatase. We do this to prevent the vector from closing up again without any insert.<br />
Put the tube into the 37°C water bath for 30 mins. After, place it in the 65°C heating block for 10 minutes. </p><br />
<p><em> Ligation Protocol </em></p><br />
<p>Take the insert out of the freezer, and add 5 μL of insert and 5 μL of vector to a new tube. Label the rest of each tube as Unligated, put the date on the tube, and stick it in the -20°C freezer incase your ligation/transformation doesn’t work. To the single tube of 10 μL mix, add 10 μL of 2x Quick Ligase Buffer, and 1 μL of Quick Ligase. Let this sit at room temperature for 5 minutes. </p><br />
<p> You are now done. If you are going to transform this construction product, add all 21μL to a tube of whichever competent bacteria you're using. </p><br />
<br />
</html>}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/agarosegelTeam:Calgary/Notebook/Protocols/agarosegel2012-10-04T01:14:11Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=Agarose Gel Electrophoresis|<br />
CONTENT=<html><br />
<br />
<p> Reagents and Materials </p><br />
<ul><br />
<li> 1X TAE </li><br />
<li> Graduated Cylinder</li><br />
<li> 125 mL flask </li><br />
<li> Agarose </li><br />
<li> Gel Pouring Tray </li><br />
<li> Tape </li><br />
<li> Gel rig </li><br />
<li> SYBR Safe </li><br />
</ul><br />
<p> Protocol </p><br />
<ol><br />
<li> Measure out 100mL of buffer </li><br />
<li> Transfer buffer to 125 mL flask </li><br />
<li> Weigh out enough agarose to make a 1% gel (in our case 1.0 g of agarose was the right amount)</li><br />
<li> Transfer agarose to 125mL flask</li><br />
<li> Melt agarose in microwave until solution is almost boiling, stirring every 15-20 seconds (should be around 2 minutes)</li><br />
<li> Allow agarose to cool (do not let it cool to the point where it is hard)</li><br />
<li> Add 4 uL of SYBR Safe to the cooling agarose</li><br />
<li> Assemble the gel pouring apparatus by inserting gate into slots.</li><br />
<li> Allow gel to cool until flask can be handled comfortably.</li><br />
<li> Place comb in the gel rig.</li><br />
<li> Pour agarose into gel tray.</li><br />
<li> Allow to solidify. While the gel is solidifying prepare the samples. Add your sample and 1 uL 10x Loading Dye, 4 uL of DNA and 5 uL of water</li><br />
<li> Pour 1X TAE over gel so that gel is covered by a 3-5mm buffer</li><br />
<li> Load samples into lane (Don't forget to load a 1kb+ ladder into one of the lanes)</li><br />
<li> Hook electrodes to gel apparatus.</li><br />
<li> Run the apparatus at 100V for 30 - 45 minutes (make sure to watch that the dye does not run off the gel)</li><br />
<li> Visualize the gel and record the results</li><br />
</ol><br />
<br />
</html>}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Project/OSCAR/UpgradingTeam:Calgary/Project/OSCAR/Upgrading2012-10-04T00:40:46Z<p>Anyakornilo: </p>
<hr />
<div>[http://www.example.com link title]{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Upgrading our Product|<br />
<br />
CONTENT={{{CONTENT|<br />
<br />
<html><br />
<p><br />
While we have been able to show that we can convert toxins such as naphthenic acids into useful hydrocarbons there is still a lot of compounds in the tailings that will decrease the value of our product as a fuel. These compounds include sulfur and nitrogen containing heterocyclic atoms such as dibenzothiophene (DBT) and carbazole. Normally these compounds are removed through chemical means, but this requires expensive machinery, extreme pressurs and temperatures, and the addition of organic solvents. By using synthetic biology to do this in a simple bioreactor we can save the energy that is required to produce these conditions and reduce the cost of the expensive processes.</p><br />
<br />
<h2>Why Use Synthetic Biology? Why Not Chemical Methods?</h2><br />
<br />
<p>The most widely used chemical method for removing nitrogen from fuel sources is called hydrodenitrogenation (HDN). This process is not very efficient, as only 77% of nitrogen containing compounds are actually removed (Zeuthen et al, 2001). It also requires harsh conditions, for example temperatures upwards of 350&deg; C and pressures up to 30 Bar. This is mostly because the nitrogen atoms in the ring must be hydrogenated before the carbon-nitrogen bond can be cleaved because this (Katzer & Sivasubramanian, 1979). The input of molybdenum based chemical catalysts required for this reaction is also very costly and can produce toxic by-products of its own (Zhu et al, 2008). Desulfurization has similar problems. Hydrodesulfurization for example, requires high temperature and pressure and also releases the sulfur in the form of hydrogen sulfide gas, a toxic compound which is then converted to elemental sulfur or sulfuric acid.</p><br />
<br />
<p></html>[[File:Ucalgary2012 upgradingOscarinapot.png|centre]]<html><br />
<br />
<br />
<br />
<p>In contrast to this, microbes have been found capable of harvesting both sulfur and nitrogen out of hydrocarbons under physiological conditions, making them a much more environmentally sound and economic approach to carry out on an industrial scale. Synthetic biology can potentially offer a new avenue to address these problems, with much more potential for innovation and new ideas. </p><br />
<br />
<p>We took two main approaches for upgrading our hydrocarbons. The first method focused on reducing the sulfur content of the product through the action of <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">desulfurization</a>. This would prevent the production of toxic sulfur gasses when the hydrocarbons are combusted in an engine. Our second approach uses <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">denitrogenation</a> to remove nitrogen containing compounds from the product. This will reduce the emissions of nitrous oxide gasses that are heavily involved in global warming.</p><br />
<br />
<h2>Click on OSCAR to learn more about what he can do!</h2><br />
<br />
<a style="margin-left: 20px;" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation"><img src="https://static.igem.org/mediawiki/2012/7/71/Calgary2012_Upgrading_Nitrogen.png"></img></a> <br />
<a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization"><img src="https://static.igem.org/mediawiki/2012/3/37/Calgary2012_Upgrading_Sulfur.png"></img></a><br />
<br />
</html>}}}<br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/DenitrogenationTeam:Calgary/Notebook/Denitrogenation2012-10-04T00:28:09Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Denitrogenation Journal|<br />
CONTENT=<br />
<html><br />
<br />
<h2>Week 1 (May 1-4)</h2><br />
<p>The following section covers wetlab aspect of our overall project focusing on microbial conversion of naphthenic acids into economically-valuable hydrocarbons. The approach taken in this endeavour will be from four strategic starting points - ring cleavage, decarboxylation, denitrogenation, and desulfurization. Overall, the 'hydrocarbons' aspect of the project is a critical one to our overall design and construction of a bioreactor capable of not only detecting but also converting naphthenic acids and other toxins to useful or at least innocuous compounds. </p><br />
<br />
<h2>Week 2-3 (May 7-18)</h2><br />
<p>In the first two weeks of iGEM our group has focused on reviewing literature regarding the bioremediation of nitrogen groups attached to naphthenic acids. The most prevalent N heterocycle is carbazole, representing 75% of total nitrogen by mass. The upper pathway of carbazole biodegradation is catalyzed by the enzymes coded for by the <i>car</i> operon, <i>CarA</i> (<i>CarAaAcAd</i>), <i>CarB</i> (<i>CarBaBb</i>), and <i>CarC</i>. These enzymes convert carbazole to anthralinic acid. The lower pathway is catalyzed by the enzymes of the <i>ant</i> operon, <i>antA, B, and C</i>, yielding catechol while releasing CO2 and NH3. The <i>car</i> and <i>ant</i> operons are both regulated by the P<sup>ant</sup> promoter which is induced by the protein, antR. <i>carAa</i> also has its own promoter which is not induced by <i>antR</i> (Diaz & Garcia, 2010). We have also investigated an alternative pathway using <i>CarA</i> combined with an amidase (<i>amdA</i>) that selectively cleaves NH2 from an intermediate of the car pathway (Xu et al, 2006). This could bypass much of the car/ant pathway and is possibly more efficient.</p><br />
<p>We have decided to use <i>Pseudomonas resinovorans</i> and <i>Rhodococcus erythropolis</i> to derive these genes from. <i>carABC</i> and <i>antABC</i> from<i> P. resinovorans</i> has been shown to have a wide range of nitrogen containing substrate specificity. <i>R. erythropolis</i> contains the <i>amdA</i> gene that we wish to use, and some evidence suggests that it may also be able to degrade sulfur rings through its <i>carABC</i> pathway.</p><br />
<br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p>This week we reviewed the primers listed in the database and also designed some new ones. Primers for <i>carAa</i>,<i> carAc</i>, and <i>carAd</i> were designed individually, while primers for <i>CarBaBbC</i> and <i>antABC</i> were designed to encompass multiple genes in a sequence. These primers were designed to be used on <i>Pseudomonas putida</i> which we decided to use as our gene source since it was available to us as opposed to ordering <i>Pseudomonas resinovorans</i>. We also designed a primer for the<i> AmdA</i> gene from <i>Rhodococcus erthyroplois</i>. In addition to ordering these primers we also ordered the nitrogen containing compounds that we will need to test these enzymes on. We decided on using carbazole to make sure the enzymes can perform their natural function as well as pyrrolidine to test them on a similar ring structure. We also ordered cyclohexamine in order to independently test the function of the alternative <i>AmdA</i> pathway. Finally, we decided to eventually order 4-Piperidine butyric acid hydrochloride to test how the enzymes will work on nitrogen containing naphthenic acids. However, we decided since it is a very expensive compound we would wait to make sure the enzyme's work on more simple compounds before ordering it.</p><br />
<p>We also started our work in the wet lab by plating colonies and making an overnight culture of -80 freezer stock <i>Psuedomonas putida</i> on Thursday. We also resuspended the primers for <i>carAa</i>, <i>carAc</i>, <i>carAd</i>,<i> antA</i>, <i>antB</i>, and <i>ant C</i> that were already in the database. We were able to use the colonies that grew on the streak plates to start a colony PCR to attempt to isolate each of these genes on Friday.</p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2><br />
<p>On Monday and Tuesday we used the database primers to attempt to isolate <i>carAa</i>, <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i>antB</i>, and <i>antC</i> from the <i>Psuedomonas putida</i> we plated last week. <i>carAd</i>, <i>antB</i>, and <i>antC</i> were all put in the gradient PCR machine to account for the wide range of their primers’ melting points, while the others were done via regular PCR. Unfortunately, no positive results were obtained from these reactions. The PCR on <i>carAc</i> and <i>antA</i> was repeated on Wednesday, still not giving positive results. Finally, we attempted to use a salt concentration gradient on the PCR reaction for <i>carAc</i> and <i>antA</i>, using concentrations that ranged from 1.0 microlitres/tube to 2.0 microlitres/tube in increments of 0.2 microlitres/tube. This helped as <i>carAc</i> showed bands in samples that had concentrations of 1.2 and 1.4 microlitres/tube. <i> antA</i> also showed bands, however they were not the correct size, indicating contamination and/or non-specific annealing of the primer. The positive control also showed bands of the correct size. Earlier in the week we also made overnight cultures of 3 environmental strains (28, 29, 30) of <i>Psuedomonas putida</i> from -80 glycerol stock. On Friday we performed a genomic prep on these cultures, and plan on attempting to amplify genes from the isolated DNA next week. </p><br />
<br />
</html>[[File:CarAa and carAc Ucalgary12.png|500px|thumb|Gel of PCR using database primers for <i>carAa</i> and <i>carAc</i> on <i>Pseudomonas putida</i>|center]]<html><br />
<br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> We started off this week by determining the DNA concentration of our genomic prep samples from last week using the nanodrop. DNA concentration for all three<i> P. putida</i> strains was at least 1000 ng/microlitre, well above what was needed for PCR. 1/2 and 1/3 dilutions were prepared for all three strains so as not to have an excess of template DNA in PCR reactions. PCR was performed on all 3 strains using primers for <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i> antB</i>, and <i>antC</i> using 6 replicates per gene. The only successful amplification appeared to be <i>antB</i> and <i>carAc</i>, both from strain 28 (with weaker bands in strain 29). We then performed another PCR, just on those two genes with an increased amount of Taq polymerase to hopefully get enough amplified DNA to move forward with. This resulted in strong bands for both at the expected size. We then performed PCR purification using the Qiaquick kit and obtained samples containing 33.5 ng/microlitre of <i>antB</i> DNA and 129 ng/microlitre of <i>carAc </i>DNA. These concentrations were both sufficient to begin a restriction digest and ligation of these parts into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. Next week we hope to verify the results of the restriction digest, continue to amplify <i>carAc</i> and <i>antB</i> from strain 28, and hopefully submit a biobrick for sequencing.</p><br />
<br />
</html>[[File:AntB and carAc Ucalgary.png|500px|thumb|Gel of PCR ran on genomic prep of <i>Pseudomonas putida</i> environmental strain 28 using primers for <i>carAc</i> and <i>antB</i>. |center]]<html><br />
<br />
<br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p>The purified PCR products for <i>carAc</i> and <i>antB</i> were digested with restriction enzymes EcoRI+SpeI, EcoRI+PstI, and XbaI+PstI. However, only <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmids that had been digested with EcoRI+SpeI and EcoRI+PstI were available to attempt ligation. Gel results were inconclusive on the restriction digest product as the plasmid size appeared much larger than expected. However, transformation was still attempted on the ligation products for both genes (both using EcoRI+SpeI ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid). The transformation products were plated onto chloroamphenicol (chlor) plates to select for colonies that had the chloro resistance gene on the <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid. Also this week another round of PCR was performed for the <i> car</i> and <i> ant</i> genes, however all, but <i>carAc</i> showed bands in the negative control lane, possibly indicating contamination or the formation of primer dimers. The <i>carAc</i> bands were fairly weak and a PCR purification resulted in very low DNA concentration, insufficient to move onto restriction digest. Next week's plan hinges heavily on the result of the transformation from this Friday. </p><br />
<br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>The results of our transformation on <i>carAc</i> and <i>antB</i> from last week showed mostly red colonies indicating that the ligation was unsuccessful and that the PSB1C3 plasmid simply closed on itself with no insert. However, there were three colonies for <i>carAc</i> that were white (cut with both X+P and E+P). Colony PCR was performed on these three colonies using biobrick primer sets to verify that the part had been inserted in these colonies, but unfortunately all PCR results were negative indicating an unsuccessful ligation.</p><br />
<p>We also performed a miniprep on <i>Pseudomonas putida</i> strain #28 this week using a home-made protocol rather than the Qiagen kit. Miniprep A had a DNA concentration of 1539.8 ng/μL and Miniprep B had DNA concentration of 1001.2 ng/μL possibly indicating that there may have been a lot of genomic DNA contamination. However miniprep A product was still used as a PCR template for a reaction attempting to isolate <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i>antB</i>, and <i>antC</i>. Special conditions for this PCR included replacing 60 μL of water with 60 μL of betaine heated to 37 degrees Celsius and running a Mg gradient ranging from 0.5 μL - 2.5 μL per tube for each gene. Of these only <i>antB</i> had bands of the right size in 3 of the lanes (Mg concentrations of 1.5, 2, and 2.5 μL). However, all of the <i>antB</i> bands (including the negative control) contained a contamination band at around 100 bases, probably due to the formation of primer dimers. This forced us to do a gel extraction rather than a PCR purification. This gel extraction did not work as the nanodrop results indicated high amounts of agarose contamination. Another PCR was performed using the same conditions, but using miniprep B instead as there was no more miniprep A product remaining. Unfortunately there were no bands for <i>antB</i>, only the 100 base pair contamination bands. </p><br />
<br />
</html>[[File:AntA and antC Ucalgary.png|500px|thumb|Gel of PCR ran using miniprep product A of <i>Pseudomonas putida</i> as template DNA in an attempt to isolate <i>antA</i> and <i>antC</i> genes.|center]]<br />
[[File:CarAc and carAd Ucalgary.png|500px|thumb|Gel of PCR ran using miniprep product A of <i>Pseudomonas putida</i> as template in an attempt to isolate <i>carAc</i> and <i>carAd</i> genes.|center]]<br />
[[File:Minipret A antB and carAc Ucalgary.png|500px|thumb|Gel of PCR ran using miniprep product A of <i>Pseudomonas putida</i> showing bands 3-5 of the correct size corresponding to antB genes and colony PCR ran using biobrick primers attempting to determine if <i>carAc</i> genes were successfully transformed into PSB1C3 plasmid.|center]] <html><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>This week we modified our PCR protocol to adjust for the homemade Taq polymerase we were using. Template DNA was boiled for 10 minutes on its own to replace the initialization step. This is because this polymerase could not tolerate the 95 degree heat during the regular initialization step. Another modification we made was to boil our primers for 5 minutes prior to adding them to the master mix. This was done to discourage the formation of primer-dimers. Finally we also attempted a touch-down PCR which started at an annealing temperature greater than the Tm of our primers and gradually worked its way down to below the Tm of the primers. This was done to increase the specificity of our reaction and also eliminate the formation of primer-dimers. Unfortunately these techniques still saw the formation of dimers, preventing us from performing a PCR purification. Again, the gel purification procedure did not lead to successful purification of our target gene. Due to the problems we have had so far our goal for next week is to perform an assay experiment to try and determine which of the <i>Pseudomonas putida</i> strains we have access to can actually degrade carbazole and therefore contain the pCAR1 plasmid. </p><br />
<br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The goal for this week was to grow different strains of<i> Pseudomonas</i> in media containing B-N media + glucose with only carbazole as a source of nitrogen. In theory only those strains which contained the pCAR1 plasmid would be able to use carbazole as a nitrogen source and thrive in this media. The strains we tested from glycerol stock included <i>P. putida</i> LD1, <i>P.putida</i> environmental strains 28-30, and <i>P. fluorescens</i> PF5. Each of these strains had two cultures grown; one with carbazole and one without it. <i>E. coli</i> grown with carbazole was used as a negative control as it does not contain the pCAR plasmid. At timepoints of 22, 46, and 117 hours of growth samples were taken to do an OD600 reading to measure bacterial growth and had an ammonia assay performed on it. The detection of ammonia acted as a proxy for detecting carbazole degradation as it is an end-product of the pathway. With this in mind, an additional control strain of LD1 was grown in media containing glucose and ammonia. This is to detect the possibility that <i>Pseudomonas</i> can use ammonia as a source of nitrogen after it has degraded carbazole which would be problematic as far as using ammonia levels to determine carbazole degradation. So far we have been having difficulties reading the results of the ammonia assay with a plate reader and may resort to testing our samples using a different assay protocol next week. The OD600 readings were initially very high at 22 hours and then dropped at 46 hours. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>This week we took our last readings from the experiment that we started last week. The OD600 readings taken at 117 hours showed minimal growth over the weekend for all strains except for environmental strain #29 (with carbazole) which increased from 0.086 to 0.313. No other strain showed an increase of 0.100 or greater. This shows that only strain #29 was able to thrive in the carbazole media, possibly indicating that it contains the pCAR plasmid enabling it to use carbazole as a nitrogen source. To verify this we used a new ammonia assay protocol to measure the production of ammonia in the different cultures and theoretically how much carbazole was degraded. Unlike the previous assay we were able to produce a standard curve with a consistent slope based on samples with known amounts of ammonia. However, the only samples that showed any elevation of ammonia levels above the blank were the LD1 samples that were cultured with ammonia chloride to control for the possibility of pseudomonas using ammonia as a nitrogen source. The ammonia levels in this sample were relatively constant which would indicate that the LD1 was not using ammonia as a nitrogen source. Unfortunately the fact that none of the test samples had elevated ammonia levels did not give us any evidence that these strains contain the pCAR plasmid. </p><br />
<br />
</html>[[File: Standard_curve_for_ammonia_assay_UCalgary2012_denitrification_group.png|500px|thumb|Standard curve for ammonia assay using OD635 absorbance.|center|]]<html><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>We received a new strain of Pseudomonas, LD2, this week that had previously been used to study the pCAR1 plasmid. It was grown up in PCA media and had its colonies used for colony PCR. We also received our new primers this week, further increasing the chances of successful PCR. Using the new strain and the new primers <i>carAa</i>, <i>carAc</i>, and <i>carAd</i> all showed at least some bands of close to the correct size. After PCR purification we obtained DNA concentrations of 21.3 ng/μL for <i>carAa</i>, 41.9 ng/ μL for <i>carAc</i>, and 32.8 ng/μL for<i> carAd</i>. These products were then restriction digested using EcoRI + PstI sites. </p><br />
<br />
</html>[[File:07.20.2012 CarAa and CarAc.jpg|500px|thumb|Gel of colony PCR from <i>Pseudomonas sp. LD2</i> for <i>carAa</i> and <i>carAc</i> genes. <i>carAa</i> expected size is 1155 bp and <i>carAc</i> is 323 bp.|center]]<br />
[[File:07.20.2012 CarAd colony PCR from LD2.jpg|500px|thumb|Gel of colony PCR from <i>Pseudomonas sp. LD2</i> for <i>carAd</i> gene. <i>carAd</i> expected size is 989 bp.|center]] <html><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The restriction digested <i>car</i> genes were ligated into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> cells. White colonies grew for<i> carAa</i> and <i>carAc</i> suggesting that ligation and transformation were successful, however no colonies grew for <i>carAd</i> suggesting that the transformation did not work. Colony PCR was performed to confirm the presence of these genes within the vectors, however only <i>carAc</i> was positive. The colonies containing the <i>carAc</i> plasmid were then mini prepped and restriction digested to isolate the plasmid and send for sequencing. Further attempts at transforming<i> carAa</i> and <i>carAd</i> were not successful. Another goal was to perform colony PCR on the <i>P. putida</i> to isolate the<i> antABC</i> genes using both the primers for the entire operon and those for the individual genes. No attempt showed any bands, although salt and temperature gradients were used as well as different types of polymerase. LD2 cultures were grown up to be plasmid prepped and genomic prepped in order to have a higher concentration of template DNA in the PCR master mix and more PCR will be attempted on these genes next week. Mutagenesis primers were also designed this week to remove the illegal restriction cut sites in the<i> carAd</i> and <i>antB</i> genes (NotI and 2xEcoRI respectively). Only 1 mutagenesis primer was designed for <i>antB</i> as one of its EcoRI sites was very close to the beginning of the gene and could be removed using a new primer. During this process it was also discovered that the original ant primers were designed for the wrong genes and therefore do not match the intended sequence. Although the new <i>antABC</i> primers were designed using the correct gene the PCR may not be working due to the length of the operon so the design of new individual primers may be necessary. Finally, we also aimed to grow up the freeze dried culture of <i>Rhodococcus erythropolis</i> (source of the<i> AmdA</i> gene) that we received from DSMZ. The reactivation media was produced and autoclaved to begin the process next week.</p><br />
<br />
</html>[[File:07.23.2012.Restriction Digest of Car genes.jpg|500px|thumb|Gel of restriction digest of car genes. A refers to <i>carAa</i>, C to <i>carAc</i>, and D to <i>carAd</i>. Ladder shows that after purification and digestion DNA is still the correct size.|center]]<br />
[[File:CarAc biobrick confirmation PCR.jpg|500px|thumb|Gel of confirmation PCR done on competent Top 10 <i>E. coli</i> cells in which a PSB1C3 vector containing the <i>carAc</i> gene had been inserted. Red X marks the 500 bp mark on the weak ladder showing the product to be the expected size (323 bp gene + 2*100 bp biobrick sites on each end).|center]] <html><br />
<br />
<h2>Week 14 (July 30 - August 3)</h2><br />
<p>We received the sequencing results this week for <i>carAc</i> which came back as a 100% match to the expected nucleotide sequence. We also successfully ligated both<i> carAa</i> and<i> carAd</i> into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into top 10 <i>E. coli</i> cells. Colony PCR verified the presence of the biobrick plasmid in these colonies. These were both miniprepped, digested and sent for sequencing by the end of the week. Using a salt gradient, addition of DMSO, and the KAPA buffer specifically designed for GC-rich amplicons we were able to amplify the ant operon from a genomic prep of LD2. However, all attempts at ligating and transforming this sequence were met with the unfortunate presence of red colonies (indicating failed insertion into the plasmid). Next week we will recut a <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> vector with Xba and Pst to attempt to mitigate this problem. We also reactivated the <i>R. erythropolis</i> following the DSMZ protocol for reviving freeze-dried cultures that can be found <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/dmszfreezedry">here</a> using DSMZ media #1 and grew it on plates made from DSMZ media #220 at 28 degrees. Colonies grew very quickly and were used for colony PCR and to make overnight cultures for downstream applications. The colony PCR using the amidase primers was unsuccessful, so a genomic prep was performed on the overnight culture. Unfortunately we lacked some of the required equipment and the genomic prep did not work, but with a few procedural modifications and the right equipment we are confident it will work next week. A glycerol stock of the<i> R. erythropoplis</i> was also made from another overnight culture. We also started construction on the <i>carAc </i>part this week by inserting a ribosome binding site (B0034) in front of the gene. Finally, since the<i> ant</i> operon was successfully amplified by PCR we designed mutagenesis primers for the 2nd EcoRI site in the operon rather than making new individual primers. We also designed a mutagenesis primer to remove the SpeI site in <i>AmdA</i>. Now that we have conclusively proven that the LD2 strain contains the pCAR1 plasmid we aim to demonstrate its ability to degrade carbazole and other nitrogen containing heterocycles using an assay experiment similar to the one performed in Week 10.</p><br />
<br />
</html>[[File:07.30.2012. CarAa, CarAd.jpg|500px|thumb|<i>carAa</i> and <i>carAd</i> amplified from genomic prep of <i>Pseudomonas sp. LD2</i>. Amplicons are larger than expected, potentially the gel ran diagonally. The 6th lane for each gene was a negative control.|center]]<br />
[[File:07.31.2012.Ant operon.jpg|500px|thumb|<i>ant</i> operon amplified from genomic prep of <i>Pseudomonas sp. LD2</i> at the expected size of 3000 bp.|center]]<br />
[[File:08.01.2012 CarAdBB.jpg|500px|thumb|Lanes 2,4,5, and 11 contain <i>carAd</i> genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent top 10 <i>E.Coli</i>.|center]]<br />
[[File:08.01.2012 CarAaBB.jpg|500px|thumb|Lane 9 contains <i>carAa</i> genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent top 10 <i>E.Coli</i>. Although larger than expected, the positive control RFP generator in lane 10 is also larger than its expected size (~1200 bp including biobrick bookends) indicating that the gel may have a slant in it.|center]]<html><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p>This week we received the sequencing results for <i>carAa</i> and <i>carAd</i>. <i>carAd</i> was a match, however <i>carAa</i> did not match at all and had misplaced biobrick restriction enzyme sites. Due to this we decided to restart the biobricking process for <i>carAa</i> all the way back to PCR from the plasmid. By the end of the week we had progressed to the point of transforming it into competent <i>E. coli</i> cells, however a thick lawn of bacteria had grown overnight making it impossible to pick colonies. A streak plate was made from this plate to attempt to get less dense growth. Since the sequencing came back positive for <i>carAd</i> the next step was to perform site directed mutagenesis on its NotI restriction enzyme site using the primers that were designed previously (for site-directed mutagenesis optimization, please refer to Desulfurization Journal Week 14). The PCR was successful, showing very bright bands at 3000 bp, and the PCR product was digested with DpnI to remove parental DNA. Transformation of this product will indicate whether the mutagenesis was successful. We were also able to successfully perform a genomic prep of <i>Rhodococcus erythropolis</i> and amplify the <i>AmdA</i> gene this week. It was ligated into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> by Friday, and a confirmation colony PCR was run on Friday evening to verify that the gene was inserted. These results will be available by Monday. After many attempts the <i>ant</i> operon was finally ligated and transformed as well this week. Confirmation PCR showed bands of about 3000 bp which is consistent with the size of the operon. By Friday the successful colonies had been grown up and miniprepped, ready to send for sequencing early next week. The <i>carAc</i>/B0034 construct was also successfully transformed this week and verified by running a PCR using the B0034 forward primer. It was also miniprepped and is ready to send for sequencing next week. </p><br />
<p>In addition to this work constructing biobricks we also started an experiment to demonstrate the function of the pCAR plasmid in LD2. Strains of LD2 were grown up in LB and then washed and placed in B-N media + glucose and various experimental additions. These additions included carbazole, 4-PBAH (a nitrogen containing naphthenic acid), ammonia chloride, and a negative control with no nitrogen source. Strains of <i>E. coli</i> were also grown under the same conditions to compare growth and ammonia production of LD2 to a strain that does not contain the pCAR plasmid. These results should act as a preview of what we expect our final biobrick containing <i>E. col</i>i species to produce as well as allowing us to establish LD2 as a positive control for this experiment. </p><br />
<br />
<br />
</html>[[File:08.07.2012 amdA pcr from genomic prep.jpg|500px|thumb|<i>AmdA</i> amplified from the genomic prep of <i>R. Erythropolis</i> shows bands just greater than 1500 base pairs in lanes 2,4-6,9, and 12-13. Lane 14 was a negative control. The expected size of <i>AmdA</i> is 1566 base pairs.|center]]<br />
[[File:08.09.2012 confirmation pcr of ant operon.jpg|500px|thumb|The two labelled lanes show amplification at 3000 base pairs from a confirmation colony PCR using the biobrick primers. The expected size of the <i>ant</i> operon with the biobrick bookends is about 3100 base pairs.|center]]<br />
[[File:08.10.2012 carad notI mutagenesis.jpg|500px|thumb|Strong amplification at just above 3000 base pairs from the <i>carAd</i> PCR using mutagenic primers. Since PSB1C3 is 2070 base pairs and <i>carAd</i> is 989 base pairs the amplification should run at about 3059 base pairs. 3 different amounts of template DNA were tested in lanes 2-7 as indicated and a positive control showed weaker amplification in lanes 8-9 at about 4000 base pairs.|center]]<br />
<html><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>The sequencing for the <i>carAc</i>/<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> construct came back with only <i>carAc</i> and no evidence of a <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> site preceding it. The confirmation PCR that was run last week using the <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> forward primer seemed to indicate that it was inserted, however since <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> is only 12 base pairs the primer may not have been specific enough. The construction on this part was restarted this week. <i>carAa</i> colony PCR on last week's transformation showed two positive colonies, however upon culturing, miniprepping, and digesting these colonies the plasmid appeared to be too large (3000 base pairs as opposed to the expected 2000). This may be due to a problem of a mislabeled plasmid tube that has been experienced by other team members, causing the gene to be inserted into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a> rather than <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. To test this, a streak plate was made from these colonies and grown on an ampicillin resistant plate. Mutagenized <i>carAd</i> was successfully transformed into Top 10 <i>E. coli</i> cells, cultured, and miniprepped. The miniprep product was digested with NotI to test whether the NotI site was successfully removed. These results were good, as the plasmid ran at 2000 base pairs and the insert at just under 1000 base pairs in the mutagenized <i>carAd</i> whereas the non-mutagenized <i>carAd</i> had its insert cut into 2 bands. This will be sent for sequencing next week to confirm the mutation. Unfortunately the sequencing results came back negative for the <i>ant</i> operon meaning we will need to restart genomic PCR on it next week. Multiple transformations and confirmation PCRs were run on <i>AmdA</i> this week, however none displayed a band size of 1700-1800 base pairs as expected. We have continued to try different digested plasmids as other team members also had difficulties this week with ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, possibly indicating a problem with our Antarctic Phosphatase stock.</p><br />
<p>The other news from this week was the final results from our carbazole degradation experiment attempting to demonstrate LD2's ability to degrade carbazole and hopefully other nitrogen containing compounds. Unfortunately, these results were very difficult to make sense of as the LD2 that were cultured with carbazole grew, but did not show elevated ammonia levels which would be indicative of carbazole degradation. One of our controls was LD2 cultured with ammonium chloride to test whether LD2 was capable of using ammonia as a nitrogen source. This would pose a problem for us as we were planning to use an increase in ammonia levels as a proxy for carbazole degradation rather than measuring carbazole degradation directly using GC-MS. However, the LD2 showed significantly less ammonia after 44 hours when compared to 26 hours indicating that it may be degraded, either by the LD2 or by an unaccounted for abiotic process. This has lead us to decide to use GC-MS and directly measure carbazole levels in our sample the next time we do this experiment.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<p>The <i>carAa</i> was re-streaked onto ampicillin plates and grew, probably showing that it was ligated into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a>, not <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, which explained the irregularity of our confirmation digest last week. It was sent for sequencing, but in case it was incorrect we also started a new <i>carAa</i> pipeline by PCRing it from the <i>Pseudomonas</i> genome again and redigesting and ligating. It was transformed into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> over the weekend. <i>carAc</i> did not see a lot of progress this week as we spent much of the time attempting to construct a RBS (B0034) in front of it, but were unsuccessful. <i>carAd</i> was also subjected to this construction with B0034 and we were able to send it for sequencing by the end of the week. Several new constructs were also started for <i>carAd</i> in case the sequencing is negative. The <i>ant</i> operon was successfully transformed and confirmed by colony PCR this week, after which it was grown up and sent for sequencing. We also began to attempt mutagenesis on one of the EcoRI sites in the operon, but had no amplification of the plasmid. We also re-digested some PCR purified product to start another ligation in case sequencing failed. After many attempts we were also able to finally successfully transform an <i>AmdA</i>/PSB1C3 plasmid into Top 10 cells this week. After a miniprep they showed bands of about 1800 base pairs on the confirmation digest gel, so they were also sent for sequencing this week and had mutagenesis started on the illegal SpeI site. Unfortunately, the sequencing sent early in the week came back with ambiguous results. The forward reaction for<i> antABC</i> was a match, but its reverse reaction matched to <i>carAa</i>. Both <i>carAa</i> reactions matched nothing. Since these two genes are over 20 000 base pairs apart on the template plasmid it is virtually impossible that they were actually combined into one plasmid (PCR extension time would not have been long enough and even if it was the product would have run much above the 3000 base pair mark). This is possibly a mistake on Eurofins end, so we are not 100% sure if we have 1 good gene, both of them, or neither. Also the sequence for mutagenized <i>carAd</i> showed only about the first 100 base pairs, which were correct however not long enough meaning it was a poor reaction and inconclusive. Finally, we also re-started our <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/carbazole">carbazole degradation assay</a> using the GC-MS to quantitatively measure the loss of carbazole at biological sciences. This process will take 2 weeks to demonstrate LD2's ability to degrade carbazole. We are forcing it to use carbazole as a carbon and a nitrogen source in one sample and as only a nitrogen source in another sample (this sample has had glucose added to it).</p><br />
<br />
</html>[[File:AmdA colonyPCR calgary12.JPG|500px|thumb|Amplification is seen in lane 7 of this PCR of top 10 colonies into which AmdA/PSB1C3 had been transformed. This indicates that <i>AmdA</i> has been successfully inserted into a biobrick vector.|center]]<br />
[[File:NewcarAagenomePCR calgary12.jpg|500px|thumb|<i>carAa</i> amplified from pCAR1.2 plasmid of <i>Pseudomonas putida</i>.|center]] <html><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<p>The new stock of <i>carAa</i> that we had PCRed from LD2 was sent for sequencing this week and came back as a match, so we have begun constructing a RBS site and a TetR promoter (<a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>) in front of it. At this point all the <i>car</i> genes have been successfully amplified, inserted into a biobrick plasmid, and sequence verified. We also continued to attempt to construct <i>carAc</i> with an RBS site (<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>) in front of it, but had no success. On a better note, we confirmed this week that<i> carAd</i> was mutated properly and we were also able to insert an RBS site in front of it. The next step for this gene will be to attempt a plasmid switch as it is currently in <a href="http://partsregistry.org/Part:pSB1A3">pSB1A3</a> and needs to be in <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> for submission to the parts registry. After sending a new batch of the ant operon for sequencing we got the same confusing results as we did last week. We therefore hypothesized that our primers were annealing to a homologous region that is similar to the<i> ant</i> genes and very close to <i>carAa</i>. This means that we need to restart PCR on this gene as all of our current product is incorrect. The sequencing results for <i>AmdA</i> that came back this week were also somewhat confusing as they did not quite match the sequence we expected. However, they were a 100% match for the <i>AmdA</i> gene in a different strain of <i>Rhodococcus erythropolis</i>. This means that DSMZ probably sent us the wrong strain of bacteria and that we were very lucky that the primers we designed based off of the expected template actually worked to amplify a slightly different gene in another strain. The good news is that this new sequence contains no illegal cut sites, which explains why mutagenesis was not working (there was nothing to mutate) and allows us to immediately begin construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> in front of <i>AmdA</i> this week. </p><br />
<br />
</html>[[File:Caraa colonyPCR calgary12.JPG|500px|thumb|Colony PCR amplification from top 10 <i>E. coli</i> cells with a PSB1C3/<i>carAa</i> construct transformed into them. Lanes 1-3, 5, and 7 were sent for sequencing with lane 5 coming back positive.|center]]<html><br />
<br />
<h2>Week 19 (September 3- September 7)</h2><br />
<p><i>carAd</i>/<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> was successfully plasmid switched into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, making it ready for submission. Construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> and <i>carAa</i> may have been successful as the digest of the miniprep looked promising. It will be sent for sequencing next week. Unfortunately there was no progress made on the constructions of <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> or <i>carAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. These will be redigested and religated for next week. We could not amplify the <i>ant</i> operon despite trying many variations of PCR using high fidelity KAPA polymerase. This is probably due to the multiple, large regions of homology on the operon that the reverse primer binds to making amplification difficult. Because the function of this gene can theoretically be replaced by <i>AmdA</i>, we may scale back efforts dedicated to it, as the chances of successful amplification appear slim. </p><br />
<br />
<h2>Weeks 20-22 (September 10 - September 30)</h2><br />
<p>Constructs that we continued to work on this week included <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, <i>carAa</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, and <i>carAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. Some sequencing was sent, but no positive results were obtained. A new approach to biobrick the <i>ant</i> operon was implemented this week. A new forward primer was designed that would anneal about 1500 base pairs upstream of the <i>ant</i> operon, avoiding the region of homology that is repeated at various points on the plasmid. Once this region is successfully amplified we could use this amplified DNA as a template for another PCR reaction which would use the normal <i>ant</i> primers. Since the template would no longer include regions of homology these primers should be free to work properly and amplify the <i>ant</i> operon. The PCR using the new primers was started this week, and although amplification at 4500 base pairs was observed as expected, there was also a lot of non-specific amplification. This means that to purify the amplified DNA that we want and avoid the other products a dreaded gel extraction had to be performed which, as usual, did not work. At this point it was decided that further attempts to amplify the <i>ant</i> operon would be futile and our efforts would be better served working on constructing the parts we do have. Unfortunately no progress was made in these weeks on construction. We have also obtained GC-MS readings from the carbazole degradation experiment that showed that <i>Pseudomonas</i> LD2 can indeed degrade carbazole, however only under low glucose conditions.</p><br />
<br />
<br />
<br />
<br />
</html><br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/DenitrogenationTeam:Calgary/Notebook/Denitrogenation2012-10-04T00:26:40Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Denitrogenation Journal|<br />
CONTENT=<br />
<html><br />
<br />
<h2>Week 1 (May 1-4)</h2><br />
<p>The following section covers wetlab aspect of our overall project focusing on microbial conversion of naphthenic acids into economically-valuable hydrocarbons. The approach taken in this endeavour will be from four strategic starting points - ring cleavage, decarboxylation, denitrogenation, and desulfurization. Overall, the 'hydrocarbons' aspect of the project is a critical one to our overall design and construction of a bioreactor capable of not only detecting but also converting naphthenic acids and other toxins to useful or at least innocuous compounds. </p><br />
<br />
<h2>Week 2-3 (May 7-18)</h2><br />
<p>In the first two weeks of iGEM our group has focused on reviewing literature regarding the bioremediation of nitrogen groups attached to naphthenic acids. The most prevalent N heterocycle is carbazole, representing 75% of total nitrogen by mass. The upper pathway of carbazole biodegradation is catalyzed by the enzymes coded for by the <i>car</i> operon, <i>CarA</i> (<i>CarAaAcAd</i>), <i>CarB</i> (<i>CarBaBb</i>), and <i>CarC</i>. These enzymes convert carbazole to anthralinic acid. The lower pathway is catalyzed by the enzymes of the <i>ant</i> operon, <i>antA, B, and C</i>, yielding catechol while releasing CO2 and NH3. The <i>car</i> and <i>ant</i> operons are both regulated by the P<sup>ant</sup> promoter which is induced by the protein, antR. <i>carAa</i> also has its own promoter which is not induced by <i>antR</i> (Diaz & Garcia, 2010). We have also investigated an alternative pathway using <i>CarA</i> combined with an amidase (<i>amdA</i>) that selectively cleaves NH2 from an intermediate of the car pathway (Xu et al, 2006). This could bypass much of the car/ant pathway and is possibly more efficient.</p><br />
<p>We have decided to use <i>Pseudomonas resinovorans</i> and <i>Rhodococcus erythropolis</i> to derive these genes from. <i>carABC</i> and <i>antABC</i> from<i> P. resinovorans</i> has been shown to have a wide range of nitrogen containing substrate specificity. <i>R. erythropolis</i> contains the <i>amdA</i> gene that we wish to use, and some evidence suggests that it may also be able to degrade sulfur rings through its <i>carABC</i> pathway.</p><br />
<br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p>This week we reviewed the primers listed in the database and also designed some new ones. Primers for <i>carAa</i>,<i> carAc</i>, and <i>carAd</i> were designed individually, while primers for <i>CarBaBbC</i> and <i>antABC</i> were designed to encompass multiple genes in a sequence. These primers were designed to be used on <i>Pseudomonas putida</i> which we decided to use as our gene source since it was available to us as opposed to ordering <i>Pseudomonas resinovorans</i>. We also designed a primer for the<i> AmdA</i> gene from <i>Rhodococcus erthyroplois</i>. In addition to ordering these primers we also ordered the nitrogen containing compounds that we will need to test these enzymes on. We decided on using carbazole to make sure the enzymes can perform their natural function as well as pyrrolidine to test them on a similar ring structure. We also ordered cyclohexamine in order to independently test the function of the alternative <i>AmdA</i> pathway. Finally, we decided to eventually order 4-Piperidine butyric acid hydrochloride to test how the enzymes will work on nitrogen containing naphthenic acids. However, we decided since it is a very expensive compound we would wait to make sure the enzyme's work on more simple compounds before ordering it.</p><br />
<p>We also started our work in the wet lab by plating colonies and making an overnight culture of -80 freezer stock <i>Psuedomonas putida</i> on Thursday. We also resuspended the primers for <i>carAa</i>, <i>carAc</i>, <i>carAd</i>,<i> antA</i>, <i>antB</i>, and <i>ant C</i> that were already in the database. We were able to use the colonies that grew on the streak plates to start a colony PCR to attempt to isolate each of these genes on Friday.</p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2><br />
<p>On Monday and Tuesday we used the database primers to attempt to isolate <i>carAa</i>, <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i>antB</i>, and <i>antC</i> from the <i>Psuedomonas putida</i> we plated last week. <i>carAd</i>, <i>antB</i>, and <i>antC</i> were all put in the gradient PCR machine to account for the wide range of their primers’ melting points, while the others were done via regular PCR. Unfortunately, no positive results were obtained from these reactions. The PCR on <i>carAc</i> and <i>antA</i> was repeated on Wednesday, still not giving positive results. Finally, we attempted to use a salt concentration gradient on the PCR reaction for <i>carAc</i> and <i>antA</i>, using concentrations that ranged from 1.0 microlitres/tube to 2.0 microlitres/tube in increments of 0.2 microlitres/tube. This helped as <i>carAc</i> showed bands in samples that had concentrations of 1.2 and 1.4 microlitres/tube. <i> antA</i> also showed bands, however they were not the correct size, indicating contamination and/or non-specific annealing of the primer. The positive control also showed bands of the correct size. Earlier in the week we also made overnight cultures of 3 environmental strains (28, 29, 30) of <i>Psuedomonas putida</i> from -80 glycerol stock. On Friday we performed a genomic prep on these cultures, and plan on attempting to amplify genes from the isolated DNA next week. </p><br />
<br />
</html>[[File:CarAa and carAc Ucalgary12.png|500px|thumb|Gel of PCR using database primers for <i>carAa</i> and <i>carAc</i> on <i>Pseudomonas putida</i>|center]]<html><br />
<br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> We started off this week by determining the DNA concentration of our genomic prep samples from last week using the nanodrop. DNA concentration for all three<i> P. putida</i> strains was at least 1000 ng/microlitre, well above what was needed for PCR. 1/2 and 1/3 dilutions were prepared for all three strains so as not to have an excess of template DNA in PCR reactions. PCR was performed on all 3 strains using primers for <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i> antB</i>, and <i>antC</i> using 6 replicates per gene. The only successful amplification appeared to be <i>antB</i> and <i>carAc</i>, both from strain 28 (with weaker bands in strain 29). We then performed another PCR, just on those two genes with an increased amount of Taq polymerase to hopefully get enough amplified DNA to move forward with. This resulted in strong bands for both at the expected size. We then performed PCR purification using the Qiaquick kit and obtained samples containing 33.5 ng/microlitre of <i>antB</i> DNA and 129 ng/microlitre of <i>carAc </i>DNA. These concentrations were both sufficient to begin a restriction digest and ligation of these parts into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. Next week we hope to verify the results of the restriction digest, continue to amplify <i>carAc</i> and <i>antB</i> from strain 28, and hopefully submit a biobrick for sequencing.</p><br />
<br />
</html>[[File:AntB and carAc Ucalgary.png|500px|thumb|Gel of PCR ran on genomic prep of <i>Pseudomonas putida</i> environmental strain 28 using primers for <i>carAc</i> and <i>antB</i>. |center]]<html><br />
<br />
<br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p>The purified PCR products for <i>carAc</i> and <i>antB</i> were digested with restriction enzymes EcoRI+SpeI, EcoRI+PstI, and XbaI+PstI. However, only <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmids that had been digested with EcoRI+SpeI and EcoRI+PstI were available to attempt ligation. Gel results were inconclusive on the restriction digest product as the plasmid size appeared much larger than expected. However, transformation was still attempted on the ligation products for both genes (both using EcoRI+SpeI ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid). The transformation products were plated onto chloroamphenicol (chlor) plates to select for colonies that had the chloro resistance gene on the <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid. Also this week another round of PCR was performed for the <i> car</i> and <i> ant</i> genes, however all, but <i>carAc</i> showed bands in the negative control lane, possibly indicating contamination or the formation of primer dimers. The <i>carAc</i> bands were fairly weak and a PCR purification resulted in very low DNA concentration, insufficient to move onto restriction digest. Next week's plan hinges heavily on the result of the transformation from this Friday. </p><br />
<br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>The results of our transformation on <i>carAc</i> and <i>antB</i> from last week showed mostly red colonies indicating that the ligation was unsuccessful and that the PSB1C3 plasmid simply closed on itself with no insert. However, there were three colonies for <i>carAc</i> that were white (cut with both X+P and E+P). Colony PCR was performed on these three colonies using biobrick primer sets to verify that the part had been inserted in these colonies, but unfortunately all PCR results were negative indicating an unsuccessful ligation.</p><br />
<p>We also performed a miniprep on <i>Pseudomonas putida</i> strain #28 this week using a home-made protocol rather than the Qiagen kit. Miniprep A had a DNA concentration of 1539.8 ng/μL and Miniprep B had DNA concentration of 1001.2 ng/μL possibly indicating that there may have been a lot of genomic DNA contamination. However miniprep A product was still used as a PCR template for a reaction attempting to isolate <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i>antB</i>, and <i>antC</i>. Special conditions for this PCR included replacing 60 μL of water with 60 μL of betaine heated to 37 degrees Celsius and running a Mg gradient ranging from 0.5 μL - 2.5 μL per tube for each gene. Of these only <i>antB</i> had bands of the right size in 3 of the lanes (Mg concentrations of 1.5, 2, and 2.5 μL). However, all of the <i>antB</i> bands (including the negative control) contained a contamination band at around 100 bases, probably due to the formation of primer dimers. This forced us to do a gel extraction rather than a PCR purification. This gel extraction did not work as the nanodrop results indicated high amounts of agarose contamination. Another PCR was performed using the same conditions, but using miniprep B instead as there was no more miniprep A product remaining. Unfortunately there were no bands for <i>antB</i>, only the 100 base pair contamination bands. </p><br />
<br />
</html>[[File:AntA and antC Ucalgary.png|500px|thumb|Gel of PCR ran using miniprep product A of <i>Pseudomonas putida</i> as template DNA in an attempt to isolate <i>antA</i> and <i>antC</i> genes.|center]]<br />
[[File:CarAc and carAd Ucalgary.png|500px|thumb|Gel of PCR ran using miniprep product A of <i>Pseudomonas putida</i> as template in an attempt to isolate <i>carAc</i> and <i>carAd</i> genes.|center]]<br />
[[File:Minipret A antB and carAc Ucalgary.png|500px|thumb|Gel of PCR ran using miniprep product A of <i>Pseudomonas putida</i> showing bands 3-5 of the correct size corresponding to antB genes and colony PCR ran using biobrick primers attempting to determine if <i>carAc</i> genes were successfully transformed into PSB1C3 plasmid.|center]] <html><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>This week we modified our PCR protocol to adjust for the homemade Taq polymerase we were using. Template DNA was boiled for 10 minutes on its own to replace the initialization step. This is because this polymerase could not tolerate the 95 degree heat during the regular initialization step. Another modification we made was to boil our primers for 5 minutes prior to adding them to the master mix. This was done to discourage the formation of primer-dimers. Finally we also attempted a touch-down PCR which started at an annealing temperature greater than the Tm of our primers and gradually worked its way down to below the Tm of the primers. This was done to increase the specificity of our reaction and also eliminate the formation of primer-dimers. Unfortunately these techniques still saw the formation of dimers, preventing us from performing a PCR purification. Again, the gel purification procedure did not lead to successful purification of our target gene. Due to the problems we have had so far our goal for next week is to perform an assay experiment to try and determine which of the <i>Pseudomonas putida</i> strains we have access to can actually degrade carbazole and therefore contain the pCAR1 plasmid. </p><br />
<br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The goal for this week was to grow different strains of<i> Pseudomonas</i> in media containing B-N media + glucose with only carbazole as a source of nitrogen. In theory only those strains which contained the pCAR1 plasmid would be able to use carbazole as a nitrogen source and thrive in this media. The strains we tested from glycerol stock included <i>P. putida</i> LD1, <i>P.putida</i> environmental strains 28-30, and <i>P. fluorescens</i> PF5. Each of these strains had two cultures grown; one with carbazole and one without it. <i>E. coli</i> grown with carbazole was used as a negative control as it does not contain the pCAR plasmid. At timepoints of 22, 46, and 117 hours of growth samples were taken to do an OD600 reading to measure bacterial growth and had an ammonia assay performed on it. The detection of ammonia acted as a proxy for detecting carbazole degradation as it is an end-product of the pathway. With this in mind, an additional control strain of LD1 was grown in media containing glucose and ammonia. This is to detect the possibility that <i>Pseudomonas</i> can use ammonia as a source of nitrogen after it has degraded carbazole which would be problematic as far as using ammonia levels to determine carbazole degradation. So far we have been having difficulties reading the results of the ammonia assay with a plate reader and may resort to testing our samples using a different assay protocol next week. The OD600 readings were initially very high at 22 hours and then dropped at 46 hours. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>This week we took our last readings from the experiment that we started last week. The OD600 readings taken at 117 hours showed minimal growth over the weekend for all strains except for environmental strain #29 (with carbazole) which increased from 0.086 to 0.313. No other strain showed an increase of 0.100 or greater. This shows that only strain #29 was able to thrive in the carbazole media, possibly indicating that it contains the pCAR plasmid enabling it to use carbazole as a nitrogen source. To verify this we used a new ammonia assay protocol to measure the production of ammonia in the different cultures and theoretically how much carbazole was degraded. Unlike the previous assay we were able to produce a standard curve with a consistent slope based on samples with known amounts of ammonia. However, the only samples that showed any elevation of ammonia levels above the blank were the LD1 samples that were cultured with ammonia chloride to control for the possibility of pseudomonas using ammonia as a nitrogen source. The ammonia levels in this sample were relatively constant which would indicate that the LD1 was not using ammonia as a nitrogen source. Unfortunately the fact that none of the test samples had elevated ammonia levels did not give us any evidence that these strains contain the pCAR plasmid. </p><br />
<br />
</html>[[File: Standard_curve_for_ammonia_assay_UCalgary2012_denitrification_group.png|500px|thumb|Standard curve for ammonia assay using OD635 absorbance.|center|]]<html><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>We received a new strain of Pseudomonas, LD2, this week that had previously been used to study the pCAR1 plasmid. It was grown up in PCA media and had its colonies used for colony PCR. We also received our new primers this week, further increasing the chances of successful PCR. Using the new strain and the new primers <i>carAa</i>, <i>carAc</i>, and <i>carAd</i> all showed at least some bands of close to the correct size. After PCR purification we obtained DNA concentrations of 21.3 ng/μL for <i>carAa</i>, 41.9 ng/ μL for <i>carAc</i>, and 32.8 ng/μL for<i> carAd</i>. These products were then restriction digested using EcoRI + PstI sites. </p><br />
<br />
</html>[[File:07.20.2012 CarAa and CarAc.jpg|500px|thumb|Gel of colony PCR from <i>Pseudomonas sp. LD2</i> for <i>carAa</i> and <i>carAc</i> genes. <i>carAa</i> expected size is 1155 bp and <i>carAc</i> is 323 bp.|center]]<br />
[[File:07.20.2012 CarAd colony PCR from LD2.jpg|500px|thumb|Gel of colony PCR from <i>Pseudomonas sp. LD2</i> for <i>carAd</i> gene. <i>carAd</i> expected size is 989 bp.|center]] <html><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The restriction digested <i>car</i> genes were ligated into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> cells. White colonies grew for<i> carAa</i> and <i>carAc</i> suggesting that ligation and transformation were successful, however no colonies grew for <i>carAd</i> suggesting that the transformation did not work. Colony PCR was performed to confirm the presence of these genes within the vectors, however only <i>carAc</i> was positive. The colonies containing the <i>carAc</i> plasmid were then mini prepped and restriction digested to isolate the plasmid and send for sequencing. Further attempts at transforming<i> carAa</i> and <i>carAd</i> were not successful. Another goal was to perform colony PCR on the <i>P. putida</i> to isolate the<i> antABC</i> genes using both the primers for the entire operon and those for the individual genes. No attempt showed any bands, although salt and temperature gradients were used as well as different types of polymerase. LD2 cultures were grown up to be plasmid prepped and genomic prepped in order to have a higher concentration of template DNA in the PCR master mix and more PCR will be attempted on these genes next week. Mutagenesis primers were also designed this week to remove the illegal restriction cut sites in the<i> carAd</i> and <i>antB</i> genes (NotI and 2xEcoRI respectively). Only 1 mutagenesis primer was designed for <i>antB</i> as one of its EcoRI sites was very close to the beginning of the gene and could be removed using a new primer. During this process it was also discovered that the original ant primers were designed for the wrong genes and therefore do not match the intended sequence. Although the new <i>antABC</i> primers were designed using the correct gene the PCR may not be working due to the length of the operon so the design of new individual primers may be necessary. Finally, we also aimed to grow up the freeze dried culture of <i>Rhodococcus erythropolis</i> (source of the<i> AmdA</i> gene) that we received from DSMZ. The reactivation media was produced and autoclaved to begin the process next week.</p><br />
<br />
</html>[[File:07.23.2012.Restriction Digest of Car genes.jpg|500px|thumb|Gel of restriction digest of car genes. A refers to <i>carAa</i>, C to <i>carAc</i>, and D to <i>carAd</i>. Ladder shows that after purification and digestion DNA is still the correct size.|center]]<br />
[[File:CarAc biobrick confirmation PCR.jpg|500px|thumb|Gel of confirmation PCR done on competent Top 10 <i>E. coli</i> cells in which a PSB1C3 vector containing the <i>carAc</i> gene had been inserted. Red X marks the 500 bp mark on the weak ladder showing the product to be the expected size (323 bp gene + 2*100 bp biobrick sites on each end).|center]] <html><br />
<br />
<h2>Week 14 (July 30 - August 3)</h2><br />
<p>We received the sequencing results this week for <i>carAc</i> which came back as a 100% match to the expected nucleotide sequence. We also successfully ligated both<i> carAa</i> and<i> carAd</i> into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into top 10 <i>E. coli</i> cells. Colony PCR verified the presence of the biobrick plasmid in these colonies. These were both miniprepped, digested and sent for sequencing by the end of the week. Using a salt gradient, addition of DMSO, and the KAPA buffer specifically designed for GC-rich amplicons we were able to amplify the ant operon from a genomic prep of LD2. However, all attempts at ligating and transforming this sequence were met with the unfortunate presence of red colonies (indicating failed insertion into the plasmid). Next week we will recut a <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> vector with Xba and Pst to attempt to mitigate this problem. We also reactivated the <i>R. erythropolis</i> following the DSMZ protocol for reviving freeze-dried cultures that can be found <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/dmszfreezedry">here</a> using DSMZ media #1 and grew it on plates made from DSMZ media #220 at 28 degrees. Colonies grew very quickly and were used for colony PCR and to make overnight cultures for downstream applications. The colony PCR using the amidase primers was unsuccessful, so a genomic prep was performed on the overnight culture. Unfortunately we lacked some of the required equipment and the genomic prep did not work, but with a few procedural modifications and the right equipment we are confident it will work next week. A glycerol stock of the<i> R. erythropoplis</i> was also made from another overnight culture. We also started construction on the <i>carAc </i>part this week by inserting a ribosome binding site (B0034) in front of the gene. Finally, since the<i> ant</i> operon was successfully amplified by PCR we designed mutagenesis primers for the 2nd EcoRI site in the operon rather than making new individual primers. We also designed a mutagenesis primer to remove the SpeI site in <i>AmdA</i>. Now that we have conclusively proven that the LD2 strain contains the pCAR1 plasmid we aim to demonstrate its ability to degrade carbazole and other nitrogen containing heterocycles using an assay experiment similar to the one performed in Week 10.</p><br />
<br />
</html>[[File:07.30.2012. CarAa, CarAd.jpg|500px|thumb|<i>carAa</i> and <i>carAd</i> amplified from genomic prep of <i>Pseudomonas sp. LD2</i>. Amplicons are larger than expected, potentially the gel ran diagonally. The 6th lane for each gene was a negative control.|center]]<br />
[[File:07.31.2012.Ant operon.jpg|500px|thumb|<i>ant</i> operon amplified from genomic prep of <i>Pseudomonas sp. LD2</i> at the expected size of 3000 bp.|center]]<br />
[[File:08.01.2012 CarAdBB.jpg|500px|thumb|Lanes 2,4,5, and 11 contain <i>carAd</i> genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent top 10 <i>E.Coli</i>.|center]]<br />
[[File:08.01.2012 CarAaBB.jpg|500px|thumb|Lane 9 contains <i>carAa</i> genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent top 10 <i>E.Coli</i>. Although larger than expected, the positive control RFP generator in lane 10 is also larger than its expected size (~1200 bp including biobrick bookends) indicating that the gel may have a slant in it.|center]]<html><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p>This week we received the sequencing results for <i>carAa</i> and <i>carAd</i>. <i>carAd</i> was a match, however <i>carAa</i> did not match at all and had misplaced biobrick restriction enzyme sites. Due to this we decided to restart the biobricking process for <i>carAa</i> all the way back to PCR from the plasmid. By the end of the week we had progressed to the point of transforming it into competent <i>E. coli</i> cells, however a thick lawn of bacteria had grown overnight making it impossible to pick colonies. A streak plate was made from this plate to attempt to get less dense growth. Since the sequencing came back positive for <i>carAd</i> the next step was to perform site directed mutagenesis on its NotI restriction enzyme site using the primers that were designed previously (for site-directed mutagenesis optimization, please refer to Desulfurization Journal Week 14). The PCR was successful, showing very bright bands at 3000 bp, and the PCR product was digested with DpnI to remove parental DNA. Transformation of this product will indicate whether the mutagenesis was successful. We were also able to successfully perform a genomic prep of <i>Rhodococcus erythropolis</i> and amplify the <i>AmdA</i> gene this week. It was ligated into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> by Friday, and a confirmation colony PCR was run on Friday evening to verify that the gene was inserted. These results will be available by Monday. After many attempts the <i>ant</i> operon was finally ligated and transformed as well this week. Confirmation PCR showed bands of about 3000 bp which is consistent with the size of the operon. By Friday the successful colonies had been grown up and miniprepped, ready to send for sequencing early next week. The <i>carAc</i>/B0034 construct was also successfully transformed this week and verified by running a PCR using the B0034 forward primer. It was also miniprepped and is ready to send for sequencing next week. </p><br />
<p>In addition to this work constructing biobricks we also started an experiment to demonstrate the function of the pCAR plasmid in LD2. Strains of LD2 were grown up in LB and then washed and placed in B-N media + glucose and various experimental additions. These additions included carbazole, 4-PBAH (a nitrogen containing naphthenic acid), ammonia chloride, and a negative control with no nitrogen source. Strains of <i>E. coli</i> were also grown under the same conditions to compare growth and ammonia production of LD2 to a strain that does not contain the pCAR plasmid. These results should act as a preview of what we expect our final biobrick containing <i>E. col</i>i species to produce as well as allowing us to establish LD2 as a positive control for this experiment. </p><br />
<br />
<br />
</html>[[File:08.07.2012 amdA pcr from genomic prep.jpg|500px|thumb|<i>AmdA</i> amplified from the genomic prep of <i>R. Erythropolis</i> shows bands just greater than 1500 base pairs in lanes 2,4-6,9, and 12-13. Lane 14 was a negative control. The expected size of <i>AmdA</i> is 1566 base pairs.|center]]<br />
[[File:08.09.2012 confirmation pcr of ant operon.jpg|500px|thumb|The two labelled lanes show amplification at 3000 base pairs from a confirmation colony PCR using the biobrick primers. The expected size of the <i>ant</i> operon with the biobrick bookends is about 3100 base pairs.|center]]<br />
[[File:08.10.2012 carad notI mutagenesis.jpg|500px|thumb|Strong amplification at just above 3000 base pairs from the <i>carAd</i> PCR using mutagenic primers. Since PSB1C3 is 2070 base pairs and <i>carAd</i> is 989 base pairs the amplification should run at about 3059 base pairs. 3 different amounts of template DNA were tested in lanes 2-7 as indicated and a positive control showed weaker amplification in lanes 8-9 at about 4000 base pairs.|center]]<br />
<html><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>The sequencing for the <i>carAc</i>/<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> construct came back with only <i>carAc</i> and no evidence of a <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> site preceding it. The confirmation PCR that was run last week using the <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> forward primer seemed to indicate that it was inserted, however since <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> is only 12 base pairs the primer may not have been specific enough. The construction on this part was restarted this week. <i>carAa</i> colony PCR on last week's transformation showed two positive colonies, however upon culturing, miniprepping, and digesting these colonies the plasmid appeared to be too large (3000 base pairs as opposed to the expected 2000). This may be due to a problem of a mislabeled plasmid tube that has been experienced by other team members, causing the gene to be inserted into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a> rather than <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. To test this, a streak plate was made from these colonies and grown on an ampicillin resistant plate. Mutagenized <i>carAd</i> was successfully transformed into Top 10 <i>E. coli</i> cells, cultured, and miniprepped. The miniprep product was digested with NotI to test whether the NotI site was successfully removed. These results were good, as the plasmid ran at 2000 base pairs and the insert at just under 1000 base pairs in the mutagenized <i>carAd</i> whereas the non-mutagenized <i>carAd</i> had its insert cut into 2 bands. This will be sent for sequencing next week to confirm the mutation. Unfortunately the sequencing results came back negative for the <i>ant</i> operon meaning we will need to restart genomic PCR on it next week. Multiple transformations and confirmation PCRs were run on <i>AmdA</i> this week, however none displayed a band size of 1700-1800 base pairs as expected. We have continued to try different digested plasmids as other team members also had difficulties this week with ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, possibly indicating a problem with our Antarctic Phosphatase stock.</p><br />
<p>The other news from this week was the final results from our carbazole degradation experiment attempting to demonstrate LD2's ability to degrade carbazole and hopefully other nitrogen containing compounds. Unfortunately, these results were very difficult to make sense of as the LD2 that were cultured with carbazole grew, but did not show elevated ammonia levels which would be indicative of carbazole degradation. One of our controls was LD2 cultured with ammonium chloride to test whether LD2 was capable of using ammonia as a nitrogen source. This would pose a problem for us as we were planning to use an increase in ammonia levels as a proxy for carbazole degradation rather than measuring carbazole degradation directly using GC-MS. However, the LD2 showed significantly less ammonia after 44 hours when compared to 26 hours indicating that it may be degraded, either by the LD2 or by an unaccounted for abiotic process. This has lead us to decide to use GC-MS and directly measure carbazole levels in our sample the next time we do this experiment.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<p>The <i>carAa</i> was re-streaked onto ampicillin plates and grew, probably showing that it was ligated into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a>, not <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, which explained the irregularity of our confirmation digest last week. It was sent for sequencing, but in case it was incorrect we also started a new <i>carAa</i> pipeline by PCRing it from the <i>Pseudomonas</i> genome again and redigesting and ligating. It was transformed into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> over the weekend. <i>carAc</i> did not see a lot of progress this week as we spent much of the time attempting to construct a RBS (B0034) in front of it, but were unsuccessful. <i>carAd</i> was also subjected to this construction with B0034 and we were able to send it for sequencing by the end of the week. Several new constructs were also started for <i>carAd</i> in case the sequencing is negative. The <i>ant</i> operon was successfully transformed and confirmed by colony PCR this week, after which it was grown up and sent for sequencing. We also began to attempt mutagenesis on one of the EcoRI sites in the operon, but had no amplification of the plasmid. We also re-digested some PCR purified product to start another ligation in case sequencing failed. After many attempts we were also able to finally successfully transform an <i>AmdA</i>/PSB1C3 plasmid into Top 10 cells this week. After a miniprep they showed bands of about 1800 base pairs on the confirmation digest gel, so they were also sent for sequencing this week and had mutagenesis started on the illegal SpeI site. Unfortunately, the sequencing sent early in the week came back with ambiguous results. The forward reaction for<i> antABC</i> was a match, but its reverse reaction matched to <i>carAa</i>. Both <i>carAa</i> reactions matched nothing. Since these two genes are over 20 000 base pairs apart on the template plasmid it is virtually impossible that they were actually combined into one plasmid (PCR extension time would not have been long enough and even if it was the product would have run much above the 3000 base pair mark). This is possibly a mistake on Eurofins end, so we are not 100% sure if we have 1 good gene, both of them, or neither. Also the sequence for mutagenized <i>carAd</i> showed only about the first 100 base pairs, which were correct however not long enough meaning it was a poor reaction and inconclusive. Finally, we also re-started our <a href=https://2012.igem.org/Team:Calgary/Notebook/Protocols/carbazole>carbazole degradation assay</a> using the GC-MS to quantitatively measure the loss of carbazole at biological sciences. This process will take 2 weeks to demonstrate LD2's ability to degrade carbazole. We are forcing it to use carbazole as a carbon and a nitrogen source in one sample and as only a nitrogen source in another sample (this sample has had glucose added to it).</p><br />
<br />
</html>[[File:AmdA colonyPCR calgary12.JPG|500px|thumb|Amplification is seen in lane 7 of this PCR of top 10 colonies into which AmdA/PSB1C3 had been transformed. This indicates that <i>AmdA</i> has been successfully inserted into a biobrick vector.|center]]<br />
[[File:NewcarAagenomePCR calgary12.jpg|500px|thumb|<i>carAa</i> amplified from pCAR1.2 plasmid of <i>Pseudomonas putida</i>.|center]] <html><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<p>The new stock of <i>carAa</i> that we had PCRed from LD2 was sent for sequencing this week and came back as a match, so we have begun constructing a RBS site and a TetR promoter (<a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>) in front of it. At this point all the <i>car</i> genes have been successfully amplified, inserted into a biobrick plasmid, and sequence verified. We also continued to attempt to construct <i>carAc</i> with an RBS site (<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>) in front of it, but had no success. On a better note, we confirmed this week that<i> carAd</i> was mutated properly and we were also able to insert an RBS site in front of it. The next step for this gene will be to attempt a plasmid switch as it is currently in <a href="http://partsregistry.org/Part:pSB1A3">pSB1A3</a> and needs to be in <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> for submission to the parts registry. After sending a new batch of the ant operon for sequencing we got the same confusing results as we did last week. We therefore hypothesized that our primers were annealing to a homologous region that is similar to the<i> ant</i> genes and very close to <i>carAa</i>. This means that we need to restart PCR on this gene as all of our current product is incorrect. The sequencing results for <i>AmdA</i> that came back this week were also somewhat confusing as they did not quite match the sequence we expected. However, they were a 100% match for the <i>AmdA</i> gene in a different strain of <i>Rhodococcus erythropolis</i>. This means that DSMZ probably sent us the wrong strain of bacteria and that we were very lucky that the primers we designed based off of the expected template actually worked to amplify a slightly different gene in another strain. The good news is that this new sequence contains no illegal cut sites, which explains why mutagenesis was not working (there was nothing to mutate) and allows us to immediately begin construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> in front of <i>AmdA</i> this week. </p><br />
<br />
</html>[[File:Caraa colonyPCR calgary12.JPG|500px|thumb|Colony PCR amplification from top 10 <i>E. coli</i> cells with a PSB1C3/<i>carAa</i> construct transformed into them. Lanes 1-3, 5, and 7 were sent for sequencing with lane 5 coming back positive.|center]]<html><br />
<br />
<h2>Week 19 (September 3- September 7)</h2><br />
<p><i>carAd</i>/<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> was successfully plasmid switched into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, making it ready for submission. Construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> and <i>carAa</i> may have been successful as the digest of the miniprep looked promising. It will be sent for sequencing next week. Unfortunately there was no progress made on the constructions of <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> or <i>carAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. These will be redigested and religated for next week. We could not amplify the <i>ant</i> operon despite trying many variations of PCR using high fidelity KAPA polymerase. This is probably due to the multiple, large regions of homology on the operon that the reverse primer binds to making amplification difficult. Because the function of this gene can theoretically be replaced by <i>AmdA</i>, we may scale back efforts dedicated to it, as the chances of successful amplification appear slim. </p><br />
<br />
<h2>Weeks 20-22 (September 10 - September 30)</h2><br />
<p>Constructs that we continued to work on this week included <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, <i>carAa</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, and <i>carAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. Some sequencing was sent, but no positive results were obtained. A new approach to biobrick the <i>ant</i> operon was implemented this week. A new forward primer was designed that would anneal about 1500 base pairs upstream of the <i>ant</i> operon, avoiding the region of homology that is repeated at various points on the plasmid. Once this region is successfully amplified we could use this amplified DNA as a template for another PCR reaction which would use the normal <i>ant</i> primers. Since the template would no longer include regions of homology these primers should be free to work properly and amplify the <i>ant</i> operon. The PCR using the new primers was started this week, and although amplification at 4500 base pairs was observed as expected, there was also a lot of non-specific amplification. This means that to purify the amplified DNA that we want and avoid the other products a dreaded gel extraction had to be performed which, as usual, did not work. At this point it was decided that further attempts to amplify the <i>ant</i> operon would be futile and our efforts would be better served working on constructing the parts we do have. Unfortunately no progress was made in these weeks on construction. We have also obtained GC-MS readings from the carbazole degradation experiment that showed that <i>Pseudomonas</i> LD2 can indeed degrade carbazole, however only under low glucose conditions.</p><br />
<br />
<br />
<br />
<br />
</html><br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Project/DataPageTeam:Calgary/Project/DataPage2012-10-04T00:23:39Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Detect and Destroy: Data Page|<br />
CONTENT=<br />
<html><br />
<head><br />
<style><br />
<br />
a.green{<br />
color: #159900 !important;<br />
}<br />
a.blue{<br />
color: #1088CC !important;<br />
}<br />
a.orange{<br />
color: #FF7A00 !important;<br />
}<br />
<br />
</style><br />
</head><br />
<br />
<body><br />
<div align="justify"><br />
<br />
<br />
<br />
</html>[[File:UCalgary2012_GraphicalAbstract.png|thumb|700px|center|thumb|Figure 1. The Calgary team has developed a dual system for the detection of toxic components in tailing ponds as well as the remediation of these compounds. Tailing ponds are large bodies of water containing waste products produced from the extraction of bitumen in the oil sands. Our biosensor involved the identification of a toxin promoter through a transposon screen. An electrochemical detector was implemented with a multiple output system allowing for the detection of multiple compounds simultaneously. This promoter/detector system was then complemented with the production of a biosensor prototype involving both a physical device and a software program for easy data analysis.<br />
<br />
Rather than just sensing toxins in the tailings ponds, a major objective was to detoxify the tailings through the reduction of toxins such as carboxylic acids (mainly naphthenic acids) and catechol, turning them into useable hydrocarbons. Purification of these hydrocarbons would contribute an added economic and industrial benefit. In order to house this system, we also aimed to design a bioreactor for our bacteria as well as optimize product output through a flux-variability based model. Finally, in order to create higher quality hydrocarbons, we explored desulfurization and denitrogenation pathways to upgrade our fuel. To do this in a safe and environmentally sound way, we designed not only structural containment mechanisms, but also genetic containment mechanisms through novel inducible ribo-killswitches.]]<html> <br />
<br />
<h2>Characterization of new parts submitted to the Registry</h2><br />
<br />
<ul><li><p>(<b><a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902000">BBa_K902000</a></b>) and (<b><a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902004">BBa_K902004, </a></b>): two novel hydrolase enzymes were submitted to the registry for the hydrolysis of two different sugar-conjugated electroactive compounds: PNPG and PDPG. Used in conjunction with the existing lacZ part in the registry (<b><a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_I732005">BBa_I732005</a></b>) which hydrolyzes CPRG, this allows for the electrochemical detection of three compounds with a single electrode. A <i>uidA</i> inducible generator (<b><a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_k902002">BBa_k902002,</a></b>) was submitted and characterized electrochemically. This data can be found on our <b><a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting </b></a> page.</li><br />
<br />
<li><p>(<b><a class="orange" href="http://partsregistry.org/wiki/index.php?title=BBa_K902008">BBa_K902008</a></b>),(<b><a href="http://partsregistry.org/wiki/index.php?title=BBa_K902023">BBa_K902023</a></b>) and (<b><a href="http://partsregistry.org/wiki/index.php?title=BBa_K902074">BBa_K902074</a></b>): three novel riboswitches sensitive to magnesium, mangenese and molybdate were submitted along with two associated promoters (<b><a href="http://partsregistry.org/wiki/index.php?title=BBa_K902009">BBa_K902009</a></b>) and <b><a href="http://partsregistry.org/wiki/index.php?title=BBa_K902073">BBa_K902073</a></b>) in addition to a rhamnose inducible, glucose repressible promoter (<b><a href="http://partsregistry.org/wiki/index.php?title=BBa_K902065">BBa_K902065</a></b>). One of these riboswitches (<b><a href="http://partsregistry.org/wiki/index.php?title=BBa_K902008">BBa_K902008</a></b>) was tested with GFP and a constitutive promoter using this construct, (<b><a href="http://partsregistry.org/wiki/index.php?title=BBa_K902021">BBa_K902021</a></b>), with its promoter and GFP using this construct (<b><a href="http://partsregistry.org/wiki/index.php?title=BBa_K902017">BBa_K902017</a></b>) and with its promoter and the S7 kill gene using this construct (<b><a href="http://partsregistry.org/wiki/index.php?title=BBa_K902018">BBa_K902018</a></b>). This data can be found on our killswitch <b><a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></b> page. </li></p><br />
<br />
<li><p>Genes for denitrogenation and desulfurization were biobricked and submitted. A novel oxidoreductase part (<b><a class="blue" href="http://partsregistry.org/wiki/index.php?title=BBa_K902058">BBa_K902058</a></b>) was also submitted and characterized for use in the desulfurization project. This data can be found on our upgrading <b><a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></b> page.</p></li></ul>. <br />
<br />
<h2>Further characterization of parts already present within the registry </h2><br />
<br />
<ul><li><p>The IPTG inducible lacI regulated promoter (<b><a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010">BBa_R0010</a></b>) was tested electorchemically to demonstrate its leakiness when not used in conjunction with strong expression of regulatory elements. This data can be found on our <b><a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</b></a> page.</li><br />
<li><p>(<b><a class="blue" href="http://partsregistry.org/wiki/index.php?title=BBa_K590025">BBa_K590025</a></b>), the PetroBrick, submitted by the Washington team in 2011, was characterized for a novel function: the conversion of naphthenic acids and 2-hydroxymuconate- a catechol break-down product from from the xylE gene (<b><a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J33204">BBa_J33204</a></b>) into hydrocarbons. This data can be found on both the <b><a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">decarboxylation</a></b> page and the <b><a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></b> page.</li><br />
<br />
<li><p> The output of (<b><a class="blue" href="http://partsregistry.org/wiki/index.php?title=BBa_K590025">BBa_K590025</a></b>) was also optimized thorugh a program we developed in MATLAB for the optimization of metabolic pathways in synthetic biology metabolic networks. The program allows you to build an artificial synthetic biology network in <i>E. coli</i> and predicts substrates that should be fed to the organism to increase production of the compound. This was characterized and validated in the wetlab with the Petrobrick. This data can be found on our <b><a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></b> page.</li><br />
<br />
<li><p> An existing <i>xylE</i> gene in the registry (<b><a class="blue" href= "http://partsregistry.org/wiki/index.php?title=BBa_J33204">BBa_J33204</a></b>) was constructed with a constitutive promoter instead of the glucose-repressible one that is available within the registry. This allows for increased output in media containing glucose, making it more suitable for a variety of applications such as our own. We validated the functionality of this part which can be found on our <b><a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Catechol Degradation</a></b> page.</p></li><br />
<br />
</ul><br />
<br />
<br />
<h2>Additional Work and Characterization </h2><br />
<br />
<ul><br />
<li><p>Developed and tested both hardware and software of a biosensor using an electrochemical sensor. The software is available on our wiki on our biosensor (<b>Robert can you help me flush this out a bit, or one of the engineers?</b></p></li><br />
<br />
<li><p> Characterization was done on one of our constitutively expressed transposon clones to test the lacZ gene electrochemically. This data can be found on our <b><a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</b></a> page.</p></li><br />
<li><p> Submitted novel parts involved in decarboxylation and validated the functionality of an additional enzyme (oleT), capable of converting fatty acids into alkenes by itself. This was done in its host organism. This data can be found in our <b><a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">decarboxylation</a></b> section, however this gene has not yet been submitted due to problems in cloning it.</p></li><br />
<br />
<li><p>Designed and prototyped a physical bioreactor for which we obtained both qualitative and quantitaive data for its functionality. This is outlined on our <b><a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></b> page. </p></li><br />
<br />
<li><p>Characterized the biodegradation of carbazole and various sulfur-containing compounds resembling naphthenic acids in the organisms from which we got our genes. This data can be found on our (<b><a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></b>) and (<b><a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">BBa_K26009</a></b>)</p></li><br />
<br />
<br />
<br />
<br />
<li><p>Resubmitted (<b><a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K206009">BBa_K26009</a></b>) an inconsistent registry composite part that we had to construct from basic parts, resubmitting as the sequence-verified (<b><a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K206009">BBa_K902016</a></b>) BBa_K902016. </p></li><br />
<br />
</body><br />
<br />
</html><br />
<br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/PartsTeam:Calgary/Parts2012-10-04T00:08:57Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/MainHeader| <html><img src="https://static.igem.org/mediawiki/2012/c/c5/UCalgary2012_HeaderLogoOrange.png"></img></html>}}<br />
{{Team:Calgary/BasicPage|parts|<br />
SECTION=Parts|<br />
SIDELIST=<br />
<html><br />
<ul><br />
<li><a href="#parts">Submitted Parts</a></li><br />
</ul><br />
</html>|<br />
<br />
<br />
TITLE=Parts|<br />
CONTENT=<br />
<html><br />
<head><br />
<style><br />
#groupparts{<br />
float: right;<br />
margin-right: 20px;<br />
}<br />
</style><br />
</head><br />
<body><br />
<img src="https://static.igem.org/mediawiki/2012/a/af/UCalgary2012_FRED_and_OSCAR_Parts.png" style="float: right; padding: 10px;"></img><br />
<p>Here is a collection of all the parts we have submitted over the course of this project. Details of each part can be found on their respective pages on both the Registry and this wiki. </p><br />
<h2>Parts of FRED</h2><p><br />
In terms of FRED, we submitted, two novel hydrolase enzymes as well as generators and composite parts for each.</p><br />
<br />
<h2>Parts of OSCAR</h2><p><br />
In terms of OSCAR, we submitted two novel genes for use in decarboxylation, previously submitted <i>xyleE</i> and catalase genes with new promoters, novel desulfurization and denitrogenation enzymes and composite parts and finally, a functional oxidoreductase enzyme expression construct.</p><br />
<br />
<br />
<br />
<h2>Killswitch Parts</h2><br />
<p><br />
In terms of our killswitch project, we submitted three novel riboswitches and their respective promoters, a novel inducible/repressible promoter as well as composite parts with each including three functional circuits with either GFP or the S7 micrococcal nuclease.</p><br />
<br />
<a name="parts"></a><br />
</body><br />
</html><br />
<br />
<groupparts>iGEM012 Calgary</groupparts><br />
<br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Project/ReferencesTeam:Calgary/Project/References2012-10-04T00:08:10Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=References|<br />
CONTENT={{{CONTENT|<br />
<html><br />
<br />
<br />
<ul><br />
<li>Asenjo J.A. (1949).Bioreactor system design. New York (NY): Marcel Dekker Inc. <br />
</p><br />
<p><br />
<li>Reit K., & Tramper J. (1991). Basic bioreactor design. New York (NY):Marcel Dekker Inc.<br />
</p><br />
<p><br />
<li>Canadian Centre for Occupational Health and Safety: Health Effects of Sulfur Dioxide, http://www.ccohs.ca/oshanswers/chemicals/chem_profiles/sulfurdi/health_sul.html (Retrieved: 09/18/2012)</li><br><br />
<br />
<li>Dana G, Kuiken T, Rejeski D & Snow A (2012) Synthetic biology: Four steps to avoid a synthetic-biology disaster. Nature 483:29.</li><br><br />
<br />
<li>Del Rio LF, Hadwin AK, Pinto LJ, MacKinnon MD, Moore MM. Degradation of naphthenic acids by sediment micro-organisms. J Appl Microbiol 2006 Nov;101(5):1049-1061. </li><br><br />
<br />
<li>Diaz, E. & Garcia, J.L. Genetics Engineering for Removal of Sulfur and Nitrogen from Fuel Heterocycles. Handbook of Hydrocarbon and Lipid Microbiology: 2144-2157. Springer, 2010. </li><br><br />
<br />
<li>Galán B, Díaz E, García JL. Enhancing desulfurization by engineering a flavin reductase-encoding gene cassette in recombinant biocatalysts. Environmental microbiology. 2000 Dec; 2(6): 687-94 </li><br><br />
<br />
<li>Herman DC, Fedorak PM, MacKinnon MD, Costerton JW. Biodegradation of naphthenic acids by microbial populations indigenous to oil sands tailings. Can J Microbiol 1994 Jun;40(6):467-477.</li><br><br />
<br />
<li>Holowenko FM, Mackinnon MD, Fedorak PM. Naphthenic acids and surrogate naphthenic acids in methanogenic microcosms. Water Res 2001 Aug;35(11):2595-2606. </li><br><br />
<br />
<li>Kamali N, Tavallaie M, Bambai B, Karkhane AA, Miri M. Site-directed mutagenesis enhances the activity of NADH-FMN oxidoreductase (DszD) activity of <i>Rhodococcus erythropolis</i>. Biotechnol Lett. 2010; 32: 921-927</li><br><br />
<br />
<li>Katzer JR, Sivasubramanian R. Process and Catalyst Needs for Hydrodenitrogenation. Catalysis Reviews: Science and Engineering 1979; 20(2):155-208. </li><br><br />
<br />
<li>Kayser, K.J. & Kilbane, J.J., II Gas Technology Institute. Method for metabolizing carbazole in petroleum, US Patent No. 6,943,006 B2. Sep 13, 2005. </li><br><br />
<br />
<li>Kilbane JJ. Microbial biocatalyst developments to upgrade fossil fuels. Current Opinion in Biotechnology 2006; 17(3):305-314.</li><br><br />
<br />
<li>Lewenza S, Falsafi RK, Winsor G, Gooderham WJ, McPhee JB, Brinkman FS, et al. Construction of a mini-Tn5-luxCDABE mutant library in Pseudomonas aeruginosa PAO1: a tool for identifying differentially regulated genes. Genome Res 2005 Apr;15(4):583-589. </li><br><br />
<br />
<li>Li GQ, Li SS, Zhang ML, Wang J, Zhu L, Liang FL, Liu RL, Ma T. Genetic rearrangement strategy for optimizing the dibenzothiophene biodesulfurization pathway in <i>Rhodococcus erythropolis</i>. Appl Environ Microbiol. 2008 Feb; 74(4): 971-6 </li><br><br />
<br />
<li>Li MZ, Squires CH, Monticello DJ, Childs JD. Genetic analysis of the dsz promoter and associated regulatory regions of <i>Rhodococcus erythropolis</i> IGTS8. J Bacteriol. 1996 Nov; 178(22): 6409-18</li><br><br />
<br />
<li>Lim HN, Lee Y, Hussein R. Fundamental relationship between operon organization and gene expression. Proc Natl Acad Sci U S A. 2011 Jun 28;108(26):10626-31. </li><br><br />
<br />
<li>Ma T. The Desulfurization Pathway in <i>Rhodococcus</i>. Microbiology Monographs 2010; 16: 207-230.</li><br><br />
<br />
<li>Morales M, Le Borgne S. Microorganisms Utilizing Nitrogen-Containing Heterocyclic Hydrocarbons. Handbook of Hydrocarbon and Lipid Microbiology: 2144-2157. Springer, 2010.<br />
<br />
<li> Nakai C, Kagamiyama H, Nozaki M. Complete nucleotide sequence of the metapyrocatechase gene on the TOL plasmid of <i>Pseudomonas putida </i> mt-2. J Biol Chem. 1983 Mar; 258(5):2923-2928.</li><br><br />
<br />
<li>Oshiro T, Ohkita R, Takikawa T, Manabe M, Lee WC, Tanokura M, Izumi Y. Improvement of 2'-hydroxybiphenyl-2-sulfinate desulfinase, an enzyme involved in the dibenzothiophene desulfurization pathway, from <i>Rhodococcus erythropolis</i> KA2-5-1 by site-directed mutagenesis. Biosci Biotechnol Biochem. 2007 Nov.; 71(11):2815-21</li><br><br />
<br />
<br />
<li>Phillips R, Kondev J, Theriot J. Physical Biology of the Cell. 1st ed. Garland Science. 2008. </li><br><br />
<br />
<li>Ramos-Padron E, Bordenave S, Lin S, Bhaskar IM, Dong X, Sensen CW, et al. Carbon and sulfur cycling by microbial communities in a gypsum-treated oil sands tailings pond. Environ Sci Technol 2011 Jan 15;45(2):439-446.</li><br><br />
<br />
<li>Reznikoff WS. Transposon Tn5. Annu Rev Genet 2008;42:269-286. </li><br><br />
<br />
<li>Schweigert N, Zehnder AJ, Eggen RI. Chemical properties of catechols and their molecular modes of toxic action in cells, from microorganisms to mammals. Environ Microbiol 2001 Feb; 3(2):81-91</li><br><br />
<br />
<li>Sheridan DL, Hughes TE. A faster way to make GFP-based biosensors: two new transposons for creating multicolored libraries of fluorescent fusion proteins. BMC Biotechnol 2004 Aug 18;4:17. </li><br><br />
<br />
<li>Shu L, Chiou Y, Orville AM, Miller MA, Lipscomb JD, Que L. X-ray absorption spectroscopic studies of the Fe(II) active site of catechol 2,3-dioxygenase. Implications for the extradiol cleavage mechanism. Biochem 1995; 34:6649-6659.</li><br><br />
<br />
<li>So J. Mini-transposon Tn5gfp constructs for differential tagging of microorganisms. Biotechnology and Bioprocess Engineering 1999;4(2):154-156. </li><br><br />
<br />
<li>Soleimani M, Bassi A, Margaritis A. Biodesulfurization of refractory organic sulfur compounds in fossil fuels. Biotechnol Adv. 2007 Nov-Dec;25(6):570-96 </li><br><br />
<br />
<li>Timms-Wilson TM, Bailey MJ. Reliable use of green fluorescent protein in fluorescent pseudomonads. J Microbiol Methods 2001 Jul 30;46(1):77-80. </li><br><br />
<br />
<br />
<li>United Nations Environment Programme: Sulfur Air Pollution, http://www.unep.org/transport/pcfv/pdf/Ethiopia-AirPollutionsulphur.pdf (Retrieved: 09/18/2012)</li><br><br />
<br />
<li>United States Environmental Protection Agency: Sulfur Dioxide, http://www.epa.gov/air/sulfurdioxide/ (Retrieved: 09/18/2012)</li><br><br />
<br />
<li>Vaillancourt FH, Bolin JT, Eltis LD. The ins and outs of ring-cleaving dioxygenases. Crit Rev Biochem Mol. 2006; 41:241-267. </li><br><br />
<br />
<li>Vogel U, Jensen KF. The RNA chain elongation rate in Escherichia coli depends on the growth rate. J Bacteriol. 1994 May;176(10):2807-13. </li><br><br />
<br />
<li>Wright JK, Overath P. Purification of the lactose:H+ carrier of Escherichia coli and characterization of galactoside binding and transport. Eur J Biochem.1984 Feb 1;138(3):497-508.</li><br><br />
<br />
<li>Xiong AS, Peng RH, Cheng ZM, Li Y, Liu JG, Zhuang J, Gao F, Xu F, Qiao YS, Zhang Z, Chen JM, Yao QH. Concurrent mutations in six amino acids in beta-glucuronidase improve its thermostability. Protein Eng Des Sel. 2007 Jul;20(7):319-25. Epub 2007 Jun 8. </li><br><br />
<br />
<li>Xu P, Yu P, Li FP, Cai XF, Ma CQ. Microbial degradation of sulfur, nitrogen and oxygen heterocycles. Trends in Microbiology 2006; 14(9):398-405.</li><br><br />
<br />
<li>Yoshimura F, Nikaido H. Permeability of Pseudomonas aeruginosa outer membrane to hydrophilic solutes. J Bacteriol. 1982 Nov;152(2):636-42. </li><br><br />
<br />
<li>Young R, Bremer H. Polypeptide-chain-elongation rate in Escherichia coli B/r as a function of growth rate. Biochem J. 1976 Nov 15;160(2):185-94. </li><br><br />
<br />
<li>Zeuthen P, Knudsen KG, Whitehurst DD.Organic nitrogen compounds in gas oil blends, their hydrotreated products and the importance to hydrotreatment. Catalysis Today Feb 2001; 65(2-4):307-314.</li><br><br />
<br />
<li>Zhang X, Wiseman S, Yu H, Liu H, Giesy JP, Hecker M. Assessing the toxicity of naphthenic acids using a microbial genome wide live cell reporter array system. Environ Sci Technol 2011 Mar 1;45(5):1984-1991.</li><br><br />
<br />
<li>Zhu G, Pang K, Parkin G. New Modes for Coordination of Aromatic Heterocyclic Nitrogen Compounds to Molybdenum: Catalytic Hydrogenation of Quinoline, Isoquinoline, and Quinoxaline by Mo(PMe<sub>3</sub>)<sub>4</sub>H<sub>4</sub>. Journal of the American Chemical Society 2008; 130(5):1564-1565. </li><br><br />
<br />
<li>2009 iGEM Calgary, 2009.igem.org/Team:Calgary/Notebook </li><br><br />
<br />
<li> He A, Li T, Daniels L, Fotheringham I, Rosazza J.P.N. Nocardia sp. Carboxylic Acid Reductase: Cloning, Expression, and Characterization of a New Aldehyde Oxidoreductase Family. Applied and Environmental Microbiology 2004 Mar;70(3):1874–1881.</li><br><br />
<br />
<li> Venkitasubramanian P, Daniels L, Rosazza J.P.N. Reduction of Carboxylic Acids by Nocardia Aldehyde Oxidoreductase Requires a Phosphopantetheinylated Enzyme. Journal of Biological Chemistry 2007 Nov 13;282(1):478-485. </li><br><br />
<br />
<li> Rude M.A, Baron T.S, Brubaker S, Alibhai M, Del Cardayre S.B, and Schirmer A. Terminal olefin (1-alkene) biosynthesis by a novel p450 fatty acid decarboxylase from Jeotgalicoccus species. Applied and Environmental Microbiology 2011 Mar;77(5):1718–1727.</li><br><br />
<br />
<li> Clemente J.S, Fedorak P.M. A review of the occurrences, analyses, toxicity, and biodegradation of naphthenic acids. Chemosphere 2005 Feb 6;60(5):585-600.</li><br><br />
<br />
<li> Frank R.A, Fischer K, Kavanagh R, Burnison B.K, Aresenault G, Headley J, Peru K.M, VanDerKraak G, Solomon K. Effect of Carboxylic Acid Content on the Acute Toxicity of Oil Sands Naphthenic Acids. EnvironSciTechnol 2009 Dec 11;43(2):266–271.</li><br><br />
<br />
<li> Slavcheva E, Shone B, Turnbull A. Review of napthenic acid corrosion in oilrefining. British Corrosion Journal 1999 Feb;34(2):125-131. </li><br><br />
<br />
<li> Behar F.H, Albrecht P. Correlations between carboxylic acids and hydrocarbons in several crude oils alteration by biodegradation. Organic Geochemistry 1984;6:597-604. </li><br><br />
<br />
<li>German Collection of Microorganisms and Cell Cultures (DSMZ): Nocardia iowensis. https://www.dsmz.de/catalogues/details/culture/DSM-45197.html (retrieved 8/28/2012)</li><br><br />
<br />
<li>UW iGEM: Diesel Production Background. https://2011.igem.org/Team:Washington/Alkanes/Background (retrieved 8/28/2012)</li><br><br />
<br />
<li>Parts Registry: The PetroBrick – Strong Constitutive Expression of ADC and AAR in pSB1C3. http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 (retrieved 8/28/2012)</li><br><br />
<br />
<li>Asenjo J.A. (1949).Bioreactor system design. New York (NY): Marcel Dekker Inc.</li></br><br />
<br />
<li>Reit K., & Tramper J. (1991). Basic bioreactor design. New York (NY):Marcel Dekker Inc.</li></br><br />
<br />
<li>Becker SA, Feist AM, Mo ML, Hannum G, Palsson BØ, Herrgard MJ. Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox. Nature Protocols 2 2007 Mar; 727-723.</li></br><br />
<br />
<li>Becker SA, Feist AM, Mo ML, Hannum G, Palsson BØ, Herrgard MJ. Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox v2.0. Nature Protocols 6 2011 Aug; 1290-1307.</li></br><br />
<br />
<li>Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BØ. A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information. Molecular Systems Biology 3 2007 Jun; 121.</li></br><br />
<br />
<li>TOMLAB Optimization, MatLab Optimization, http://tomopt.com/tomlab/</li></br><br />
<br />
<li>Gopinath AV, Russell D. An Inexpensive Field-Portable Programmable Potentiostat. Chem.Educator.2006 July;11:23-28.</li></br><br />
<br />
<li>Biran I, Levcov K, Hengge-Aronis R, Ron EZ, Rishpon J. On-line monitoring of gene expression. Microbiology.1999 April;145:2129-2133.</li></br><br />
<br />
<li>Waters LS, Sandoval M, and Storz G. <i>The Escherichia coli MntR miniregulon includes genes encoding a small protein and an efflux pump required for manganese homeostasis.</i> J Bacteriol 2011 Nov; 193(21) 5887-97.</li></br><br />
<br />
<li>Ramesh A, Wakeman CA, and Winkler WC. <i>Insights into metalloregulation by M-box riboswitch RNAs via structural analysis of manganese-bound complexes.</i> J Mol Biol 2011 Apr 8; 407(4) 556-70. </li></br><br />
<br />
<li>Helmann J. <i>Measuring metals with RNA.</i> J Mol Cell 2007 Sept 21; 27(6) 859-860. </li></br><br />
<br />
<li>Daus B, Mattusch J, Paschke A, Wennrich R, and Weiss H. <i>Kinetics of the arsenite oxidation in seepage water from a tin mill tailings pond.</i> Talanta 2000 May 5; 51(6) 1087-95. </li></br><br />
<br />
<li>Cromie MJ, Groisman EA. <i>Promoter and riboswitch control of the Mg2+ transporter MgtA from Salmonella enterica. </i>J Bacteriol 2010 Jan;192(2):604-607.</li></br><br />
<br />
<li>Vitreschak AG, Rodionov DA, Mironov AA, Gelfand MS. Riboswitches: the oldest mechanism for the regulation of gene expression? Trends Genet 2004 Jan;20(1):44-50.</li></br><br />
<br />
<li>Waters LS, Sandoval M, Storz G. <i>The Escherichia coli MntR miniregulon includes genes encoding a small protein and an efflux pump required for manganese homeostasis.</i> J Bacteriol 2011 Nov;193(21):5887-5897. </li></br><br />
<br />
</ul><br />
<br />
<br />
</html>}}}<br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Project/OSCARTeam:Calgary/Project/OSCAR2012-10-04T00:05:29Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=OSCAR|<br />
<br />
CONTENT=<html><br />
<head><br />
<style> <br />
#hub_oscar1{<br />
background: #5BB5E8;<br />
}<br />
#hub_oscar2{<br />
background: #50A2D0;<br />
}<br />
#hub_oscar3{<br />
background: #4292BF;<br />
}<br />
#hub_oscar4{<br />
background: #3285B4;<br />
}<br />
#hub_oscar5{<br />
background: #2580B3;<br />
}<br />
#hub_oscar1:hover, #hub_oscar2:hover, #hub_oscar3:hover, #hub_oscar4:hover, #hub_oscar5:hover{<br />
background: #7DD7FF;<br />
}<br />
<br />
</style><br />
</head><br />
<body><br />
</html>[[IMAGE:UCalgary2012_OSCAR+definition.png|268px|right]]<html><br />
<br />
<p>The <b>O</b>ptimized <b>S</b>ystem for <b>C</b>arboxylic <br />
<br />
<b>A</b>cid <b>R</b>emediation, or OSCAR, is the <br />
<br />
<i><b>Destroy</i></b> component to our iGEM 2012 Calgary project. <br />
<br />
Building on <a href="https://2011.igem.org/Team:Calgary">last <br />
<br />
year's biosensor</a>, OSCAR converts toxic compounds, such as <br />
<br />
naphthenic acids and catechol, into hydrocarbons by removing <br />
<br />
unwanted carboxylic acid and hydroxyl groups. <br />
<br />
</p><br />
<br />
<p> By conversion to hydrocarbons we can not only detoxify tailing <br />
<br />
waters but provide an economically viable method for doing so. By <br />
<br />
using flux balance analysis we developed a system to optimize the <br />
<br />
output of carboxylic acid removal system which we validated in the <br />
<br />
wetlab. Furthermore we developed a bioreactor prototype to <br />
<br />
demonstrate the applicability of our system using novel <br />
<br />
hydrocarbon collection methodologies. Finally, we developed <br />
<br />
constructs and genetic circuits to upgrade these hydrocarbons to <br />
<br />
reduce sulfur and nitrogen content. Altogether, OSCAR provides a <br />
<br />
method to upgrade naphthenic acids and other toxic components from <br />
<br />
waste products into useable fuels.</p><br />
<br />
<h2>What is OSCAR composed of?</h2><br />
<br />
<br />
<br />
<a class="hublink" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation"><br />
<div class="hubbox" id="hub_oscar1"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f1/UCalgary2012_IconOSCAR1.png"></img><br />
<h2>Decarboxylation</h2><br />
<p>The major goal of OSCAR was to be able to remediate toxic components in oil sand tailings ponds, tuning them into useable hydrocarbons. We targeted </html>'''carboxylic acid remediation'''<html>, such as naphthenic acids, as they are some of the most toxic components of the ponds. We had a couple approaches to this, with some promising results!</p><br />
</div><br />
</a><br />
<br />
<br />
<a class="hublink" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation"><br />
<div class="hubbox" id="hub_oscar2"><br />
<img src="https://static.igem.org/mediawiki/2012/0/0c/UCalgary2012_IconOSCAR4.png"></img><br />
<h2>Catechol Degradation</h2><br />
<p>Another toxin we wanted to target was catechol, as it is the common breakdown product of many different toxic compounds. Through modifications to existing registry parts, we showed that we could </html>'''degrade catechol'''<html> into 2HMS and then further degrade it into hydrocarbons.</p><br />
</div><br />
</a><br />
<br />
<a class="hublink" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis"><br />
<div class="hubbox" id="hub_oscar3"><br />
<img src="https://static.igem.org/mediawiki/2012/4/40/UCalgary2012_IconOSCAR6.png"></img><br />
<h2>Flux Analysis</h2><br />
<p>Once we had our bacteria producing hydrocarbons out of tailings pond toxins, we wanted to figure out how to optimize their efficiency. We developed a program to optimize the metabolic network of our synthetic organism using a </html>'''mathematical model'''<html> which predicts compounds that could be fed to the organism to increase hydrocarbon production. Once complete, we validated it in the wetlab, and developed a graphical user interface using the matlab platform, allowing all iGEM teams to use it in their application.</p><br />
</div><br />
</a><br />
<br />
<a class="hublink" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor"><br />
<div class="hubbox" id="hub_oscar4"><br />
<img src="https://static.igem.org/mediawiki/2012/3/3e/UCalgary2012_IconOSCAR5.png"></img><br />
<h2>Bioreactor</h2><br />
<p>With our biological systems showing some promising results in terms of decarboxylation, we needed to think about where OSCAR could live. He needed a house: a </html> '''bioreactor!''' <html>We first used Maya to create a model animation, and then designed and prototyped it. We tested its functionality in terms of a few different parameters, trying to find the most efficient design. </p><br />
</div><br />
</a><br />
<br />
<a class="hublink" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading"><br />
<div class="hubbox" id="hub_oscar5"><br />
<img src="https://static.igem.org/mediawiki/2012/4/44/UCalgary2012_IconOSCAR7.png"></img><br />
<h2>Oil Upgrading</h2><br />
<p>Once we knew that we could turn carboxylic acids into hydrocarbons, we wanted to explore how to upgrade them. Sulfur and nitrogen are both compounds abundant in the tailings ponds and that reduce the quality of fuels produced. They also produce toxic compounds when burned, making them undesirable. We explored ways to remove these components through </html> '''desulfurization''' and '''denitrogenation'''<html>. We targeted pathways in a variety of organisms and submitted biobricks for each.</p><br />
</div><br />
</a><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</body><br />
</html><br />
<br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/DenitrogenationTeam:Calgary/Notebook/Denitrogenation2012-10-03T23:57:28Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Denitrogenation Journal|<br />
CONTENT=<br />
<html><br />
<br />
<h2>Week 1 (May 1-4)</h2><br />
<p>The following section covers wetlab aspect of our overall project focusing on microbial conversion of naphthenic acids into economically-valuable hydrocarbons. The approach taken in this endeavour will be from four strategic starting points - ring cleavage, decarboxylation, denitrogenation, and desulfurization. Overall, the 'hydrocarbons' aspect of the project is a critical one to our overall design and construction of a bioreactor capable of not only detecting but also converting naphthenic acids and other toxins to useful or at least innocuous compounds. </p><br />
<br />
<h2>Week 2-3 (May 7-18)</h2><br />
<p>In the first two weeks of iGEM our group has focused on reviewing literature regarding the bioremediation of nitrogen groups attached to naphthenic acids. The most prevalent N heterocycle is carbazole, representing 75% of total nitrogen by mass. The upper pathway of carbazole biodegradation is catalyzed by the enzymes coded for by the <i>car</i> operon, <i>CarA</i> (<i>CarAaAcAd</i>), <i>CarB</i> (<i>CarBaBb</i>), and <i>CarC</i>. These enzymes convert carbazole to anthralinic acid. The lower pathway is catalyzed by the enzymes of the <i>ant</i> operon, <i>antA, B, and C</i>, yielding catechol while releasing CO2 and NH3. The <i>car</i> and <i>ant</i> operons are both regulated by the P<sup>ant</sup> promoter which is induced by the protein, antR. <i>carAa</i> also has its own promoter which is not induced by <i>antR</i> (Diaz & Garcia, 2010). We have also investigated an alternative pathway using <i>CarA</i> combined with an amidase (<i>amdA</i>) that selectively cleaves NH2 from an intermediate of the car pathway (Xu et al, 2006). This could bypass much of the car/ant pathway and is possibly more efficient.</p><br />
<p>We have decided to use <i>Pseudomonas resinovorans</i> and <i>Rhodococcus erythropolis</i> to derive these genes from. <i>carABC</i> and <i>antABC</i> from<i> P. resinovorans</i> has been shown to have a wide range of nitrogen containing substrate specificity. <i>R. erythropolis</i> contains the <i>amdA</i> gene that we wish to use, and some evidence suggests that it may also be able to degrade sulfur rings through its <i>carABC</i> pathway.</p><br />
<br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p>This week we reviewed the primers listed in the database and also designed some new ones. Primers for <i>carAa</i>,<i> carAc</i>, and <i>carAd</i> were designed individually, while primers for <i>CarBaBbC</i> and <i>antABC</i> were designed to encompass multiple genes in a sequence. These primers were designed to be used on <i>Pseudomonas putida</i> which we decided to use as our gene source since it was available to us as opposed to ordering <i>Pseudomonas resinovorans</i>. We also designed a primer for the<i> AmdA</i> gene from <i>Rhodococcus erthyroplois</i>. In addition to ordering these primers we also ordered the nitrogen containing compounds that we will need to test these enzymes on. We decided on using carbazole to make sure the enzymes can perform their natural function as well as pyrrolidine to test them on a similar ring structure. We also ordered cyclohexamine in order to independently test the function of the alternative <i>AmdA</i> pathway. Finally, we decided to eventually order 4-Piperidine butyric acid hydrochloride to test how the enzymes will work on nitrogen containing naphthenic acids. However, we decided since it is a very expensive compound we would wait to make sure the enzyme's work on more simple compounds before ordering it.</p><br />
<p>We also started our work in the wet lab by plating colonies and making an overnight culture of -80 freezer stock <i>Psuedomonas putida</i> on Thursday. We also resuspended the primers for <i>carAa</i>, <i>carAc</i>, <i>carAd</i>,<i> antA</i>, <i>antB</i>, and <i>ant C</i> that were already in the database. We were able to use the colonies that grew on the streak plates to start a colony PCR to attempt to isolate each of these genes on Friday.</p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2><br />
<p>On Monday and Tuesday we used the database primers to attempt to isolate <i>carAa</i>, <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i>antB</i>, and <i>antC</i> from the <i>Psuedomonas putida</i> we plated last week. <i>carAd</i>, <i>antB</i>, and <i>antC</i> were all put in the gradient PCR machine to account for the wide range of their primers’ melting points, while the others were done via regular PCR. Unfortunately, no positive results were obtained from these reactions. The PCR on <i>carAc</i> and <i>antA</i> was repeated on Wednesday, still not giving positive results. Finally, we attempted to use a salt concentration gradient on the PCR reaction for <i>carAc</i> and <i>antA</i>, using concentrations that ranged from 1.0 microlitres/tube to 2.0 microlitres/tube in increments of 0.2 microlitres/tube. This helped as <i>carAc</i> showed bands in samples that had concentrations of 1.2 and 1.4 microlitres/tube. <i> antA</i> also showed bands, however they were not the correct size, indicating contamination and/or non-specific annealing of the primer. The positive control also showed bands of the correct size. Earlier in the week we also made overnight cultures of 3 environmental strains (28, 29, 30) of <i>Psuedomonas putida</i> from -80 glycerol stock. On Friday we performed a genomic prep on these cultures, and plan on attempting to amplify genes from the isolated DNA next week. </p><br />
<br />
</html>[[File:CarAa and carAc Ucalgary12.png|500px|thumb|Gel of PCR using database primers for <i>carAa</i> and <i>carAc</i> on <i>Pseudomonas putida</i>|center]]<html><br />
<br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> We started off this week by determining the DNA concentration of our genomic prep samples from last week using the nanodrop. DNA concentration for all three<i> P. putida</i> strains was at least 1000 ng/microlitre, well above what was needed for PCR. 1/2 and 1/3 dilutions were prepared for all three strains so as not to have an excess of template DNA in PCR reactions. PCR was performed on all 3 strains using primers for <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i> antB</i>, and <i>antC</i> using 6 replicates per gene. The only successful amplification appeared to be <i>antB</i> and <i>carAc</i>, both from strain 28 (with weaker bands in strain 29). We then performed another PCR, just on those two genes with an increased amount of Taq polymerase to hopefully get enough amplified DNA to move forward with. This resulted in strong bands for both at the expected size. We then performed PCR purification using the Qiaquick kit and obtained samples containing 33.5 ng/microlitre of <i>antB</i> DNA and 129 ng/microlitre of <i>carAc </i>DNA. These concentrations were both sufficient to begin a restriction digest and ligation of these parts into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. Next week we hope to verify the results of the restriction digest, continue to amplify <i>carAc</i> and <i>antB</i> from strain 28, and hopefully submit a biobrick for sequencing.</p><br />
<br />
</html>[[File:AntB and carAc Ucalgary.png|500px|thumb|Gel of PCR ran on genomic DNA of <i>Pseudomonas putida</i> environmental strain 28 using primers for <i>carAc</i> and <i>antB</i>. |center]]<html><br />
<br />
<br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p>The purified PCR products for <i>carAc</i> and <i>antB</i> were digested with restriction enzymes EcoRI+SpeI, EcoRI+PstI, and XbaI+PstI. However, only <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmids that had been digested with EcoRI+SpeI and EcoRI+PstI were available to attempt ligation. Gel results were inconclusive on the restriction digest product as the plasmid size appeared much larger than expected. However, transformation was still attempted on the ligation products for both genes (both using EcoRI+SpeI ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid). The transformation products were plated onto chloroamphenicol (chlor) plates to select for colonies that had the chloro resistance gene on the <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid. Also this week another round of PCR was performed for the <i> car</i> and <i> ant</i> genes, however all, but <i>carAc</i> showed bands in the negative control lane, possibly indicating contamination or the formation of primer dimers. The <i>carAc</i> bands were fairly weak and a PCR purification resulted in very low DNA concentration, insufficient to move onto restriction digest. Next week's plan hinges heavily on the result of the transformation from this Friday. </p><br />
<br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>The results of our transformation on <i>carAc</i> and <i>antB</i> from last week showed mostly red colonies indicating that the ligation was unsuccessful and that the PSB1C3 plasmid simply closed on itself with no insert. However, there were three colonies for <i>carAc</i> that were white (cut with both X+P and E+P). Colony PCR was performed on these three colonies using biobrick primer sets to verify that the part had been inserted in these colonies, but unfortunately all PCR results were negative indicating an unsuccessful ligation.</p><br />
<p>We also performed a miniprep on <i>Pseudomonas putida</i> strain #28 this week using a home-made protocol rather than the Qiagen kit. Miniprep A had a DNA concentration of 1539.8 ng/μL and Miniprep B had DNA concentration of 1001.2 ng/μL possibly indicating that there may have been a lot of genomic DNA contamination. However miniprep A product was still used as a PCR template for a reaction attempting to isolate <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i>antB</i>, and <i>antC</i>. Special conditions for this PCR included replacing 60 μL of water with 60 μL of betaine heated to 37 degrees Celsius and running a Mg gradient ranging from 0.5 μL - 2.5 μL per tube for each gene. Of these only <i>antB</i> had bands of the right size in 3 of the lanes (Mg concentrations of 1.5, 2, and 2.5 μL). However, all of the <i>antB</i> bands (including the negative control) contained a contamination band at around 100 bases, probably due to the formation of primer dimers. This forced us to do a gel extraction rather than a PCR purification. This gel extraction did not work as the nanodrop results indicated high amounts of agarose contamination. Another PCR was performed using the same conditions, but using miniprep B instead as there was no more miniprep A product remaining. Unfortunately there were no bands for <i>antB</i>, only the 100 base pair contamination bands. </p><br />
<br />
</html>[[File:AntA and antC Ucalgary.png|500px|thumb|Gel of PCR ran on miniprep A of <i>Pseudomonas putida</i> attempting to isolate <i>antA</i> and <i>antC</i> genes.|center]]<br />
[[File:CarAc and carAd Ucalgary.png|500px|thumb|Gel of PCR ran on miniprep A of <i>Pseudomonas putida</i> attempting to isolate <i>carAc</i> and <i>carAd</i> genes.|center]]<br />
[[File:Minipret A antB and carAc Ucalgary.png|500px|thumb|Gel of PCR ran on miniprep A showing bands 3-5 of the correct size corresponding to antB genes and colony PCR ran using biobrick primers attempting to determine if <i>carAc</i> genes were successfully transformed into PSB1C3 plasmid.|center]] <html><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>This week we modified our PCR protocol to adjust for the homemade Taq polymerase we were using. Template DNA was boiled for 10 minutes on its own to replace the initialization step. This is because this polymerase could not tolerate the 95 degree heat during the regular initialization step. Another modification we made was to boil our primers for 5 minutes prior to adding them to the master mix. This was done to discourage the formation of primer-dimers. Finally we also attempted a touch-down PCR which started at an annealing temperature greater than the Tm of our primers and gradually worked its way down to below the Tm of the primers. This was done to increase the specificity of our reaction and also eliminate the formation of primer-dimers. Unfortunately these techniques still saw the formation of dimers, preventing us from performing a PCR purification. Again, the gel purification procedure did not lead to successful purification of our target gene. Due to the problems we have had so far our goal for next week is to perform an assay experiment to try and determine which of the <i>Pseudomonas putida</i> strains we have access to can actually degrade carbazole and therefore contain the pCAR1 plasmid. </p><br />
<br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The goal for this week was to grow different strains of<i> Pseudomonas</i> in media containing B-N media + glucose with only carbazole as a source of nitrogen. In theory only those strains which contained the pCAR1 plasmid would be able to use carbazole as a nitrogen source and thrive in this media. The strains we tested from glycerol stock included <i>P. putida</i> LD1, <i>P.putida</i> environmental strains 28-30, and <i>P. fluorescens</i> PF5. Each of these strains had two cultures grown; one with carbazole and one without it. <i>E. coli</i> grown with carbazole was used as a negative control as it does not contain the pCAR plasmid. At timepoints of 22, 46, and 117 hours of growth samples were taken to do an OD600 reading to measure bacterial growth and had an ammonia assay performed on it. The detection of ammonia acted as a proxy for detecting carbazole degradation as it is an end-product of the pathway. With this in mind, an additional control strain of LD1 was grown in media containing glucose and ammonia. This is to detect the possibility that <i>Pseudomonas</i> can use ammonia as a source of nitrogen after it has degraded carbazole which would be problematic as far as using ammonia levels to determine carbazole degradation. So far we have been having difficulties reading the results of the ammonia assay with a plate reader and may resort to testing our samples using a different assay protocol next week. The OD600 readings were initially very high at 22 hours and then dropped at 46 hours. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>This week we took our last readings from the experiment that we started last week. The OD600 readings taken at 117 hours showed minimal growth over the weekend for all strains except for environmental strain #29 (with carbazole) which increased from 0.086 to 0.313. No other strain showed an increase of 0.100 or greater. This shows that only strain #29 was able to thrive in the carbazole media, possibly indicating that it contains the pCAR plasmid enabling it to use carbazole as a nitrogen source. To verify this we used a new ammonia assay protocol to measure the production of ammonia in the different cultures and theoretically how much carbazole was degraded. Unlike the previous assay we were able to produce a standard curve with a consistent slope based on samples with known amounts of ammonia. However, the only samples that showed any elevation of ammonia levels above the blank were the LD1 samples that were cultured with ammonia chloride to control for the possibility of pseudomonas using ammonia as a nitrogen source. The ammonia levels in this sample were relatively constant which would indicate that the LD1 was not using ammonia as a nitrogen source. Unfortunately the fact that none of the test samples had elevated ammonia levels did not give us any evidence that these strains contain the pCAR plasmid. </p><br />
<br />
</html>[[File: Standard_curve_for_ammonia_assay_UCalgary2012_denitrification_group.png|500px|thumb|Standard curve for ammonia assay using OD635 absorbance.|center|]]<html><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>We received a new strain of Pseudomonas, LD2, this week that had previously been used to study the pCAR1 plasmid. It was grown up in PCA media and had its colonies used for colony PCR. We also received our new primers this week, further increasing the chances of successful PCR. Using the new strain and the new primers <i>carAa</i>, <i>carAc</i>, and <i>carAd</i> all showed at least some bands of close to the correct size. After PCR purification we obtained DNA concentrations of 21.3 ng/μL for <i>carAa</i>, 41.9 ng/ μL for <i>carAc</i>, and 32.8 ng/μL for<i> carAd</i>. These products were then restriction digested using EcoRI + PstI sites. </p><br />
<br />
</html>[[File:07.20.2012 CarAa and CarAc.jpg|500px|thumb|Gel of colony PCR from <i>Pseudomonas sp. LD2</i> for <i>carAa</i> and <i>carAc</i> genes. <i>carAa</i> expected size is 1155 bp and <i>carAc</i> is 323 bp.|center]]<br />
[[File:07.20.2012 CarAd colony PCR from LD2.jpg|500px|thumb|Gel of colony PCR from <i>Pseudomonas sp. LD2</i> for <i>carAd</i> gene. <i>carAd</i> expected size is 989 bp.|center]] <html><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The restriction digested <i>car</i> genes were ligated into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> cells. White colonies grew for<i> carAa</i> and <i>carAc</i> suggesting that ligation and transformation were successful, however no colonies grew for <i>carAd</i> suggesting that the transformation did not work. Colony PCR was performed to confirm the presence of these genes within the vectors, however only <i>carAc</i> was positive. The colonies containing the <i>carAc</i> plasmid were then mini prepped and restriction digested to isolate the plasmid and send for sequencing. Further attempts at transforming<i> carAa</i> and <i>carAd</i> were not successful. Another goal was to perform colony PCR on the <i>P. putida</i> to isolate the<i> antABC</i> genes using both the primers for the entire operon and those for the individual genes. No attempt showed any bands, although salt and temperature gradients were used as well as different types of polymerase. LD2 cultures were grown up to be plasmid prepped and genomic prepped in order to have a higher concentration of template DNA in the PCR master mix and more PCR will be attempted on these genes next week. Mutagenesis primers were also designed this week to remove the illegal restriction cut sites in the<i> carAd</i> and <i>antB</i> genes (NotI and 2xEcoRI respectively). Only 1 mutagenesis primer was designed for <i>antB</i> as one of its EcoRI sites was very close to the beginning of the gene and could be removed using a new primer. During this process it was also discovered that the original ant primers were designed for the wrong genes and therefore do not match the intended sequence. Although the new <i>antABC</i> primers were designed using the correct gene the PCR may not be working due to the length of the operon so the design of new individual primers may be necessary. Finally, we also aimed to grow up the freeze dried culture of <i>Rhodococcus erythropolis</i> (source of the<i> AmdA</i> gene) that we received from DSMZ. The reactivation media was produced and autoclaved to begin the process next week.</p><br />
<br />
</html>[[File:07.23.2012.Restriction Digest of Car genes.jpg|500px|thumb|Gel of restriction digest of car genes. A refers to <i>carAa</i>, C to <i>carAc</i>, and D to <i>carAd</i>. Ladder shows that after purification and digestion DNA is still the correct size.|center]]<br />
[[File:CarAc biobrick confirmation PCR.jpg|500px|thumb|Gel of confirmation PCR done on competent Top 10 <i>E. coli</i> cells in which a PSB1C3 vector containing the <i>carAc</i> gene had been inserted. Red X marks the 500 bp mark on the weak ladder showing the product to be the expected size (323 bp gene + 2*100 bp biobrick sites on each end).|center]] <html><br />
<br />
<h2>Week 14 (July 30 - August 3)</h2><br />
<p>We received the sequencing results this week for <i>carAc</i> which came back as a 100% match to the expected nucleotide sequence. We also successfully ligated both<i> carAa</i> and<i> carAd</i> into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into top 10 <i>E. coli</i> cells. Colony PCR verified the presence of the biobrick plasmid in these colonies. These were both miniprepped, digested and sent for sequencing by the end of the week. Using a salt gradient, addition of DMSO, and the KAPA buffer specifically designed for GC-rich amplicons we were able to amplify the ant operon from a genomic prep of LD2. However, all attempts at ligating and transforming this sequence were met with the unfortunate presence of red colonies (indicating failed insertion into the plasmid). Next week we will recut a <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> vector with Xba and Pst to attempt to mitigate this problem. We also reactivated the <i>R. erythropolis</i> following the DSMZ protocol for reviving freeze-dried cultures that can be found <a href=https://2012.igem.org/Team:Calgary/Notebook/Protocols/dmszfreezedry>here</a> using DSMZ media #1 and grew it on plates made from DSMZ media #220 at 28 degrees. Colonies grew very quickly and were used for colony PCR and to make overnight cultures for downstream applications. The colony PCR using the amidase primers was unsuccessful, so a genomic prep was performed on the overnight culture. Unfortunately we lacked some of the required equipment and the genomic prep did not work, but with a few procedural modifications and the right equipment we are confident it will work next week. A glycerol stock of the<i> R. erythropoplis</i> was also made from another overnight culture. We also started construction on the <i>carAc </i>part this week by inserting a ribosome binding site (B0034) in front of the gene. Finally, since the<i> ant</i> operon was successfully amplified by PCR we designed mutagenesis primers for the 2nd EcoRI site in the operon rather than making new individual primers. We also designed a mutagenesis primer to remove the SpeI site in <i>AmdA</i>. Now that we have conclusively proven that the LD2 strain contains the pCAR1 plasmid we aim to demonstrate its ability to degrade carbazole and other nitrogen containing heterocycles using an assay experiment similar to the one performed in Week 10.</p><br />
<br />
</html>[[File:07.30.2012. CarAa, CarAd.jpg|500px|thumb|<i>carAa</i> and <i>carAd</i> amplified from genomic prep of <i>Pseudomonas sp. LD2</i>. Amplicons are larger than expected, potentially the gel ran diagonally. The 6th lane for each gene was a negative control.|center]]<br />
[[File:07.31.2012.Ant operon.jpg|500px|thumb|<i>ant</i> operon amplified from genomic prep of <i>Pseudomonas sp. LD2</i> at the expected size of 3000 bp.|center]]<br />
[[File:08.01.2012 CarAdBB.jpg|500px|thumb|Lanes 2,4,5, and 11 contain <i>carAd</i> genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent top 10 <i>E.Coli</i>.|center]]<br />
[[File:08.01.2012 CarAaBB.jpg|500px|thumb|Lane 9 contains <i>carAa</i> genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent top 10 <i>E.Coli</i>. Although larger than expected, the positive control RFP generator in lane 10 is also larger than its expected size (~1200 bp including biobrick bookends) indicating that the gel may have a slant in it.|center]]<html><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p>This week we received the sequencing results for <i>carAa</i> and <i>carAd</i>. <i>carAd</i> was a match, however <i>carAa</i> did not match at all and had misplaced biobrick restriction enzyme sites. Due to this we decided to restart the biobricking process for <i>carAa</i> all the way back to PCR from the plasmid. By the end of the week we had progressed to the point of transforming it into competent <i>E. coli</i> cells, however a thick lawn of bacteria had grown overnight making it impossible to pick colonies. A streak plate was made from this plate to attempt to get less dense growth. Since the sequencing came back positive for <i>carAd</i> the next step was to perform site directed mutagenesis on its NotI restriction enzyme site using the primers that were designed previously (for site-directed mutagenesis optimization, please refer to Desulfurization Journal Week 14). The PCR was successful, showing very bright bands at 3000 bp, and the PCR product was digested with DpnI to remove parental DNA. Transformation of this product will indicate whether the mutagenesis was successful. We were also able to successfully perform a genomic prep of <i>Rhodococcus erythropolis</i> and amplify the <i>AmdA</i> gene this week. It was ligated into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> by Friday, and a confirmation colony PCR was run on Friday evening to verify that the gene was inserted. These results will be available by Monday. After many attempts the <i>ant</i> operon was finally ligated and transformed as well this week. Confirmation PCR showed bands of about 3000 bp which is consistent with the size of the operon. By Friday the successful colonies had been grown up and miniprepped, ready to send for sequencing early next week. The <i>carAc</i>/B0034 construct was also successfully transformed this week and verified by running a PCR using the B0034 forward primer. It was also miniprepped and is ready to send for sequencing next week. </p><br />
<p>In addition to this work constructing biobricks we also started an experiment to demonstrate the function of the pCAR plasmid in LD2. Strains of LD2 were grown up in LB and then washed and placed in B-N media + glucose and various experimental additions. These additions included carbazole, 4-PBAH (a nitrogen containing naphthenic acid), ammonia chloride, and a negative control with no nitrogen source. Strains of <i>E. coli</i> were also grown under the same conditions to compare growth and ammonia production of LD2 to a strain that does not contain the pCAR plasmid. These results should act as a preview of what we expect our final biobrick containing <i>E. col</i>i species to produce as well as allowing us to establish LD2 as a positive control for this experiment. </p><br />
<br />
<br />
</html>[[File:08.07.2012 amdA pcr from genomic prep.jpg|500px|thumb|<i>AmdA</i> amplified from the genomic prep of <i>R. Erythropolis</i> shows bands just greater than 1500 base pairs in lanes 2,4-6,9, and 12-13. Lane 14 was a negative control. The expected size of <i>AmdA</i> is 1566 base pairs.|center]]<br />
[[File:08.09.2012 confirmation pcr of ant operon.jpg|500px|thumb|The two labelled lanes show amplification at 3000 base pairs from a confirmation colony PCR using the biobrick primers. The expected size of the <i>ant</i> operon with the biobrick bookends is about 3100 base pairs.|center]]<br />
[[File:08.10.2012 carad notI mutagenesis.jpg|500px|thumb|Strong amplification at just above 3000 base pairs from the <i>carAd</i> PCR using mutagenic primers. Since PSB1C3 is 2070 base pairs and <i>carAd</i> is 989 base pairs the amplification should run at about 3059 base pairs. 3 different amounts of template DNA were tested in lanes 2-7 as indicated and a positive control showed weaker amplification in lanes 8-9 at about 4000 base pairs.|center]]<br />
<html><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>The sequencing for the <i>carAc</i>/<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> construct came back with only <i>carAc</i> and no evidence of a <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> site preceding it. The confirmation PCR that was run last week using the <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> forward primer seemed to indicate that it was inserted, however since <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> is only 12 base pairs the primer may not have been specific enough. The construction on this part was restarted this week. <i>carAa</i> colony PCR on last week's transformation showed two positive colonies, however upon culturing, miniprepping, and digesting these colonies the plasmid appeared to be too large (3000 base pairs as opposed to the expected 2000). This may be due to a problem of a mislabeled plasmid tube that has been experienced by other team members, causing the gene to be inserted into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a> rather than <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. To test this, a streak plate was made from these colonies and grown on an ampicillin resistant plate. Mutagenized <i>carAd</i> was successfully transformed into Top 10 <i>E. coli</i> cells, cultured, and miniprepped. The miniprep product was digested with NotI to test whether the NotI site was successfully removed. These results were good, as the plasmid ran at 2000 base pairs and the insert at just under 1000 base pairs in the mutagenized <i>carAd</i> whereas the non-mutagenized <i>carAd</i> had its insert cut into 2 bands. This will be sent for sequencing next week to confirm the mutation. Unfortunately the sequencing results came back negative for the <i>ant</i> operon meaning we will need to restart genomic PCR on it next week. Multiple transformations and confirmation PCRs were run on <i>AmdA</i> this week, however none displayed a band size of 1700-1800 base pairs as expected. We have continued to try different digested plasmids as other team members also had difficulties this week with ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, possibly indicating a problem with our Antarctic Phosphatase stock.</p><br />
<p>The other news from this week was the final results from our carbazole degradation experiment attempting to demonstrate LD2's ability to degrade carbazole and hopefully other nitrogen containing compounds. Unfortunately, these results were very difficult to make sense of as the LD2 that were cultured with carbazole grew, but did not show elevated ammonia levels which would be indicative of carbazole degradation. One of our controls was LD2 cultured with ammonium chloride to test whether LD2 was capable of using ammonia as a nitrogen source. This would pose a problem for us as we were planning to use an increase in ammonia levels as a proxy for carbazole degradation rather than measuring carbazole degradation directly using GC-MS. However, the LD2 showed significantly less ammonia after 44 hours when compared to 26 hours indicating that it may be degraded, either by the LD2 or by an unaccounted for abiotic process. This has lead us to decide to use GC-MS and directly measure carbazole levels in our sample the next time we do this experiment.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<p>The <i>carAa</i> was re-streaked onto ampicillin plates and grew, probably showing that it was ligated into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a>, not <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, which explained the irregularity of our confirmation digest last week. It was sent for sequencing, but in case it was incorrect we also started a new <i>carAa</i> pipeline by PCRing it from the <i>Pseudomonas</i> genome again and redigesting and ligating. It was transformed into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> over the weekend. <i>carAc</i> did not see a lot of progress this week as we spent much of the time attempting to construct a RBS (B0034) in front of it, but were unsuccessful. <i>carAd</i> was also subjected to this construction with B0034 and we were able to send it for sequencing by the end of the week. Several new constructs were also started for <i>carAd</i> in case the sequencing is negative. The <i>ant</i> operon was successfully transformed and confirmed by colony PCR this week, after which it was grown up and sent for sequencing. We also began to attempt mutagenesis on one of the EcoRI sites in the operon, but had no amplification of the plasmid. We also re-digested some PCR purified product to start another ligation in case sequencing failed. After many attempts we were also able to finally successfully transform an <i>AmdA</i>/PSB1C3 plasmid into Top 10 cells this week. After a miniprep they showed bands of about 1800 base pairs on the confirmation digest gel, so they were also sent for sequencing this week and had mutagenesis started on the illegal SpeI site. Unfortunately, the sequencing sent early in the week came back with ambiguous results. The forward reaction for<i> antABC</i> was a match, but its reverse reaction matched to <i>carAa</i>. Both <i>carAa</i> reactions matched nothing. Since these two genes are over 20 000 base pairs apart on the template plasmid it is virtually impossible that they were actually combined into one plasmid (PCR extension time would not have been long enough and even if it was the product would have run much above the 3000 base pair mark). This is possibly a mistake on Eurofins end, so we are not 100% sure if we have 1 good gene, both of them, or neither. Also the sequence for mutagenized <i>carAd</i> showed only about the first 100 base pairs, which were correct however not long enough meaning it was a poor reaction and inconclusive. Finally, we also re-started our <a href=https://2012.igem.org/Team:Calgary/Notebook/Protocols/carbazole>carbazole degradation assay</a> using the GC-MS to quantitatively measure the loss of carbazole at biological sciences. This process will take 2 weeks to demonstrate LD2's ability to degrade carbazole. We are forcing it to use carbazole as a carbon and a nitrogen source in one sample and as only a nitrogen source in another sample (this sample has had glucose added to it).</p><br />
<br />
</html>[[File:AmdA colonyPCR calgary12.JPG|500px|thumb|Amplification is seen in lane 7 of this PCR of top 10 colonies into which AmdA/PSB1C3 had been transformed. This indicates that <i>AmdA</i> has been successfully inserted into a biobrick vector.|center]]<br />
[[File:NewcarAagenomePCR calgary12.jpg|500px|thumb|<i>carAa</i> amplified from pCAR1.2 plasmid of <i>Pseudomonas putida</i>.|center]] <html><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<p>The new stock of <i>carAa</i> that we had PCRed from LD2 was sent for sequencing this week and came back as a match, so we have begun constructing a RBS site and a TetR promoter (<a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>) in front of it. At this point all the <i>car</i> genes have been successfully amplified, inserted into a biobrick plasmid, and sequence verified. We also continued to attempt to construct <i>carAc</i> with an RBS site (<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>) in front of it, but had no success. On a better note, we confirmed this week that<i> carAd</i> was mutated properly and we were also able to insert an RBS site in front of it. The next step for this gene will be to attempt a plasmid switch as it is currently in <a href="http://partsregistry.org/Part:pSB1A3">pSB1A3</a> and needs to be in <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> for submission to the parts registry. After sending a new batch of the ant operon for sequencing we got the same confusing results as we did last week. We therefore hypothesized that our primers were annealing to a homologous region that is similar to the<i> ant</i> genes and very close to <i>carAa</i>. This means that we need to restart PCR on this gene as all of our current product is incorrect. The sequencing results for <i>AmdA</i> that came back this week were also somewhat confusing as they did not quite match the sequence we expected. However, they were a 100% match for the <i>AmdA</i> gene in a different strain of <i>Rhodococcus erythropolis</i>. This means that DSMZ probably sent us the wrong strain of bacteria and that we were very lucky that the primers we designed based off of the expected template actually worked to amplify a slightly different gene in another strain. The good news is that this new sequence contains no illegal cut sites, which explains why mutagenesis was not working (there was nothing to mutate) and allows us to immediately begin construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> in front of <i>AmdA</i> this week. </p><br />
<br />
</html>[[File:Caraa colonyPCR calgary12.JPG|500px|thumb|Colony PCR amplification from top 10 <i>E. coli</i> cells with a PSB1C3/<i>carAa</i> construct transformed into them. Lanes 1-3, 5, and 7 were sent for sequencing with lane 5 coming back positive.|center]]<html><br />
<br />
<h2>Week 19 (September 3- September 7)</h2><br />
<p><i>carAd</i>/<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> was successfully plasmid switched into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, making it ready for submission. Construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> and <i>carAa</i> may have been successful as the digest of the miniprep looked promising. It will be sent for sequencing next week. Unfortunately there was no progress made on the constructions of <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> or <i>carAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. These will be redigested and religated for next week. We could not amplify the <i>ant</i> operon despite trying many variations of PCR using high fidelity KAPA polymerase. This is probably due to the multiple, large regions of homology on the operon that the reverse primer binds to making amplification difficult. Because the function of this gene can theoretically be replaced by <i>AmdA</i>, we may scale back efforts dedicated to it, as the chances of successful amplification appear slim. </p><br />
<br />
<h2>Weeks 20-22 (September 10 - September 30)</h2><br />
<p>Constructs that we continued to work on this week included <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, <i>carAa</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, and <i>carAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. Some sequencing was sent, but no positive results were obtained. A new approach to biobrick the <i>ant</i> operon was implemented this week. A new forward primer was designed that would anneal about 1500 base pairs upstream of the <i>ant</i> operon, avoiding the region of homology that is repeated at various points on the plasmid. Once this region is successfully amplified we could use this amplified DNA as a template for another PCR reaction which would use the normal <i>ant</i> primers. Since the template would no longer include regions of homology these primers should be free to work properly and amplify the <i>ant</i> operon. The PCR using the new primers was started this week, and although amplification at 4500 base pairs was observed as expected, there was also a lot of non-specific amplification. This means that to purify the amplified DNA that we want and avoid the other products a dreaded gel extraction had to be performed which, as usual, did not work. At this point it was decided that further attempts to amplify the <i>ant</i> operon would be futile and our efforts would be better served working on constructing the parts we do have. Unfortunately no progress was made in these weeks on construction. We have also obtained GC-MS readings from the carbazole degradation experiment that showed that <i>Pseudomonas</i> LD2 can indeed degrade carbazole, however only under low glucose conditions.</p><br />
<br />
<br />
<br />
<br />
</html><br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/DenitrogenationTeam:Calgary/Notebook/Denitrogenation2012-10-03T23:56:23Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Denitrogenation Journal|<br />
CONTENT=<br />
<html><br />
<br />
<h2>Week 1 (May 1-4)</h2><br />
<p>The following section covers wetlab aspect of our overall project focusing on microbial conversion of naphthenic acids into economically-valuable hydrocarbons. The approach taken in this endeavour will be from four strategic starting points - ring cleavage, decarboxylation, denitrogenation, and desulfurization. Overall, the 'hydrocarbons' aspect of the project is a critical one to our overall design and construction of a bioreactor capable of not only detecting but also converting naphthenic acids and other toxins to useful or at least innocuous compounds. </p><br />
<br />
<h2>Week 2-3 (May 7-18)</h2><br />
<p>In the first two weeks of iGEM our group has focused on reviewing literature regarding the bioremediation of nitrogen groups attached to naphthenic acids. The most prevalent N heterocycle is carbazole, representing 75% of total nitrogen by mass. The upper pathway of carbazole biodegradation is catalyzed by the enzymes coded for by the <i>car</i> operon, <i>CarA</i> (<i>CarAaAcAd</i>), <i>CarB</i> (<i>CarBaBb</i>), and <i>CarC</i>. These enzymes convert carbazole to anthralinic acid. The lower pathway is catalyzed by the enzymes of the <i>ant</i> operon, <i>antA, B, and C</i>, yielding catechol while releasing CO2 and NH3. The <i>car</i> and <i>ant</i> operons are both regulated by the P<sup>ant</sup> promoter which is induced by the protein, antR. <i>carAa</i> also has its own promoter which is not induced by <i>antR</i> (Diaz & Garcia, 2010). We have also investigated an alternative pathway using <i>CarA</i> combined with an amidase (<i>amdA</i>) that selectively cleaves NH2 from an intermediate of the car pathway (Xu et al, 2006). This could bypass much of the car/ant pathway and is possibly more efficient.</p><br />
<p>We have decided to use <i>Pseudomonas resinovorans</i> and <i>Rhodococcus erythropolis</i> to derive these genes from. <i>carABC</i> and <i>antABC</i> from<i> P. resinovorans</i> has been shown to have a wide range of nitrogen containing substrate specificity. <i>R. erythropolis</i> contains the <i>amdA</i> gene that we wish to use, and some evidence suggests that it may also be able to degrade sulfur rings through its <i>carABC</i> pathway.</p><br />
<br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p>This week we reviewed the primers listed in the database and also designed some new ones. Primers for <i>carAa</i>,<i> carAc</i>, and <i>carAd</i> were designed individually, while primers for <i>CarBaBbC</i> and <i>antABC</i> were designed to encompass multiple genes in a sequence. These primers were designed to be used on <i>Pseudomonas putida</i> which we decided to use as our gene source since it was available to us as opposed to ordering <i>Pseudomonas resinovorans</i>. We also designed a primer for the<i> AmdA</i> gene from <i>Rhodococcus erthyroplois</i>. In addition to ordering these primers we also ordered the nitrogen containing compounds that we will need to test these enzymes on. We decided on using carbazole to make sure the enzymes can perform their natural function as well as pyrrolidine to test them on a similar ring structure. We also ordered cyclohexamine in order to independently test the function of the alternative <i>AmdA</i> pathway. Finally, we decided to eventually order 4-Piperidine butyric acid hydrochloride to test how the enzymes will work on nitrogen containing naphthenic acids. However, we decided since it is a very expensive compound we would wait to make sure the enzyme's work on more simple compounds before ordering it.</p><br />
<p>We also started our work in the wet lab by plating colonies and making an overnight culture of -80 freezer stock <i>Psuedomonas putida</i> on Thursday. We also resuspended the primers for <i>carAa</i>, <i>carAc</i>, <i>carAd</i>,<i> antA</i>, <i>antB</i>, and <i>ant C</i> that were already in the database. We were able to use the colonies that grew on the streak plates to start a colony PCR to attempt to isolate each of these genes on Friday.</p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2><br />
<p>On Monday and Tuesday we used the database primers to attempt to isolate <i>carAa</i>, <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i>antB</i>, and <i>antC</i> from the <i>Psuedomonas putida</i> we plated last week. <i>carAd</i>, <i>antB</i>, and <i>antC</i> were all put in the gradient PCR machine to account for the wide range of their primers’ melting points, while the others were done via regular PCR. Unfortunately, no positive results were obtained from these reactions. The PCR on <i>carAc</i> and <i>antA</i> was repeated on Wednesday, still not giving positive results. Finally, we attempted to use a salt concentration gradient on the PCR reaction for <i>carAc</i> and <i>antA</i>, using concentrations that ranged from 1.0 microlitres/tube to 2.0 microlitres/tube in increments of 0.2 microlitres/tube. This helped as <i>carAc</i> showed bands in samples that had concentrations of 1.2 and 1.4 microlitres/tube. <i> antA</i> also showed bands, however they were not the correct size, indicating contamination and/or non-specific annealing of the primer. The positive control also showed bands of the correct size. Earlier in the week we also made overnight cultures of 3 environmental strains (28, 29, 30) of <i>Psuedomonas putida</i> from -80 glycerol stock. On Friday we performed a genomic prep on these cultures, and plan on attempting to amplify genes from the isolated DNA next week. </p><br />
<br />
</html>[[File:CarAa and carAc Ucalgary12.png|500px|thumb|Gel of PCR using database primers for <i>carAa</i> and <i>carAc</i> on <i>Pseudomonas putida</i>|center]]<html><br />
<br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> We started off this week by determining the DNA concentration of our genomic prep samples from last week using the nanodrop. DNA concentration for all three<i> P. putida</i> strains was at least 1000 ng/microlitre, well above what was needed for PCR. 1/2 and 1/3 dilutions were prepared for all three strains so as not to have an excess of template DNA in PCR reactions. PCR was performed on all 3 strains using primers for <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i> antB</i>, and <i>antC</i> using 6 replicates per gene. The only successful amplification appeared to be <i>antB</i> and <i>carAc</i>, both from strain 28 (with weaker bands in strain 29). We then performed another PCR, just on those two genes with an increased amount of Taq polymerase to hopefully get enough amplified DNA to move forward with. This resulted in strong bands for both at the expected size. We then performed PCR purification using the Qiaquick kit and obtained samples containing 33.5 ng/microlitre of <i>antB</i> DNA and 129 ng/microlitre of <i>carAc </i>DNA. These concentrations were both sufficient to begin a restriction digest and ligation of these parts into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. Next week we hope to verify the results of the restriction digest, continue to amplify <i>carAc</i> and <i>antB</i> from strain 28, and hopefully submit a biobrick for sequencing.</p><br />
<br />
</html>[[File:AntB and carAc Ucalgary.png|500px|thumb|Gel of PCR ran on genomic DNA of <i>Pseudomonas putida</i> environmental strain 28 using primers for <i>carAc</i> and <i>antB</i>. |center]]<html><br />
<br />
<br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p>The purified PCR products for <i>carAc</i> and <i>antB</i> were digested with restriction enzymes EcoRI+SpeI, EcoRI+PstI, and XbaI+PstI. However, only <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmids that had been digested with EcoRI+SpeI and EcoRI+PstI were available to attempt ligation. Gel results were inconclusive on the restriction digest product as the plasmid size appeared much larger than expected. However, transformation was still attempted on the ligation products for both genes (both using EcoRI+SpeI ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid). The transformation products were plated onto chloroamphenicol (chlor) plates to select for colonies that had the chloro resistance gene on the <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid. Also this week another round of PCR was performed for the <i> car</i> and <i> ant</i> genes, however all, but <i>carAc</i> showed bands in the negative control lane, possibly indicating contamination or the formation of primer dimers. The <i>carAc</i> bands were fairly weak and a PCR purification resulted in very low DNA concentration, insufficient to move onto restriction digest. Next week's plan hinges heavily on the result of the transformation from this Friday. </p><br />
<br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>The results of our transformation on <i>carAc</i> and <i>antB</i> from last week showed mostly red colonies indicating that the ligation was unsuccessful and that the PSB1C3 plasmid simply closed on itself with no insert. However, there were three colonies for <i>carAc</i> that were white (cut with both X+P and E+P). Colony PCR was performed on these three colonies using biobrick primer sets to verify that the part had been inserted in these colonies, but unfortunately all PCR results were negative indicating an unsuccessful ligation.</p><br />
<p>We also performed a miniprep on <i>Pseudomonas putida</i> strain #28 this week using a home-made protocol rather than the Qiagen kit. Miniprep A had a DNA concentration of 1539.8 ng/μL and Miniprep B had DNA concentration of 1001.2 ng/μL possibly indicating that there may have been a lot of genomic DNA contamination. However miniprep A product was still used as a PCR template for a reaction attempting to isolate <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i>antB</i>, and <i>antC</i>. Special conditions for this PCR included replacing 60 μL of water with 60 μL of betaine heated to 37 degrees Celsius and running a Mg gradient ranging from 0.5 μL - 2.5 μL per tube for each gene. Of these only <i>antB</i> had bands of the right size in 3 of the lanes (Mg concentrations of 1.5, 2, and 2.5 μL). However, all of the <i>antB</i> bands (including the negative control) contained a contamination band at around 100 bases, probably due to the formation of primer dimers. This forced us to do a gel extraction rather than a PCR purification. This gel extraction did not work as the nanodrop results indicated high amounts of agarose contamination. Another PCR was performed using the same conditions, but using miniprep B instead as there was no more miniprep A product remaining. Unfortunately there were no bands for <i>antB</i>, only the 100 base pair contamination bands. </p><br />
<br />
</html>[[File:AntA and antC Ucalgary.png|500px|thumb|Gel of PCR ran on miniprep A of <i>Pseudomonas putida</i> attempting to isolate <i>antA</i> and <i>antC</i> genes.|center]]<br />
[[File:CarAc and carAd Ucalgary.png|500px|thumb|Gel of PCR ran on miniprep A of <i>Pseudomonas putida</i> attempting to isolate <i>carAc</i> and <i>carAd</i> genes.|center]]<br />
[[File:Minipret A antB and carAc Ucalgary.png|500px|thumb|Gel of PCR ran on miniprep A showing bands 3-5 of the correct size corresponding to antB genes and colony PCR ran using biobrick primers attempting to determine if <i>carAc</i> genes were successfully transformed into PSB1C3 plasmid.|center]] <html><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>This week we modified our PCR protocol to adjust for the homemade Taq polymerase we were using. Template DNA was boiled for 10 minutes on its own to replace the initialization step. This is because this polymerase could not tolerate the 95 degree heat during the regular initialization step. Another modification we made was to boil our primers for 5 minutes prior to adding them to the master mix. This was done to discourage the formation of primer-dimers. Finally we also attempted a touch-down PCR which started at an annealing temperature greater than the Tm of our primers and gradually worked its way down to below the Tm of the primers. This was done to increase the specificity of our reaction and also eliminate the formation of primer-dimers. Unfortunately these techniques still saw the formation of dimers, preventing us from performing a PCR purification. Again, the gel purification procedure did not lead to successful purification of our target gene. Due to the problems we have had so far our goal for next week is to perform an assay experiment to try and determine which of the <i>Pseudomonas putida</i> strains we have access to can actually degrade carbazole and therefore contain the pCAR1 plasmid. </p><br />
<br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The goal for this week was to grow different strains of<i> Pseudomonas</i> in media containing B-N media + glucose with only carbazole as a source of nitrogen. In theory only those strains which contained the pCAR1 plasmid would be able to use carbazole as a nitrogen source and thrive in this media. The strains we tested from glycerol stock included <i>P. putida</i> LD1, <i>P.putida</i> environmental strains 28-30, and <i>P. fluorescens</i> PF5. Each of these strains had two cultures grown; one with carbazole and one without it. <i>E. coli</i> grown with carbazole was used as a negative control as it does not contain the pCAR plasmid. At timepoints of 22, 46, and 117 hours of growth samples were taken to do an OD600 reading to measure bacterial growth and had an ammonia assay performed on it. The detection of ammonia acted as a proxy for detecting carbazole degradation as it is an end-product of the pathway. With this in mind, an additional control strain of LD1 was grown in media containing glucose and ammonia. This is to detect the possibility that <i>Pseudomonas</i> can use ammonia as a source of nitrogen after it has degraded carbazole which would be problematic as far as using ammonia levels to determine carbazole degradation. So far we have been having difficulties reading the results of the ammonia assay with a plate reader and may resort to testing our samples using a different assay protocol next week. The OD600 readings were initially very high at 22 hours and then dropped at 46 hours. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>This week we took our last readings from the experiment that we started last week. The OD600 readings taken at 117 hours showed minimal growth over the weekend for all strains except for environmental strain #29 (with carbazole) which increased from 0.086 to 0.313. No other strain showed an increase of 0.100 or greater. This shows that only strain #29 was able to thrive in the carbazole media, possibly indicating that it contains the pCAR plasmid enabling it to use carbazole as a nitrogen source. To verify this we used a new ammonia assay protocol to measure the production of ammonia in the different cultures and theoretically how much carbazole was degraded. Unlike the previous assay we were able to produce a standard curve with a consistent slope based on samples with known amounts of ammonia. However, the only samples that showed any elevation of ammonia levels above the blank were the LD1 samples that were cultured with ammonia chloride to control for the possibility of pseudomonas using ammonia as a nitrogen source. The ammonia levels in this sample were relatively constant which would indicate that the LD1 was not using ammonia as a nitrogen source. Unfortunately the fact that none of the test samples had elevated ammonia levels did not give us any evidence that these strains contain the pCAR plasmid. </p><br />
<br />
</html>[[File: Standard_curve_for_ammonia_assay_UCalgary2012_denitrification_group.png|500px|thumb|Standard curve for ammonia assay using OD635 absorbance.|center|]]<html><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>We received a new strain of Pseudomonas, LD2, this week that had previously been used to study the pCAR1 plasmid. It was grown up in PCA media and had its colonies used for colony PCR. We also received our new primers this week, further increasing the chances of successful PCR. Using the new strain and the new primers <i>carAa</i>, <i>carAc</i>, and <i>carAd</i> all showed at least some bands of close to the correct size. After PCR purification we obtained DNA concentrations of 21.3 ng/μL for <i>carAa</i>, 41.9 ng/ μL for <i>carAc</i>, and 32.8 ng/μL for<i> carAd</i>. These products were then restriction digested using EcoRI + PstI sites. </p><br />
<br />
</html>[[File:07.20.2012 CarAa and CarAc.jpg|500px|thumb|Gel of colony PCR from <i>Pseudomonas sp. LD2</i> for <i>carAa</i> and <i>carAc</i> genes. <i>carAa</i> expected size is 1155 bp and <i>carAc</i> is 323 bp.|center]]<br />
[[File:07.20.2012 CarAd colony PCR from LD2.jpg|500px|thumb|Gel of colony PCR from <i>Pseudomonas sp. LD2</i> for <i>carAd</i> gene. <i>carAd</i> expected size is 989 bp.|center]] <html><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The restriction digested <i>car</i> genes were ligated into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> cells. White colonies grew for<i> carAa</i> and <i>carAc</i> suggesting that ligation and transformation were successful, however no colonies grew for <i>carAd</i> suggesting that the transformation did not work. Colony PCR was performed to confirm the presence of these genes within the vectors, however only <i>carAc</i> was positive. The colonies containing the <i>carAc</i> plasmid were then mini prepped and restriction digested to isolate the plasmid and send for sequencing. Further attempts at transforming<i> carAa</i> and <i>carAd</i> were not successful. Another goal was to perform colony PCR on the <i>P. putida</i> to isolate the<i> antABC</i> genes using both the primers for the entire operon and those for the individual genes. No attempt showed any bands, although salt and temperature gradients were used as well as different types of polymerase. LD2 cultures were grown up to be plasmid prepped and genomic prepped in order to have a higher concentration of template DNA in the PCR master mix and more PCR will be attempted on these genes next week. Mutagenesis primers were also designed this week to remove the illegal restriction cut sites in the<i> carAd</i> and <i>antB</i> genes (NotI and 2xEcoRI respectively). Only 1 mutagenesis primer was designed for <i>antB</i> as one of its EcoRI sites was very close to the beginning of the gene and could be removed using a new primer. During this process it was also discovered that the original ant primers were designed for the wrong genes and therefore do not match the intended sequence. Although the new <i>antABC</i> primers were designed using the correct gene the PCR may not be working due to the length of the operon so the design of new individual primers may be necessary. Finally, we also aimed to grow up the freeze dried culture of <i>Rhodococcus erythropolis</i> (source of the<i> AmdA</i> gene) that we received from DSMZ. The reactivation media was produced and autoclaved to begin the process next week.</p><br />
<br />
</html>[[File:07.23.2012.Restriction Digest of Car genes.jpg|500px|thumb|Gel of restriction digest of car genes. A refers to <i>carAa</i>, C to <i>carAc</i>, and D to <i>carAd</i>. Ladder shows that after purification and digestion DNA is still the correct size.|center]]<br />
[[File:CarAc biobrick confirmation PCR.jpg|500px|thumb|Gel of confirmation PCR done on competent Top 10 <i>E. coli</i> cells in which a PSB1C3 vector containing the <i>carAc</i> gene had been inserted. Red X marks the 500 bp mark on the weak ladder showing the product to be the expected size (323 bp gene + 2*100 bp biobrick sites on each end).|center]] <html><br />
<br />
<h2>Week 14 (July 30 - August 3)</h2><br />
<p>We received the sequencing results this week for <i>carAc</i> which came back as a 100% match to the expected nucleotide sequence. We also successfully ligated both<i> carAa</i> and<i> carAd</i> into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into top 10 <i>E. coli</i> cells. Colony PCR verified the presence of the biobrick plasmid in these colonies. These were both miniprepped, digested and sent for sequencing by the end of the week. Using a salt gradient, addition of DMSO, and the KAPA buffer specifically designed for GC-rich amplicons we were able to amplify the ant operon from a genomic prep of LD2. However, all attempts at ligating and transforming this sequence were met with the unfortunate presence of red colonies (indicating failed insertion into the plasmid). Next week we will recut a <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> vector with Xba and Pst to attempt to mitigate this problem. We also reactivated the <i>R. erythropolis</i> following the DSMZ protocol for reviving freeze-dried cultures that can be found <a href=https://2012.igem.org/Team:Calgary/Notebook/Protocols/dmszfreezedry>here</a> using DSMZ media #1 and grew it on plates made from DSMZ media #220 at 28 degrees. Colonies grew very quickly and were used for colony PCR and to make overnight cultures for downstream applications. The colony PCR using the amidase primers was unsuccessful, so a genomic prep was performed on the overnight culture. Unfortunately we lacked some of the required equipment and the genomic prep did not work, but with a few procedural modifications and the right equipment we are confident it will work next week. A glycerol stock of the<i> R. erythropoplis</i> was also made from another overnight culture. We also started construction on the <i>carAc </i>part this week by inserting a ribosome binding site (B0034) in front of the gene. Finally, since the<i> ant</i> operon was successfully amplified by PCR we designed mutagenesis primers for the 2nd EcoRI site in the operon rather than making new individual primers. We also designed a mutagenesis primer to remove the SpeI site in <i>AmdA</i>. Now that we have conclusively proven that the LD2 strain contains the pCAR1 plasmid we aim to demonstrate its ability to degrade carbazole and other nitrogen containing heterocycles using an assay experiment similar to the one performed in Week 10.</p><br />
<br />
</html>[[File:07.30.2012. CarAa, CarAd.jpg|500px|thumb|<i>carAa</i> and <i>carAd</i> amplified from genomic prep of <i>Pseudomonas sp. LD2</i>. Amplicons are larger than expected, potentially the gel ran diagonally. The 6th lane for each gene was a negative control.|center]]<br />
[[File:07.31.2012.Ant operon.jpg|500px|thumb|<i>ant</i> operon amplified from genomic prep of <i>Pseudomonas sp. LD2</i> at the expected size of 3000 bp.|center]]<br />
[[File:08.01.2012 CarAdBB.jpg|500px|thumb|Lanes 2,4,5, and 11 contain <i>carAd</i> genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent top 10 <i>E.Coli</i>.|center]]<br />
[[File:08.01.2012 CarAaBB.jpg|500px|thumb|Lane 9 contains <i>carAa</i> genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent top 10 <i>E.Coli</i>. Although larger than expected, the positive control RFP generator in lane 10 is also larger than its expected size (~1200 bp including biobrick bookends) indicating that the gel may have a slant in it.|center]]<html><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p>This week we received the sequencing results for <i>carAa</i> and <i>carAd</i>. <i>carAd</i> was a match, however <i>carAa</i> did not match at all and had misplaced biobrick restriction enzyme sites. Due to this we decided to restart the biobricking process for <i>carAa</i> all the way back to PCR from the plasmid. By the end of the week we had progressed to the point of transforming it into competent <i>E. coli</i> cells, however a thick lawn of bacteria had grown overnight making it impossible to pick colonies. A streak plate was made from this plate to attempt to get less dense growth. Since the sequencing came back positive for <i>carAd</i> the next step was to perform site directed mutagenesis on its NotI restriction enzyme site using the primers that were designed previously (for site-directed mutagenesis optimization, please refer to Desulfurization Journal Week 14). The PCR was successful, showing very bright bands at 3000 bp, and the PCR product was digested with DpnI to remove parental DNA. Transformation of this product will indicate whether the mutagenesis was successful. We were also able to successfully perform a genomic prep of <i>Rhodococcus erythropolis</i> and amplify the <i>AmdA</i> gene this week. It was ligated into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> by Friday, and a confirmation colony PCR was run on Friday evening to verify that the gene was inserted. These results will be available by Monday. After many attempts the <i>ant</i> operon was finally ligated and transformed as well this week. Confirmation PCR showed bands of about 3000 bp which is consistent with the size of the operon. By Friday the successful colonies had been grown up and miniprepped, ready to send for sequencing early next week. The <i>carAc</i>/B0034 construct was also successfully transformed this week and verified by running a PCR using the B0034 forward primer. It was also miniprepped and is ready to send for sequencing next week. </p><br />
<p>In addition to this work constructing biobricks we also started an experiment to demonstrate the function of the pCAR plasmid in LD2. Strains of LD2 were grown up in LB and then washed and placed in B-N media + glucose and various experimental additions. These additions included carbazole, 4-PBAH (a nitrogen containing naphthenic acid), ammonia chloride, and a negative control with no nitrogen source. Strains of <i>E. coli</i> were also grown under the same conditions to compare growth and ammonia production of LD2 to a strain that does not contain the pCAR plasmid. These results should act as a preview of what we expect our final biobrick containing <i>E. col</i>i species to produce as well as allowing us to establish LD2 as a positive control for this experiment. </p><br />
<br />
<br />
</html>[[File:08.07.2012 amdA pcr from genomic prep.jpg|500px|thumb|<i>AmdA</i> amplified from the genomic prep of <i>R. Erythropolis</i> shows bands just greater than 1500 base pairs in lanes 2,4-6,9, and 12-13. Lane 14 was a negative control. The expected size of <i>AmdA</i> is 1566 base pairs.|center]]<br />
[[File:08.09.2012 confirmation pcr of ant operon.jpg|500px|thumb|The two labelled lanes show amplification at 3000 base pairs from a confirmation colony PCR using the biobrick primers. The expected size of the <i>ant</i> operon with the biobrick bookends is about 3100 base pairs.|center]]<br />
[[File:08.10.2012 carad notI mutagenesis.jpg|500px|thumb|Strong amplification at just above 3000 base pairs from the <i>carAd</i> PCR using mutagenic primers. Since PSB1C3 is 2070 base pairs and <i>carAd</i> is 989 base pairs the amplification should run at about 3059 base pairs. 3 different amounts of template DNA were tested in lanes 2-7 as indicated and a positive control showed weaker amplification in lanes 8-9 at about 4000 base pairs.|center]]<br />
<html><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>The sequencing for the <i>carAc</i>/<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> construct came back with only <i>carAc</i> and no evidence of a <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> site preceding it. The confirmation PCR that was run last week using the <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> forward primer seemed to indicate that it was inserted, however since <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> is only 12 base pairs the primer may not have been specific enough. The construction on this part was restarted this week. <i>carAa</i> colony PCR on last week's transformation showed two positive colonies, however upon culturing, miniprepping, and digesting these colonies the plasmid appeared to be too large (3000 base pairs as opposed to the expected 2000). This may be due to a problem of a mislabeled plasmid tube that has been experienced by other team members, causing the gene to be inserted into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a> rather than <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. To test this, a streak plate was made from these colonies and grown on an ampicillin resistant plate. Mutagenized <i>carAd</i> was successfully transformed into Top 10 <i>E. coli</i> cells, cultured, and miniprepped. The miniprep product was digested with NotI to test whether the NotI site was successfully removed. These results were good, as the plasmid ran at 2000 base pairs and the insert at just under 1000 base pairs in the mutagenized <i>carAd</i> whereas the non-mutagenized <i>carAd</i> had its insert cut into 2 bands. This will be sent for sequencing next week to confirm the mutation. Unfortunately the sequencing results came back negative for the <i>ant</i> operon meaning we will need to restart genomic PCR on it next week. Multiple transformations and confirmation PCRs were run on <i>AmdA</i> this week, however none displayed a band size of 1700-1800 base pairs as expected. We have continued to try different digested plasmids as other team members also had difficulties this week with ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, possibly indicating a problem with our Antarctic Phosphatase stock.</p><br />
<p>The other news from this week was the final results from our carbazole degradation experiment attempting to demonstrate LD2's ability to degrade carbazole and hopefully other nitrogen containing compounds. Unfortunately, these results were very difficult to make sense of as the LD2 that were cultured with carbazole grew, but did not show elevated ammonia levels which would be indicative of carbazole degradation. One of our controls was LD2 cultured with ammonium chloride to test whether LD2 was capable of using ammonia as a nitrogen source. This would pose a problem for us as we were planning to use an increase in ammonia levels as a proxy for carbazole degradation rather than measuring carbazole degradation directly using GC-MS. However, the LD2 showed significantly less ammonia after 44 hours when compared to 26 hours indicating that it may be degraded, either by the LD2 or by an unaccounted for abiotic process. This has lead us to decide to use GC-MS and directly measure carbazole levels in our sample the next time we do this experiment.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<p>The <i>carAa</i> was re-streaked onto ampicillin plates and grew, probably showing that it was ligated into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a>, not <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, which explained the irregularity of our confirmation digest last week. It was sent for sequencing, but in case it was incorrect we also started a new <i>carAa</i> pipeline by PCRing it from the <i>Pseudomonas</i> genome again and redigesting and ligating. It was transformed into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> over the weekend. <i>carAc</i> did not see a lot of progress this week as we spent much of the time attempting to construct a RBS (B0034) in front of it, but were unsuccessful. <i>carAd</i> was also subjected to this construction with B0034 and we were able to send it for sequencing by the end of the week. Several new constructs were also started for <i>carAd</i> in case the sequencing is negative. The <i>ant</i> operon was successfully transformed and confirmed by colony PCR this week, after which it was grown up and sent for sequencing. We also began to attempt mutagenesis on one of the EcoRI sites in the operon, but had no amplification of the plasmid. We also re-digested some PCR purified product to start another ligation in case sequencing failed. After many attempts we were also able to finally successfully transform an <i>AmdA</i>/PSB1C3 plasmid into Top 10 cells this week. After a miniprep they showed bands of about 1800 base pairs on the confirmation digest gel, so they were also sent for sequencing this week and had mutagenesis started on the illegal SpeI site. Unfortunately, the sequencing sent early in the week came back with ambiguous results. The forward reaction for<i> antABC</i> was a match, but its reverse reaction matched to <i>carAa</i>. Both <i>carAa</i> reactions matched nothing. Since these two genes are over 20 000 base pairs apart on the template plasmid it is virtually impossible that they were actually combined into one plasmid (PCR extension time would not have been long enough and even if it was the product would have run much above the 3000 base pair mark). This is possibly a mistake on Eurofins end, so we are not 100% sure if we have 1 good gene, both of them, or neither. Also the sequence for mutagenized <i>carAd</i> showed only about the first 100 base pairs, which were correct however not long enough meaning it was a poor reaction and inconclusive. Finally, we also re-started our <a href=https://2012.igem.org/Team:Calgary/Notebook/Protocols/carbazole>carbazole degradation assay</a> using the GC-MS to quantitatively measure the loss of carbazole at biological sciences. This process will take 2 weeks to demonstrate LD2's ability to degrade carbazole. We are forcing it to use carbazole as a carbon and a nitrogen source in one sample and as only a nitrogen source in another sample (this sample has had glucose added to it).</p><br />
<br />
</html>[[File:AmdA colonyPCR calgary12.JPG|500px|thumb|Amplification is seen in lane 7 of this PCR of top 10 colonies into which AmdA/PSB1C3 had been transformed. This indicates that <i>AmdA</i> has been successfully inserted into a biobrick vector.|center]]<br />
[[File:NewcarAagenomePCR calgary12.jpg|500px|thumb|<i>carAa</i> amplified from pCAR1.2 plasmid of <i>Pseudomonas putida</i>.|center]] <html><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<p>The new stock of <i>carAa</i> that we had PCRed from LD2 was sent for sequencing this week and came back as a match, so we have begun constructing a RBS site and a TetR promoter (<a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>) in front of it. At this point all the <i>car</i> genes have been successfully amplified, inserted into a biobrick plasmid, and sequence verified. We also continued to attempt to construct <i>carAc</i> with an RBS site (<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>) in front of it, but had no success. On a better note, we confirmed this week that<i> carAd</i> was mutated properly and we were also able to insert an RBS site in front of it. The next step for this gene will be to attempt a plasmid switch as it is currently in <a href="http://partsregistry.org/Part:pSB1A3">pSB1A3</a> and needs to be in <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> for submission to the parts registry. After sending a new batch of the ant operon for sequencing we got the same confusing results as we did last week. We therefore hypothesized that our primers were annealing to a homologous region that is similar to the<i> ant</i> genes and very close to <i>carAa</i>. This means that we need to restart PCR on this gene as all of our current product is incorrect. The sequencing results for <i>AmdA</i> that came back this week were also somewhat confusing as they did not quite match the sequence we expected. However, they were a 100% match for the <i>AmdA</i> gene in a different strain of <i>Rhodococcus erythropolis</i>. This means that DSMZ probably sent us the wrong strain of bacteria and that we were very lucky that the primers we designed based off of the expected template actually worked to amplify a slightly different gene in another strain. The good news is that this new sequence contains no illegal cut sites, which explains why mutagenesis was not working (there was nothing to mutate) and allows us to immediately begin construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> in front of <i>AmdA</i> this week. </p><br />
<br />
</html>[[File:Caraa colonyPCR calgary12.JPG|500px|thumb|Colony PCR amplification from top 10 <i>E. coli</i> cells with a PSB1C3/<i>carAa</i> construct transformed into them. Lanes 1-3, 5, and 7 were sent for sequencing with lane 5 coming back positive.|center]]<html><br />
<br />
<h2>Week 19 (September 3- September 7)</h2><br />
<p><i>carAd</i>/<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> was successfully plasmid switched into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, making it ready for submission. Construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> and <i>carAa</i> may have been successful as the digest of the miniprep looked promising. It will be sent for sequencing next week. Unfortunately there was no progress made on the constructions of <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> or <i>carAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. These will be redigested and religated for next week. We could not amplify the <i>ant</i> operon despite trying many variations of PCR using high fidelity KAPA polymerase. This is probably due to the multiple, large regions of homology on the operon that the reverse primer binds to making amplification difficult. Because the function of this gene can theoretically be replaced by <i>AmdA</i>, we may scale back efforts dedicated to it, as the chances of successful amplification appear slim. </p><br />
<br />
<h2>Weeks 20-22 (September 10 - September 30)</h2><br />
<p>Constructs that we continued to work on this week included <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, <i>carAa</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, and <i>carAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. Some sequencing was sent, but no positive results were obtained. A new approach to biobrick the <i>ant</i> operon was implemented this week. A new forward primer was designed that would anneal about 1500 base pairs upstream of the <i>ant</i> operon, avoiding the region of homology that is repeated at various points on the plasmid. Once this region is successfully amplified we could use this amplified DNA as a template for another PCR reaction which would use the normal <i>ant</i> primers. Since the template would no longer include regions of homology these primers should be free to work properly and amplify the <i>ant</i> operon. The PCR using the new primers was started this week, and although amplification at 4500 base pairs was observed as expected, there was also a lot of non-specific amplification. This means that to purify the amplified DNA that we want and avoid the other products a dreaded gel extraction had to be performed which, as usual, did not work. At this point it was decided that further attempts to amplify the <i>ant</i> operon would be futile and our efforts would be better served working on constructing the parts we do have. Unfortunately no progress was made in these weeks on construction. We have also obtained GC-MS readings from the carbazole degradation experiment that showed that <i>Pseudomonas</i> LD2 can indeed degrade carbazole under low glucose conditions.</p><br />
<br />
<br />
<br />
<br />
</html><br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/DenitrogenationTeam:Calgary/Notebook/Denitrogenation2012-10-03T23:46:36Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Denitrogenation Journal|<br />
CONTENT=<br />
<html><br />
<br />
<h2>Week 1 (May 1-4)</h2><br />
<p>The following section covers wetlab aspect of our overall project focusing on microbial conversion of naphthenic acids into economically-valuable hydrocarbons. The approach taken in this endeavour will be from four strategic starting points - ring cleavage, decarboxylation, denitrogenation, and desulfurization. Overall, the 'hydrocarbons' aspect of the project is a critical one to our overall design and construction of a bioreactor capable of not only detecting but also converting naphthenic acids and other toxins to useful or at least innocuous compounds. </p><br />
<br />
<h2>Week 2-3 (May 7-18)</h2><br />
<p>In the first two weeks of iGEM our group has focused on reviewing literature regarding the bioremediation of nitrogen groups attached to naphthenic acids. The most prevalent N heterocycle is carbazole, representing 75% of total nitrogen by mass. The upper pathway of carbazole biodegradation is catalyzed by the enzymes coded for by the <i>car</i> operon, <i>CarA</i> (<i>CarAaAcAd</i>), <i>CarB</i> (<i>CarBaBb</i>), and <i>CarC</i>. These enzymes convert carbazole to anthralinic acid. The lower pathway is catalyzed by the enzymes of the <i>ant</i> operon, <i>antA, B, and C</i>, yielding catechol while releasing CO2 and NH3. The <i>car</i> and <i>ant</i> operons are both regulated by the P<sup>ant</sup> promoter which is induced by the protein, antR. <i>carAa</i> also has its own promoter which is not induced by <i>antR</i> (Diaz & Garcia, 2010). We have also investigated an alternative pathway using <i>CarA</i> combined with an amidase (<i>amdA</i>) that selectively cleaves NH2 from an intermediate of the car pathway (Xu et al, 2006). This could bypass much of the car/ant pathway and is possibly more efficient.</p><br />
<p>We have decided to use <i>Pseudomonas resinovorans</i> and <i>Rhodococcus erythropolis</i> to derive these genes from. <i>carABC</i> and <i>antABC</i> from<i> P. resinovorans</i> has been shown to have a wide range of nitrogen containing substrate specificity. <i>R. erythropolis</i> contains the <i>amdA</i> gene that we wish to use, and some evidence suggests that it may also be able to degrade sulfur rings through its <i>carABC</i> pathway.</p><br />
<br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p>This week we reviewed the primers listed in the database and also designed some new ones. Primers for <i>carAa</i>,<i> carAc</i>, and <i>carAd</i> were designed individually, while primers for <i>CarBaBbC</i> and <i>antABC</i> were designed to encompass multiple genes in a sequence. These primers were designed to be used on <i>Pseudomonas putida</i> which we decided to use as our gene source since it was available to us as opposed to ordering <i>Pseudomonas resinovorans</i>. We also designed a primer for the<i> AmdA</i> gene from <i>Rhodococcus erthyroplois</i>. In addition to ordering these primers we also ordered the nitrogen containing compounds that we will need to test these enzymes on. We decided on using carbazole to make sure the enzymes can perform their natural function as well as pyrrolidine to test them on a similar ring structure. We also ordered cyclohexamine in order to independently test the function of the alternative <i>AmdA</i> pathway. Finally, we decided to eventually order 4-Piperidine butyric acid hydrochloride to test how the enzymes will work on nitrogen containing naphthenic acids. However, we decided since it is a very expensive compound we would wait to make sure the enzyme's work on more simple compounds before ordering it.</p><br />
<p>We also started our work in the wet lab by plating colonies and making an overnight culture of -80 freezer stock <i>Psuedomonas putida</i> on Thursday. We also resuspended the primers for <i>carAa</i>, <i>carAc</i>, <i>carAd</i>,<i> antA</i>, <i>antB</i>, and <i>ant C</i> that were already in the database. We were able to use the colonies that grew on the streak plates to start a colony PCR to attempt to isolate each of these genes on Friday.</p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2><br />
<p>On Monday and Tuesday we used the database primers to attempt to isolate <i>carAa</i>, <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i>antB</i>, and <i>antC</i> from the <i>Psuedomonas putida</i> we plated last week. <i>carAd</i>, <i>antB</i>, and <i>antC</i> were all put in the gradient PCR machine to account for the wide range of their primers’ melting points, while the others were done via regular PCR. Unfortunately, no positive results were obtained from these reactions. The PCR on <i>carAc</i> and <i>antA</i> was repeated on Wednesday, still not giving positive results. Finally, we attempted to use a salt concentration gradient on the PCR reaction for <i>carAc</i> and <i>antA</i>, using concentrations that ranged from 1.0 microlitres/tube to 2.0 microlitres/tube in increments of 0.2 microlitres/tube. This helped as <i>carAc</i> showed bands in samples that had concentrations of 1.2 and 1.4 microlitres/tube. <i> antA</i> also showed bands, however they were not the correct size, indicating contamination and/or non-specific annealing of the primer. The positive control also showed bands of the correct size. Earlier in the week we also made overnight cultures of 3 environmental strains (28, 29, 30) of <i>Psuedomonas putida</i> from -80 glycerol stock. On Friday we performed a genomic prep on these cultures, and plan on attempting to amplify genes from the isolated DNA next week. </p><br />
<br />
</html>[[File:CarAa and carAc Ucalgary12.png|500px|thumb|Gel of PCR using database primers for <i>carAa</i> and <i>carAc</i> on <i>Pseudomonas putida</i>|center]]<html><br />
<br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> We started off this week by determining the DNA concentration of our genomic prep samples from last week using the nanodrop. DNA concentration for all three<i> P. putida</i> strains was at least 1000 ng/microlitre, well above what was needed for PCR. 1/2 and 1/3 dilutions were prepared for all three strains so as not to have an excess of template DNA in PCR reactions. PCR was performed on all 3 strains using primers for <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i> antB</i>, and <i>antC</i> using 6 replicates per gene. The only successful amplification appeared to be <i>antB</i> and <i>carAc</i>, both from strain 28 (with weaker bands in strain 29). We then performed another PCR, just on those two genes with an increased amount of Taq polymerase to hopefully get enough amplified DNA to move forward with. This resulted in strong bands for both at the expected size. We then performed PCR purification using the Qiaquick kit and obtained samples containing 33.5 ng/microlitre of <i>antB</i> DNA and 129 ng/microlitre of <i>carAc </i>DNA. These concentrations were both sufficient to begin a restriction digest and ligation of these parts into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. Next week we hope to verify the results of the restriction digest, continue to amplify <i>carAc</i> and <i>antB</i> from strain 28, and hopefully submit a biobrick for sequencing.</p><br />
<br />
</html>[[File:AntB and carAc Ucalgary.png|500px|thumb|Gel of PCR ran on genomic DNA of <i>Pseudomonas putida</i> environmental strain 28 using primers for <i>carAc</i> and <i>antB</i>. |center]]<html><br />
<br />
<br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p>The purified PCR products for <i>carAc</i> and <i>antB</i> were digested with restriction enzymes EcoRI+SpeI, EcoRI+PstI, and XbaI+PstI. However, only <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmids that had been digested with EcoRI+SpeI and EcoRI+PstI were available to attempt ligation. Gel results were inconclusive on the restriction digest product as the plasmid size appeared much larger than expected. However, transformation was still attempted on the ligation products for both genes (both using EcoRI+SpeI ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid). The transformation products were plated onto chloroamphenicol (chlor) plates to select for colonies that had the chloro resistance gene on the <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid. Also this week another round of PCR was performed for the <i> car</i> and <i> ant</i> genes, however all, but <i>carAc</i> showed bands in the negative control lane, possibly indicating contamination or the formation of primer dimers. The <i>carAc</i> bands were fairly weak and a PCR purification resulted in very low DNA concentration, insufficient to move onto restriction digest. Next week's plan hinges heavily on the result of the transformation from this Friday. </p><br />
<br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>The results of our transformation on <i>carAc</i> and <i>antB</i> from last week showed mostly red colonies indicating that the ligation was unsuccessful and that the PSB1C3 plasmid simply closed on itself with no insert. However, there were three colonies for <i>carAc</i> that were white (cut with both X+P and E+P). Colony PCR was performed on these three colonies using biobrick primer sets to verify that the part had been inserted in these colonies, but unfortunately all PCR results were negative indicating an unsuccessful ligation.</p><br />
<p>We also performed a miniprep on <i>Pseudomonas putida</i> strain #28 this week using a home-made protocol rather than the Qiagen kit. Miniprep A had a DNA concentration of 1539.8 ng/μL and Miniprep B had DNA concentration of 1001.2 ng/μL possibly indicating that there may have been a lot of genomic DNA contamination. However miniprep A product was still used as a PCR template for a reaction attempting to isolate <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i>antB</i>, and <i>antC</i>. Special conditions for this PCR included replacing 60 μL of water with 60 μL of betaine heated to 37 degrees Celsius and running a Mg gradient ranging from 0.5 μL - 2.5 μL per tube for each gene. Of these only <i>antB</i> had bands of the right size in 3 of the lanes (Mg concentrations of 1.5, 2, and 2.5 μL). However, all of the <i>antB</i> bands (including the negative control) contained a contamination band at around 100 bases, probably due to the formation of primer dimers. This forced us to do a gel extraction rather than a PCR purification. This gel extraction did not work as the nanodrop results indicated high amounts of agarose contamination. Another PCR was performed using the same conditions, but using miniprep B instead as there was no more miniprep A product remaining. Unfortunately there were no bands for <i>antB</i>, only the 100 base pair contamination bands. </p><br />
<br />
</html>[[File:AntA and antC Ucalgary.png|500px|thumb|Gel of PCR ran on miniprep A of <i>Pseudomonas putida</i> attempting to isolate <i>antA</i> and <i>antC</i> genes.|center]]<br />
[[File:CarAc and carAd Ucalgary.png|500px|thumb|Gel of PCR ran on miniprep A of <i>Pseudomonas putida</i> attempting to isolate <i>carAc</i> and <i>carAd</i> genes.|center]]<br />
[[File:Minipret A antB and carAc Ucalgary.png|500px|thumb|Gel of PCR ran on miniprep A showing bands 3-5 of the correct size corresponding to antB genes and colony PCR ran using biobrick primers attempting to determine if <i>carAc</i> genes were successfully transformed into PSB1C3 plasmid.|center]] <html><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>This week we modified our PCR protocol to adjust for the homemade Taq polymerase we were using. Template DNA was boiled for 10 minutes on its own to replace the initialization step. This is because this polymerase could not tolerate the 95 degree heat during the regular initialization step. Another modification we made was to boil our primers for 5 minutes prior to adding them to the master mix. This was done to discourage the formation of primer-dimers. Finally we also attempted a touch-down PCR which started at an annealing temperature greater than the Tm of our primers and gradually worked its way down to below the Tm of the primers. This was done to increase the specificity of our reaction and also eliminate the formation of primer-dimers. Unfortunately these techniques still saw the formation of dimers, preventing us from performing a PCR purification. Again, the gel purification procedure did not lead to successful purification of our target gene. Due to the problems we have had so far our goal for next week is to perform an assay experiment to try and determine which of the <i>Pseudomonas putida</i> strains we have access to can actually degrade carbazole and therefore contain the pCAR1 plasmid. </p><br />
<br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The goal for this week was to grow different strains of<i> Pseudomonas</i> in media containing B-N media + glucose with only carbazole as a source of nitrogen. In theory only those strains which contained the pCAR1 plasmid would be able to use carbazole as a nitrogen source and thrive in this media. The strains we tested from glycerol stock included <i>P. putida</i> LD1, <i>P.putida</i> environmental strains 28-30, and <i>P. fluorescens</i> PF5. Each of these strains had two cultures grown; one with carbazole and one without it. <i>E. coli</i> grown with carbazole was used as a negative control as it does not contain the pCAR plasmid. At timepoints of 22, 46, and 117 hours of growth samples were taken to do an OD600 reading to measure bacterial growth and had an ammonia assay performed on it. The detection of ammonia acted as a proxy for detecting carbazole degradation as it is an end-product of the pathway. With this in mind, an additional control strain of LD1 was grown in media containing glucose and ammonia. This is to detect the possibility that <i>Pseudomonas</i> can use ammonia as a source of nitrogen after it has degraded carbazole which would be problematic as far as using ammonia levels to determine carbazole degradation. So far we have been having difficulties reading the results of the ammonia assay with a plate reader and may resort to testing our samples using a different assay protocol next week. The OD600 readings were initially very high at 22 hours and then dropped at 46 hours. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>This week we took our last readings from the experiment that we started last week. The OD600 readings taken at 117 hours showed minimal growth over the weekend for all strains except for environmental strain #29 (with carbazole) which increased from 0.086 to 0.313. No other strain showed an increase of 0.100 or greater. This shows that only strain #29 was able to thrive in the carbazole media, possibly indicating that it contains the pCAR plasmid enabling it to use carbazole as a nitrogen source. To verify this we used a new ammonia assay protocol to measure the production of ammonia in the different cultures and theoretically how much carbazole was degraded. Unlike the previous assay we were able to produce a standard curve with a consistent slope based on samples with known amounts of ammonia. However, the only samples that showed any elevation of ammonia levels above the blank were the LD1 samples that were cultured with ammonia chloride to control for the possibility of pseudomonas using ammonia as a nitrogen source. The ammonia levels in this sample were relatively constant which would indicate that the LD1 was not using ammonia as a nitrogen source. Unfortunately the fact that none of the test samples had elevated ammonia levels did not give us any evidence that these strains contain the pCAR plasmid. </p><br />
<br />
</html>[[File: Standard_curve_for_ammonia_assay_UCalgary2012_denitrification_group.png|500px|thumb|Standard curve for ammonia assay using OD635 absorbance.|center|]]<html><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>We received a new strain of Pseudomonas, LD2, this week that had previously been used to study the pCAR1 plasmid. It was grown up in PCA media and had its colonies used for colony PCR. We also received our new primers this week, further increasing the chances of successful PCR. Using the new strain and the new primers <i>carAa</i>, <i>carAc</i>, and <i>carAd</i> all showed at least some bands of close to the correct size. After PCR purification we obtained DNA concentrations of 21.3 ng/μL for <i>carAa</i>, 41.9 ng/ μL for <i>carAc</i>, and 32.8 ng/μL for<i> carAd</i>. These products were then restriction digested using EcoRI + PstI sites. </p><br />
<br />
</html>[[File:07.20.2012 CarAa and CarAc.jpg|500px|thumb|Gel of colony PCR from <i>Pseudomonas sp. LD2</i> for <i>carAa</i> and <i>carAc</i> genes. <i>carAa</i> expected size is 1155 bp and <i>carAc</i> is 323 bp.|center]]<br />
[[File:07.20.2012 CarAd colony PCR from LD2.jpg|500px|thumb|Gel of colony PCR from <i>Pseudomonas sp. LD2</i> for <i>carAd</i> gene. <i>carAd</i> expected size is 989 bp.|center]] <html><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The restriction digested <i>car</i> genes were ligated into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> cells. White colonies grew for<i> carAa</i> and <i>carAc</i> suggesting that ligation and transformation were successful, however no colonies grew for <i>carAd</i> suggesting that the transformation did not work. Colony PCR was performed to confirm the presence of these genes within the vectors, however only <i>carAc</i> was positive. The colonies containing the <i>carAc</i> plasmid were then mini prepped and restriction digested to isolate the plasmid and send for sequencing. Further attempts at transforming<i> carAa</i> and <i>carAd</i> were not successful. Another goal was to perform colony PCR on the <i>P. putida</i> to isolate the<i> antABC</i> genes using both the primers for the entire operon and those for the individual genes. No attempt showed any bands, although salt and temperature gradients were used as well as different types of polymerase. LD2 cultures were grown up to be plasmid prepped and genomic prepped in order to have a higher concentration of template DNA in the PCR master mix and more PCR will be attempted on these genes next week. Mutagenesis primers were also designed this week to remove the illegal restriction cut sites in the<i> carAd</i> and <i>antB</i> genes (NotI and 2xEcoRI respectively). Only 1 mutagenesis primer was designed for <i>antB</i> as one of its EcoRI sites was very close to the beginning of the gene and could be removed using a new primer. During this process it was also discovered that the original ant primers were designed for the wrong genes and therefore do not match the intended sequence. Although the new <i>antABC</i> primers were designed using the correct gene the PCR may not be working due to the length of the operon so the design of new individual primers may be necessary. Finally, we also aimed to grow up the freeze dried culture of <i>Rhodococcus erythropolis</i> (source of the<i> AmdA</i> gene) that we received from DSMZ. The reactivation media was produced and autoclaved to begin the process next week.</p><br />
<br />
</html>[[File:07.23.2012.Restriction Digest of Car genes.jpg|500px|thumb|Gel of restriction digest of car genes. A refers to <i>carAa</i>, C to <i>carAc</i>, and D to <i>carAd</i>. Ladder shows that after purification and digestion DNA is still the correct size.|center]]<br />
[[File:CarAc biobrick confirmation PCR.jpg|500px|thumb|Gel of confirmation PCR done on competent Top 10 <i>E. coli</i> cells in which a PSB1C3 vector containing the <i>carAc</i> gene had been inserted. Red X marks the 500 bp mark on the weak ladder showing the product to be the expected size (323 bp gene + 2*100 bp biobrick sites on each end).|center]] <html><br />
<br />
<h2>Week 14 (July 30 - August 3)</h2><br />
<p>We received the sequencing results this week for <i>carAc</i> which came back as a 100% match to the expected nucleotide sequence. We also successfully ligated both<i> carAa</i> and<i> carAd</i> into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into top 10 <i>E. coli</i> cells. Colony PCR verified the presence of the biobrick plasmid in these colonies. These were both miniprepped, digested and sent for sequencing by the end of the week. Using a salt gradient, addition of DMSO, and the KAPA buffer specifically designed for GC-rich amplicons we were able to amplify the ant operon from a genomic prep of LD2. However, all attempts at ligating and transforming this sequence were met with the unfortunate presence of red colonies (indicating failed insertion into the plasmid). Next week we will recut a <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> vector with Xba and Pst to attempt to mitigate this problem. We also reactivated the <i>R. erythropolis</i> following the DSMZ protocol for reviving freeze-dried cultures that can be found <a href=https://2012.igem.org/Team:Calgary/Notebook/Protocols/dmszfreezedry>here</a> using DSMZ media #1 and grew it on plates made from DSMZ media #220 at 28 degrees. Colonies grew very quickly and were used for colony PCR and to make overnight cultures for downstream applications. The colony PCR using the amidase primers was unsuccessful, so a genomic prep was performed on the overnight culture. Unfortunately we lacked some of the required equipment and the genomic prep did not work, but with a few procedural modifications and the right equipment we are confident it will work next week. A glycerol stock of the<i> R. erythropoplis</i> was also made from another overnight culture. We also started construction on the <i>carAc </i>part this week by inserting a ribosome binding site (B0034) in front of the gene. Finally, since the<i> ant</i> operon was successfully amplified by PCR we designed mutagenesis primers for the 2nd EcoRI site in the operon rather than making new individual primers. We also designed a mutagenesis primer to remove the SpeI site in <i>AmdA</i>. Now that we have conclusively proven that the LD2 strain contains the pCAR1 plasmid we aim to demonstrate its ability to degrade carbazole and other nitrogen containing heterocycles using an assay experiment similar to the one performed in Week 10.</p><br />
<br />
</html>[[File:07.30.2012. CarAa, CarAd.jpg|500px|thumb|<i>carAa</i> and <i>carAd</i> amplified from genomic prep of <i>Pseudomonas sp. LD2</i>. Amplicons are larger than expected, potentially the gel ran diagonally. The 6th lane for each gene was a negative control.|center]]<br />
[[File:07.31.2012.Ant operon.jpg|500px|thumb|<i>ant</i> operon amplified from genomic prep of <i>Pseudomonas sp. LD2</i> at the expected size of 3000 bp.|center]]<br />
[[File:08.01.2012 CarAdBB.jpg|500px|thumb|Lanes 2,4,5, and 11 contain <i>carAd</i> genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent top 10 <i>E.Coli</i>.|center]]<br />
[[File:08.01.2012 CarAaBB.jpg|500px|thumb|Lane 9 contains <i>carAa</i> genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent top 10 <i>E.Coli</i>. Although larger than expected, the positive control RFP generator in lane 10 is also larger than its expected size (~1200 bp including biobrick bookends) indicating that the gel may have a slant in it.|center]]<html><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p>This week we received the sequencing results for <i>carAa</i> and <i>carAd</i>. <i>carAd</i> was a match, however <i>carAa</i> did not match at all and had misplaced biobrick restriction enzyme sites. Due to this we decided to restart the biobricking process for <i>carAa</i> all the way back to PCR from the plasmid. By the end of the week we had progressed to the point of transforming it into competent <i>E. coli</i> cells, however a thick lawn of bacteria had grown overnight making it impossible to pick colonies. A streak plate was made from this plate to attempt to get less dense growth. Since the sequencing came back positive for <i>carAd</i> the next step was to perform site directed mutagenesis on its NotI restriction enzyme site using the primers that were designed previously (for site-directed mutagenesis optimization, please refer to Desulfurization Journal Week 14). The PCR was successful, showing very bright bands at 3000 bp, and the PCR product was digested with DpnI to remove parental DNA. Transformation of this product will indicate whether the mutagenesis was successful. We were also able to successfully perform a genomic prep of <i>Rhodococcus erythropolis</i> and amplify the <i>AmdA</i> gene this week. It was ligated into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> by Friday, and a confirmation colony PCR was run on Friday evening to verify that the gene was inserted. These results will be available by Monday. After many attempts the <i>ant</i> operon was finally ligated and transformed as well this week. Confirmation PCR showed bands of about 3000 bp which is consistent with the size of the operon. By Friday the successful colonies had been grown up and miniprepped, ready to send for sequencing early next week. The <i>carAc</i>/B0034 construct was also successfully transformed this week and verified by running a PCR using the B0034 forward primer. It was also miniprepped and is ready to send for sequencing next week. </p><br />
<p>In addition to this work constructing biobricks we also started an experiment to demonstrate the function of the pCAR plasmid in LD2. Strains of LD2 were grown up in LB and then washed and placed in B-N media + glucose and various experimental additions. These additions included carbazole, 4-PBAH (a nitrogen containing naphthenic acid), ammonia chloride, and a negative control with no nitrogen source. Strains of <i>E. coli</i> were also grown under the same conditions to compare growth and ammonia production of LD2 to a strain that does not contain the pCAR plasmid. These results should act as a preview of what we expect our final biobrick containing <i>E. col</i>i species to produce as well as allowing us to establish LD2 as a positive control for this experiment. </p><br />
<br />
<br />
</html>[[File:08.07.2012 amdA pcr from genomic prep.jpg|500px|thumb|<i>AmdA</i> amplified from the genomic prep of <i>R. Erythropolis</i> shows bands just greater than 1500 base pairs in lanes 2,4-6,9, and 12-13. Lane 14 was a negative control. The expected size of <i>AmdA</i> is 1566 base pairs.|center]]<br />
[[File:08.09.2012 confirmation pcr of ant operon.jpg|500px|thumb|The two labelled lanes show amplification at 3000 base pairs from a confirmation colony PCR using the biobrick primers. The expected size of the <i>ant</i> operon with the biobrick bookends is about 3100 base pairs.|center]]<br />
[[File:08.10.2012 carad notI mutagenesis.jpg|500px|thumb|Strong amplification at just above 3000 base pairs from the <i>carAd</i> PCR using mutagenic primers. Since PSB1C3 is 2070 base pairs and <i>carAd</i> is 989 base pairs the amplification should run at about 3059 base pairs. 3 different amounts of template DNA were tested in lanes 2-7 as indicated and a positive control showed weaker amplification in lanes 8-9 at about 4000 base pairs.|center]]<br />
<html><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>The sequencing for the <i>carAc</i>/<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> construct came back with only <i>carAc</i> and no evidence of a <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> site preceding it. The confirmation PCR that was run last week using the <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> forward primer seemed to indicate that it was inserted, however since <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> is only 12 base pairs the primer may not have been specific enough. The construction on this part was restarted this week. <i>carAa</i> colony PCR on last week's transformation showed two positive colonies, however upon culturing, miniprepping, and digesting these colonies the plasmid appeared to be too large (3000 base pairs as opposed to the expected 2000). This may be due to a problem of a mislabeled plasmid tube that has been experienced by other team members, causing the gene to be inserted into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a> rather than <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. To test this, a streak plate was made from these colonies and grown on an ampicillin resistant plate. Mutagenized <i>carAd</i> was successfully transformed into Top 10 <i>E. coli</i> cells, cultured, and miniprepped. The miniprep product was digested with NotI to test whether the NotI site was successfully removed. These results were good, as the plasmid ran at 2000 base pairs and the insert at just under 1000 base pairs in the mutagenized <i>carAd</i> whereas the non-mutagenized <i>carAd</i> had its insert cut into 2 bands. This will be sent for sequencing next week to confirm the mutation. Unfortunately the sequencing results came back negative for the <i>ant</i> operon meaning we will need to restart genomic PCR on it next week. Multiple transformations and confirmation PCRs were run on <i>AmdA</i> this week, however none displayed a band size of 1700-1800 base pairs as expected. We have continued to try different digested plasmids as other team members also had difficulties this week with ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, possibly indicating a problem with our Antarctic Phosphatase stock.</p><br />
<p>The other news from this week was the final results from our carbazole degradation experiment attempting to demonstrate LD2's ability to degrade carbazole and hopefully other nitrogen containing compounds. Unfortunately, these results were very difficult to make sense of as the LD2 that were cultured with carbazole grew, but did not show elevated ammonia levels which would be indicative of carbazole degradation. One of our controls was LD2 cultured with ammonium chloride to test whether LD2 was capable of using ammonia as a nitrogen source. This would pose a problem for us as we were planning to use an increase in ammonia levels as a proxy for carbazole degradation rather than measuring carbazole degradation directly using GC-MS. However, the LD2 showed significantly less ammonia after 44 hours when compared to 26 hours indicating that it may be degraded, either by the LD2 or by an unaccounted for abiotic process. This has lead us to decide to use GC-MS and directly measure carbazole levels in our sample the next time we do this experiment.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<p>The <i>carAa</i> was re-streaked onto ampicillin plates and grew, probably showing that it was ligated into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a>, not <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, which explained the irregularity of our confirmation digest last week. It was sent for sequencing, but in case it was incorrect we also started a new <i>carAa</i> pipeline by PCRing it from the <i>Pseudomonas</i> genome again and redigesting and ligating. It was transformed into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> over the weekend. <i>carAc</i> did not see a lot of progress this week as we spent much of the time attempting to construct a RBS (B0034) in front of it, but were unsuccessful. <i>carAd</i> was also subjected to this construction with B0034 and we were able to send it for sequencing by the end of the week. Several new constructs were also started for <i>carAd</i> in case the sequencing is negative. The <i>ant</i> operon was successfully transformed and confirmed by colony PCR this week, after which it was grown up and sent for sequencing. We also began to attempt mutagenesis on one of the EcoRI sites in the operon, but had no amplification of the plasmid. We also re-digested some PCR purified product to start another ligation in case sequencing failed. After many attempts we were also able to finally successfully transform an <i>AmdA</i>/PSB1C3 plasmid into Top 10 cells this week. After a miniprep they showed bands of about 1800 base pairs on the confirmation digest gel, so they were also sent for sequencing this week and had mutagenesis started on the illegal SpeI site. Unfortunately, the sequencing sent early in the week came back with ambiguous results. The forward reaction for<i> antABC</i> was a match, but its reverse reaction matched to <i>carAa</i>. Both <i>carAa</i> reactions matched nothing. Since these two genes are over 20 000 base pairs apart on the template plasmid it is virtually impossible that they were actually combined into one plasmid (PCR extension time would not have been long enough and even if it was the product would have run much above the 3000 base pair mark). This is possibly a mistake on Eurofins end, so we are not 100% sure if we have 1 good gene, both of them, or neither. Also the sequence for mutagenized <i>carAd</i> showed only about the first 100 base pairs, which were correct however not long enough meaning it was a poor reaction and inconclusive. Finally, we also re-started our <a href=https://2012.igem.org/Team:Calgary/Notebook/Protocols/carbazole>carbazole degradation assay</a> using the GC-MS to quantitatively measure the loss of carbazole at biological sciences. This process will take 2 weeks to demonstrate LD2's ability to degrade carbazole. We are forcing it to use carbazole as a carbon and a nitrogen source in one sample and as only a nitrogen source in another sample (this sample has had glucose added to it).</p><br />
<br />
</html>[[File:AmdA colonyPCR calgary12.JPG|500px|thumb|Amplification is seen in lane 7 of this PCR of top 10 colonies into which AmdA/PSB1C3 had been transformed. This indicates that <i>AmdA</i> has been successfully inserted into a biobrick vector.|center]]<br />
[[File:NewcarAagenomePCR calgary12.jpg|500px|thumb|<i>carAa</i> amplified from pCAR1.2 plasmid of <i>Pseudomonas putida</i>.|center]] <html><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<p>The new stock of <i>carAa</i> that we had PCRed from LD2 was sent for sequencing this week and came back as a match, so we have begun constructing a RBS site and a TetR promoter (<a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>) in front of it. At this point all the <i>car</i> genes have been successfully amplified, inserted into a biobrick plasmid, and sequence verified. We also continued to attempt to construct <i>carAc</i> with an RBS site (<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>) in front of it, but had no success. On a better note, we confirmed this week that<i> carAd</i> was mutated properly and we were also able to insert an RBS site in front of it. The next step for this gene will be to attempt a plasmid switch as it is currently in <a href="http://partsregistry.org/Part:pSB1A3">pSB1A3</a> and needs to be in <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> for submission to the parts registry. After sending a new batch of the ant operon for sequencing we got the same confusing results as we did last week. We therefore hypothesized that our primers were annealing to a homologous region that is similar to the<i> ant</i> genes and very close to <i>carAa</i>. This means that we need to restart PCR on this gene as all of our current product is incorrect. The sequencing results for <i>AmdA</i> that came back this week were also somewhat confusing as they did not quite match the sequence we expected. However, they were a 100% match for the <i>AmdA</i> gene in a different strain of <i>Rhodococcus erythropolis</i>. This means that DSMZ probably sent us the wrong strain of bacteria and that we were very lucky that the primers we designed based off of the expected template actually worked to amplify a slightly different gene in another strain. The good news is that this new sequence contains no illegal cut sites, which explains why mutagenesis was not working (there was nothing to mutate) and allows us to immediately begin construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> in front of <i>AmdA</i> this week. </p><br />
<br />
</html>[[File:Caraa colonyPCR calgary12.JPG|500px|thumb|Colony PCR amplification from top 10 <i>E. coli</i> cells with a PSB1C3/<i>carAa</i> construct transformed into them. Lanes 1-3, 5, and 7 were sent for sequencing with lane 5 coming back positive.|center]]<html><br />
<br />
<h2>Week 19 (September 3- September 7)</h2><br />
<p><i>carAd</i>/<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> was successfully plasmid switched into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, making it ready for submission. Construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> and <i>carAa</i> may have been successful as the digest of the miniprep looked promising. It will be sent for sequencing next week. Unfortunately there was no progress made on the constructions of <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> or <i>carAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. These will be redigested and religated for next week. We could not amplify the <i>ant</i> operon despite trying many variations of PCR using high fidelity KAPA polymerase. This is probably due to the multiple, large regions of homology on the operon that the reverse primer binds to making amplification difficult. Because the function of this gene can theoretically be replaced by <i>AmdA</i>, we may scale back efforts dedicated to it, as the chances of successful amplification appear slim. </p><br />
<br />
<h2>Weeks 20-22 (September 10 - September 30)</h2><br />
<p>Constructs that we continued to work on this week included <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, <i>carAa</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, and <i>carAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. Some sequencing was sent, but no positive results were obtained. A new approach to biobrick the <i>ant</i> operon was implemented this week. A new forward primer was designed that would anneal about 1500 base pairs upstream of the <i>ant</i> operon, avoiding the region of homology that is repeated at various points on the plasmid. Once this region is successfully amplified we could use this amplified DNA as a template for another PCR reaction which would use the normal <i>ant</i> primers. Since the template would no longer include regions of homology these primers should be free to work properly and amplify the <i>ant</i> operon. The PCR using the new primers was started this week, and although amplification at 4500 base pairs was observed as expected, there was also a lot of non-specific amplification. This means that to purify the amplified DNA that we want and avoid the other products a dreaded gel extraction had to be performed which, as usual, did not work. At this point it was decided that further attempts to amplify the <i>ant</i> operon would be futile and our efforts would be better served working on constructing the parts we do have. Unfortunately no progress was made in these weeks on construction. </p><br />
<br />
<br />
<br />
<br />
</html><br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/DenitrogenationTeam:Calgary/Notebook/Denitrogenation2012-10-03T23:45:23Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Denitrogenation Journal|<br />
CONTENT=<br />
<html><br />
<br />
<h2>Week 1 (May 1-4)</h2><br />
<p>The following section covers wetlab aspect of our overall project focusing on microbial conversion of naphthenic acids into economically-valuable hydrocarbons. The approach taken in this endeavour will be from four strategic starting points - ring cleavage, decarboxylation, denitrogenation, and desulfurization. Overall, the 'hydrocarbons' aspect of the project is a critical one to our overall design and construction of a bioreactor capable of not only detecting but also converting naphthenic acids and other toxins to useful or at least innocuous compounds. </p><br />
<br />
<h2>Week 2-3 (May 7-18)</h2><br />
<p>In the first two weeks of iGEM our group has focused on reviewing literature regarding the bioremediation of nitrogen groups attached to naphthenic acids. The most prevalent N heterocycle is carbazole, representing 75% of total nitrogen by mass. The upper pathway of carbazole biodegradation is catalyzed by the enzymes coded for by the <i>car</i> operon, <i>CarA</i> (<i>CarAaAcAd</i>), <i>CarB</i> (<i>CarBaBb</i>), and <i>CarC</i>. These enzymes convert carbazole to anthralinic acid. The lower pathway is catalyzed by the enzymes of the <i>ant</i> operon, <i>antA, B, and C</i>, yielding catechol while releasing CO2 and NH3. The <i>car</i> and <i>ant</i> operons are both regulated by the P<sup>ant</sup> promoter which is induced by the protein, antR. <i>carAa</i> also has its own promoter which is not induced by <i>antR</i> (Diaz & Garcia, 2010). We have also investigated an alternative pathway using <i>CarA</i> combined with an amidase (<i>amdA</i>) that selectively cleaves NH2 from an intermediate of the car pathway (Xu et al, 2006). This could bypass much of the car/ant pathway and is possibly more efficient.</p><br />
<p>We have decided to use <i>Pseudomonas resinovorans</i> and <i>Rhodococcus erythropolis</i> to derive these genes from. <i>carABC</i> and <i>antABC</i> from<i> P. resinovorans</i> has been shown to have a wide range of nitrogen containing substrate specificity. <i>R. erythropolis</i> contains the <i>amdA</i> gene that we wish to use, and some evidence suggests that it may also be able to degrade sulfur rings through its <i>carABC</i> pathway.</p><br />
<br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p>This week we reviewed the primers listed in the database and also designed some new ones. Primers for <i>carAa</i>,<i> carAc</i>, and <i>carAd</i> were designed individually, while primers for <i>CarBaBbC</i> and <i>antABC</i> were designed to encompass multiple genes in a sequence. These primers were designed to be used on <i>Pseudomonas putida</i> which we decided to use as our gene source since it was available to us as opposed to ordering <i>Pseudomonas resinovorans</i>. We also designed a primer for the<i> AmdA</i> gene from <i>Rhodococcus erthyroplois</i>. In addition to ordering these primers we also ordered the nitrogen containing compounds that we will need to test these enzymes on. We decided on using carbazole to make sure the enzymes can perform their natural function as well as pyrrolidine to test them on a similar ring structure. We also ordered cyclohexamine in order to independently test the function of the alternative <i>AmdA</i> pathway. Finally, we decided to eventually order 4-Piperidine butyric acid hydrochloride to test how the enzymes will work on nitrogen containing naphthenic acids. However, we decided since it is a very expensive compound we would wait to make sure the enzyme's work on more simple compounds before ordering it.</p><br />
<p>We also started our work in the wet lab by plating colonies and making an overnight culture of -80 freezer stock <i>Psuedomonas putida</i> on Thursday. We also resuspended the primers for <i>carAa</i>, <i>carAc</i>, <i>carAd</i>,<i> antA</i>, <i>antB</i>, and <i>ant C</i> that were already in the database. We were able to use the colonies that grew on the streak plates to start a colony PCR to attempt to isolate each of these genes on Friday.</p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2><br />
<p>On Monday and Tuesday we used the database primers to attempt to isolate <i>carAa</i>, <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i>antB</i>, and <i>antC</i> from the <i>Psuedomonas putida</i> we plated last week. <i>carAd</i>, <i>antB</i>, and <i>antC</i> were all put in the gradient PCR machine to account for the wide range of their primers’ melting points, while the others were done via regular PCR. Unfortunately, no positive results were obtained from these reactions. The PCR on <i>carAc</i> and <i>antA</i> was repeated on Wednesday, still not giving positive results. Finally, we attempted to use a salt concentration gradient on the PCR reaction for <i>carAc</i> and <i>antA</i>, using concentrations that ranged from 1.0 microlitres/tube to 2.0 microlitres/tube in increments of 0.2 microlitres/tube. This helped as <i>carAc</i> showed bands in samples that had concentrations of 1.2 and 1.4 microlitres/tube. <i> antA</i> also showed bands, however they were not the correct size, indicating contamination and/or non-specific annealing of the primer. The positive control also showed bands of the correct size. Earlier in the week we also made overnight cultures of 3 environmental strains (28, 29, 30) of <i>Psuedomonas putida</i> from -80 glycerol stock. On Friday we performed a genomic prep on these cultures, and plan on attempting to amplify genes from the isolated DNA next week. </p><br />
<br />
</html>[[File:CarAa and carAc Ucalgary12.png|500px|thumb|Gel of PCR using database primers for <i>carAa</i> and <i>carAc</i> on <i>Pseudomonas putida</i>|center]]<html><br />
<br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> We started off this week by determining the DNA concentration of our genomic prep samples from last week using the nanodrop. DNA concentration for all three<i> P. putida</i> strains was at least 1000 ng/microlitre, well above what was needed for PCR. 1/2 and 1/3 dilutions were prepared for all three strains so as not to have an excess of template DNA in PCR reactions. PCR was performed on all 3 strains using primers for <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i> antB</i>, and <i>antC</i> using 6 replicates per gene. The only successful amplification appeared to be <i>antB</i> and <i>carAc</i>, both from strain 28 (with weaker bands in strain 29). We then performed another PCR, just on those two genes with an increased amount of Taq polymerase to hopefully get enough amplified DNA to move forward with. This resulted in strong bands for both at the expected size. We then performed PCR purification using the Qiaquick kit and obtained samples containing 33.5 ng/microlitre of <i>antB</i> DNA and 129 ng/microlitre of <i>carAc </i>DNA. These concentrations were both sufficient to begin a restriction digest and ligation of these parts into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. Next week we hope to verify the results of the restriction digest, continue to amplify <i>carAc</i> and <i>antB</i> from strain 28, and hopefully submit a biobrick for sequencing.</p><br />
<br />
</html>[[File:AntB and carAc Ucalgary.png|500px|thumb|Gel of PCR ran on genomic DNA of <i>Pseudomonas putida</i> environmental strain 28 using primers for <i>carAc</i> and <i>antB</i>. |center]]<html><br />
<br />
<br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p>The purified PCR products for <i>carAc</i> and <i>antB</i> were digested with restriction enzymes EcoRI+SpeI, EcoRI+PstI, and XbaI+PstI. However, only <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmids that had been digested with EcoRI+SpeI and EcoRI+PstI were available to attempt ligation. Gel results were inconclusive on the restriction digest product as the plasmid size appeared much larger than expected. However, transformation was still attempted on the ligation products for both genes (both using EcoRI+SpeI ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid). The transformation products were plated onto chloroamphenicol (chlor) plates to select for colonies that had the chloro resistance gene on the <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid. Also this week another round of PCR was performed for the <i> car</i> and <i> ant</i> genes, however all, but <i>carAc</i> showed bands in the negative control lane, possibly indicating contamination or the formation of primer dimers. The <i>carAc</i> bands were fairly weak and a PCR purification resulted in very low DNA concentration, insufficient to move onto restriction digest. Next week's plan hinges heavily on the result of the transformation from this Friday. </p><br />
<br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>The results of our transformation on <i>carAc</i> and <i>antB</i> from last week showed mostly red colonies indicating that the ligation was unsuccessful and that the PSB1C3 plasmid simply closed on itself with no insert. However, there were three colonies for <i>carAc</i> that were white (cut with both X+P and E+P). Colony PCR was performed on these three colonies using biobrick primer sets to verify that the part had been inserted in these colonies, but unfortunately all PCR results were negative indicating an unsuccessful ligation.</p><br />
<p>We also performed a miniprep on <i>Pseudomonas putida</i> strain #28 this week using a home-made protocol rather than the Qiagen kit. Miniprep A had a DNA concentration of 1539.8 ng/μL and Miniprep B had DNA concentration of 1001.2 ng/μL possibly indicating that there may have been a lot of genomic DNA contamination. However miniprep A product was still used as a PCR template for a reaction attempting to isolate <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i>antB</i>, and <i>antC</i>. Special conditions for this PCR included replacing 60 μL of water with 60 μL of betaine heated to 37 degrees Celsius and running a Mg gradient ranging from 0.5 μL - 2.5 μL per tube for each gene. Of these only <i>antB</i> had bands of the right size in 3 of the lanes (Mg concentrations of 1.5, 2, and 2.5 μL). However, all of the <i>antB</i> bands (including the negative control) contained a contamination band at around 100 bases, probably due to the formation of primer dimers. This forced us to do a gel extraction rather than a PCR purification. This gel extraction did not work as the nanodrop results indicated high amounts of agarose contamination. Another PCR was performed using the same conditions, but using miniprep B instead as there was no more miniprep A product remaining. Unfortunately there were no bands for <i>antB</i>, only the 100 base pair contamination bands. </p><br />
<br />
</html>[[File:AntA and antC Ucalgary.png|500px|thumb|Gel of PCR ran on miniprep A of <i>Pseudomonas putida</i> attempting to isolate <i>antA</i> and <i>antC</i> genes.|center]]<br />
[[File:CarAc and carAd Ucalgary.png|500px|thumb|Gel of PCR ran on miniprep A of <i>Pseudomonas putida</i> attempting to isolate <i>carAc</i> and <i>carAd</i> genes.|center]]<br />
[[File:Minipret A antB and carAc Ucalgary.png|500px|thumb|Gel of PCR ran on miniprep A showing bands 3-5 of the correct size corresponding to antB genes and colony PCR ran using biobrick primers attempting to determine if <i>carAc</i> genes were successfully transformed into PSB1C3 plasmid.|center]] <html><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>This week we modified our PCR protocol to adjust for the homemade Taq polymerase we were using. Template DNA was boiled for 10 minutes on its own to replace the initialization step. This is because this polymerase could not tolerate the 95 degree heat during the regular initialization step. Another modification we made was to boil our primers for 5 minutes prior to adding them to the master mix. This was done to discourage the formation of primer-dimers. Finally we also attempted a touch-down PCR which started at an annealing temperature greater than the Tm of our primers and gradually worked its way down to below the Tm of the primers. This was done to increase the specificity of our reaction and also eliminate the formation of primer-dimers. Unfortunately these techniques still saw the formation of dimers, preventing us from performing a PCR purification. Again, the gel purification procedure did not lead to successful purification of our target gene. Due to the problems we have had so far our goal for next week is to perform an assay experiment to try and determine which of the <i>Pseudomonas putida</i> strains we have access to can actually degrade carbazole and therefore contain the pCAR1 plasmid. </p><br />
<br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The goal for this week was to grow different strains of<i> Pseudomonas</i> in media containing B-N media + glucose with only carbazole as a source of nitrogen. In theory only those strains which contained the pCAR1 plasmid would be able to use carbazole as a nitrogen source and thrive in this media. The strains we tested from glycerol stock included <i>P. putida</i> LD1, <i>P.putida</i> environmental strains 28-30, and <i>P. fluorescens</i> PF5. Each of these strains had two cultures grown; one with carbazole and one without it. <i>E. coli</i> grown with carbazole was used as a negative control as it does not contain the pCAR plasmid. At timepoints of 22, 46, and 117 hours of growth samples were taken to do an OD600 reading to measure bacterial growth and had an ammonia assay performed on it. The detection of ammonia acted as a proxy for detecting carbazole degradation as it is an end-product of the pathway. With this in mind, an additional control strain of LD1 was grown in media containing glucose and ammonia. This is to detect the possibility that <i>Pseudomonas</i> can use ammonia as a source of nitrogen after it has degraded carbazole which would be problematic as far as using ammonia levels to determine carbazole degradation. So far we have been having difficulties reading the results of the ammonia assay with a plate reader and may resort to testing our samples using a different assay protocol next week. The OD600 readings were initially very high at 22 hours and then dropped at 46 hours. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>This week we took our last readings from the experiment that we started last week. The OD600 readings taken at 117 hours showed minimal growth over the weekend for all strains except for environmental strain #29 (with carbazole) which increased from 0.086 to 0.313. No other strain showed an increase of 0.100 or greater. This shows that only strain #29 was able to thrive in the carbazole media, possibly indicating that it contains the pCAR plasmid enabling it to use carbazole as a nitrogen source. To verify this we used a new ammonia assay protocol to measure the production of ammonia in the different cultures and theoretically how much carbazole was degraded. Unlike the previous assay we were able to produce a standard curve with a consistent slope based on samples with known amounts of ammonia. However, the only samples that showed any elevation of ammonia levels above the blank were the LD1 samples that were cultured with ammonia chloride to control for the possibility of pseudomonas using ammonia as a nitrogen source. The ammonia levels in this sample were relatively constant which would indicate that the LD1 was not using ammonia as a nitrogen source. Unfortunately the fact that none of the test samples had elevated ammonia levels did not give us any evidence that these strains contain the pCAR plasmid. </p><br />
<br />
</html>[[File: Standard_curve_for_ammonia_assay_UCalgary2012_denitrification_group.png|500px|thumb|Standard curve for ammonia assay using OD635 absorbance.|center|]]<html><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>We received a new strain of Pseudomonas, LD2, this week that had previously been used to study the pCAR1 plasmid. It was grown up in PCA media and had its colonies used for colony PCR. We also received our new primers this week, further increasing the chances of successful PCR. Using the new strain and the new primers <i>carAa</i>, <i>carAc</i>, and <i>carAd</i> all showed at least some bands of close to the correct size. After PCR purification we obtained DNA concentrations of 21.3 ng/μL for <i>carAa</i>, 41.9 ng/ μL for <i>carAc</i>, and 32.8 ng/μL for<i> carAd</i>. These products were then restriction digested using EcoRI + PstI sites. </p><br />
<br />
</html>[[File:07.20.2012 CarAa and CarAc.jpg|500px|thumb|Gel of colony PCR from <i>Pseudomonas sp. LD2</i> for <i>carAa</i> and <i>carAc</i> genes. <i>carAa</i> expected size is 1155 bp and <i>carAc</i> is 323 bp.|center]]<br />
[[File:07.20.2012 CarAd colony PCR from LD2.jpg|500px|thumb|Gel of colony PCR from <i>Pseudomonas sp. LD2</i> for <i>carAd</i> gene. <i>carAd</i> expected size is 989 bp.|center]] <html><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The restriction digested <i>car</i> genes were ligated into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> cells. White colonies grew for<i> carAa</i> and <i>carAc</i> suggesting that ligation and transformation were successful, however no colonies grew for <i>carAd</i> suggesting that the transformation did not work. Colony PCR was performed to confirm the presence of these genes within the vectors, however only <i>carAc</i> was positive. The colonies containing the <i>carAc</i> plasmid were then mini prepped and restriction digested to isolate the plasmid and send for sequencing. Further attempts at transforming<i> carAa</i> and <i>carAd</i> were not successful. Another goal was to perform colony PCR on the <i>P. putida</i> to isolate the<i> antABC</i> genes using both the primers for the entire operon and those for the individual genes. No attempt showed any bands, although salt and temperature gradients were used as well as different types of polymerase. LD2 cultures were grown up to be plasmid prepped and genomic prepped in order to have a higher concentration of template DNA in the PCR master mix and more PCR will be attempted on these genes next week. Mutagenesis primers were also designed this week to remove the illegal restriction cut sites in the<i> carAd</i> and <i>antB</i> genes (NotI and 2xEcoRI respectively). Only 1 mutagenesis primer was designed for <i>antB</i> as one of its EcoRI sites was very close to the beginning of the gene and could be removed using a new primer. During this process it was also discovered that the original ant primers were designed for the wrong genes and therefore do not match the intended sequence. Although the new <i>antABC</i> primers were designed using the correct gene the PCR may not be working due to the length of the operon so the design of new individual primers may be necessary. Finally, we also aimed to grow up the freeze dried culture of <i>Rhodococcus erythropolis</i> (source of the<i> AmdA</i> gene) that we received from DSMZ. The reactivation media was produced and autoclaved to begin the process next week.</p><br />
<br />
</html>[[File:07.23.2012.Restriction Digest of Car genes.jpg|500px|thumb|Gel of restriction digest of car genes. A refers to <i>carAa</i>, C to <i>carAc</i>, and D to <i>carAd</i>. Ladder shows that after purification and digestion DNA is still the correct size.|center]]<br />
[[File:CarAc biobrick confirmation PCR.jpg|500px|thumb|Gel of confirmation PCR done on competent Top 10 <i>E. coli</i> cells in which a PSB1C3 vector containing the <i>carAc</i> gene had been inserted. Red X marks the 500 bp mark on the weak ladder showing the product to be the expected size (323 bp gene + 2*100 bp biobrick sites on each end).|center]] <html><br />
<br />
<h2>Week 14 (July 30 - August 3)</h2><br />
<p>We received the sequencing results this week for <i>carAc</i> which came back as a 100% match to the expected nucleotide sequence. We also successfully ligated both<i> carAa</i> and<i> carAd</i> into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into top 10 <i>E. coli</i> cells. Colony PCR verified the presence of the biobrick plasmid in these colonies. These were both miniprepped, digested and sent for sequencing by the end of the week. Using a salt gradient, addition of DMSO, and the KAPA buffer specifically designed for GC-rich amplicons we were able to amplify the ant operon from a genomic prep of LD2. However, all attempts at ligating and transforming this sequence were met with the unfortunate presence of red colonies (indicating failed insertion into the plasmid). Next week we will recut a <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> vector with Xba and Pst to attempt to mitigate this problem. We also reactivated the <i>R. erythropolis</i> following the DSMZ protocol for reviving freeze-dried cultures that can be found <a href=https://2012.igem.org/Team:Calgary/Notebook/Protocols/dmszfreezedry>here</a> using DSMZ media #1 and grew it on plates made from DSMZ media #220 at 28 degrees. Colonies grew very quickly and were used for colony PCR and to make overnight cultures for downstream applications. The colony PCR using the amidase primers was unsuccessful, so a genomic prep was performed on the overnight culture. Unfortunately we lacked some of the required equipment and the genomic prep did not work, but with a few procedural modifications and the right equipment we are confident it will work next week. A glycerol stock of the<i> R. erythropoplis</i> was also made from another overnight culture. We also started construction on the <i>carAc </i>part this week by inserting a ribosome binding site (B0034) in front of the gene. Finally, since the<i> ant</i> operon was successfully amplified by PCR we designed mutagenesis primers for the 2nd EcoRI site in the operon rather than making new individual primers. We also designed a mutagenesis primer to remove the SpeI site in <i>AmdA</i>. Now that we have conclusively proven that the LD2 strain contains the pCAR1 plasmid we aim to demonstrate its ability to degrade carbazole and other nitrogen containing heterocycles using an assay experiment similar to the one performed in Week 10.</p><br />
<br />
</html>[[File:07.30.2012. CarAa, CarAd.jpg|500px|thumb|<i>carAa</i> and <i>carAd</i> amplified from genomic prep of <i>Pseudomonas sp. LD2</i>. Amplicons are larger than expected, potentially the gel ran diagonally. The 6th lane for each gene was a negative control.|center]]<br />
[[File:07.31.2012.Ant operon.jpg|500px|thumb|<i>ant</i> operon amplified from genomic prep of <i>Pseudomonas sp. LD2</i> at the expected size of 3000 bp.|center]]<br />
[[File:08.01.2012 CarAdBB.jpg|500px|thumb|Lanes 2,4,5, and 11 contain <i>carAd</i> genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent top 10 <i>E.Coli</i>.|center]]<br />
[[File:08.01.2012 CarAaBB.jpg|500px|thumb|Lane 9 contains <i>carAa</i> genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent top 10 <i>E.Coli</i>. Although larger than expected, the positive control RFP generator in lane 10 is also larger than its expected size (~1200 bp including biobrick bookends) indicating that the gel may have a slant in it.|center]]<html><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p>This week we received the sequencing results for <i>carAa</i> and <i>carAd</i>. <i>carAd</i> was a match, however <i>carAa</i> did not match at all and had misplaced biobrick restriction enzyme sites. Due to this we decided to restart the biobricking process for <i>carAa</i> all the way back to PCR from the plasmid. By the end of the week we had progressed to the point of transforming it into competent <i>E. coli</i> cells, however a thick lawn of bacteria had grown overnight making it impossible to pick colonies. A streak plate was made from this plate to attempt to get less dense growth. Since the sequencing came back positive for <i>carAd</i> the next step was to perform site directed mutagenesis on its NotI restriction enzyme site using the primers that were designed previously (for site-directed mutagenesis optimization, please refer to Desulfurization Journal Week 14). The PCR was successful, showing very bright bands at 3000 bp, and the PCR product was digested with DpnI to remove parental DNA. Transformation of this product will indicate whether the mutagenesis was successful. We were also able to successfully perform a genomic prep of <i>Rhodococcus erythropolis</i> and amplify the <i>AmdA</i> gene this week. It was ligated into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> by Friday, and a confirmation colony PCR was run on Friday evening to verify that the gene was inserted. These results will be available by Monday. After many attempts the <i>ant</i> operon was finally ligated and transformed as well this week. Confirmation PCR showed bands of about 3000 bp which is consistent with the size of the operon. By Friday the successful colonies had been grown up and miniprepped, ready to send for sequencing early next week. The <i>carAc</i>/B0034 construct was also successfully transformed this week and verified by running a PCR using the B0034 forward primer. It was also miniprepped and is ready to send for sequencing next week. </p><br />
<p>In addition to this work constructing biobricks we also started an experiment to demonstrate the function of the pCAR plasmid in LD2. Strains of LD2 were grown up in LB and then washed and placed in B-N media + glucose and various experimental additions. These additions included carbazole, 4-PBAH (a nitrogen containing naphthenic acid), ammonia chloride, and a negative control with no nitrogen source. Strains of <i>E. coli</i> were also grown under the same conditions to compare growth and ammonia production of LD2 to a strain that does not contain the pCAR plasmid. These results should act as a preview of what we expect our final biobrick containing <i>E. col</i>i species to produce as well as allowing us to establish LD2 as a positive control for this experiment. </p><br />
<br />
<br />
</html>[[File:08.07.2012 amdA pcr from genomic prep.jpg|500px|thumb|<i>AmdA</i> amplified from the genomic prep of <i>R. Erythropolis</i> shows bands just greater than 1500 base pairs in lanes 2,4-6,9, and 12-13. Lane 14 was a negative control. The expected size of <i>AmdA</i> is 1566 base pairs.|center]]<br />
[[File:08.09.2012 confirmation pcr of ant operon.jpg|500px|thumb|The two labelled lanes show amplification at 3000 base pairs from a confirmation colony PCR using the biobrick primers. The expected size of the <i>ant</i> operon with the biobrick bookends is about 3100 base pairs.|center]]<br />
[[File:08.10.2012 carad notI mutagenesis.jpg|500px|thumb|Strong amplification at just above 3000 base pairs from the <i>carAd</i> PCR using mutagenic primers. Since PSB1C3 is 2070 base pairs and <i>carAd</i> is 989 base pairs the amplification should run at about 3059 base pairs. 3 different amounts of template DNA were tested in lanes 2-7 as indicated and a positive control showed weaker amplification in lanes 8-9 at about 4000 base pairs.|center]]<br />
<html><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>The sequencing for the <i>carAc</i>/<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> construct came back with only <i>carAc</i> and no evidence of a <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> site preceding it. The confirmation PCR that was run last week using the <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> forward primer seemed to indicate that it was inserted, however since <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> is only 12 base pairs the primer may not have been specific enough. The construction on this part was restarted this week. <i>carAa</i> colony PCR on last week's transformation showed two positive colonies, however upon culturing, miniprepping, and digesting these colonies the plasmid appeared to be too large (3000 base pairs as opposed to the expected 2000). This may be due to a problem of a mislabeled plasmid tube that has been experienced by other team members, causing the gene to be inserted into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a> rather than <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. To test this, a streak plate was made from these colonies and grown on an ampicillin resistant plate. Mutagenized <i>carAd</i> was successfully transformed into Top 10 <i>E. coli</i> cells, cultured, and miniprepped. The miniprep product was digested with NotI to test whether the NotI site was successfully removed. These results were good, as the plasmid ran at 2000 base pairs and the insert at just under 1000 base pairs in the mutagenized <i>carAd</i> whereas the non-mutagenized <i>carAd</i> had its insert cut into 2 bands. This will be sent for sequencing next week to confirm the mutation. Unfortunately the sequencing results came back negative for the <i>ant</i> operon meaning we will need to restart genomic PCR on it next week. Multiple transformations and confirmation PCRs were run on <i>AmdA</i> this week, however none displayed a band size of 1700-1800 base pairs as expected. We have continued to try different digested plasmids as other team members also had difficulties this week with ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, possibly indicating a problem with our Antarctic Phosphatase stock.</p><br />
<p>The other news from this week was the final results from our carbazole degradation experiment attempting to demonstrate LD2's ability to degrade carbazole and hopefully other nitrogen containing compounds. Unfortunately, these results were very difficult to make sense of as the LD2 that were cultured with carbazole grew, but did not show elevated ammonia levels which would be indicative of carbazole degradation. One of our controls was LD2 cultured with ammonium chloride to test whether LD2 was capable of using ammonia as a nitrogen source. This would pose a problem for us as we were planning to use an increase in ammonia levels as a proxy for carbazole degradation rather than measuring carbazole degradation directly using GC-MS. However, the LD2 showed significantly less ammonia after 44 hours when compared to 26 hours indicating that it may be degraded, either by the LD2 or by an unaccounted for abiotic process. This has lead us to decide to use GC-MS and directly measure carbazole levels in our sample the next time we do this experiment.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<p>The <i>carAa</i> was re-streaked onto ampicillin plates and grew, probably showing that it was ligated into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a>, not <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, which explained the irregularity of our confirmation digest last week. It was sent for sequencing, but in case it was incorrect we also started a new <i>carAa</i> pipeline by PCRing it from the <i>Pseudomonas</i> genome again and redigesting and ligating. It was transformed into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> over the weekend. <i>carAc</i> did not see a lot of progress this week as we spent much of the time attempting to construct a RBS (B0034) in front of it, but were unsuccessful. <i>carAd</i> was also subjected to this construction with B0034 and we were able to send it for sequencing by the end of the week. Several new constructs were also started for <i>carAd</i> in case the sequencing is negative. The <i>ant</i> operon was successfully transformed and confirmed by colony PCR this week, after which it was grown up and sent for sequencing. We also began to attempt mutagenesis on one of the EcoRI sites in the operon, but had no amplification of the plasmid. We also re-digested some PCR purified product to start another ligation in case sequencing failed. After many attempts we were also able to finally successfully transform an <i>AmdA</i>/PSB1C3 plasmid into Top 10 cells this week. After a miniprep they showed bands of about 1800 base pairs on the confirmation digest gel, so they were also sent for sequencing this week and had mutagenesis started on the illegal SpeI site. Unfortunately, the sequencing sent early in the week came back with ambiguous results. The forward reaction for<i> antABC</i> was a match, but its reverse reaction matched to <i>carAa</i>. Both <i>carAa</i> reactions matched nothing. Since these two genes are over 20 000 base pairs apart on the template plasmid it is virtually impossible that they were actually combined into one plasmid (PCR extension time would not have been long enough and even if it was the product would have run much above the 3000 base pair mark). This is possibly a mistake on Eurofins end, so we are not 100% sure if we have 1 good gene, both of them, or neither. Also the sequence for mutagenized <i>carAd</i> showed only about the first 100 base pairs, which were correct however not long enough meaning it was a poor reaction and inconclusive. Finally, we also re-started our <a href=”https://2012.igem.org/Team:Calgary/Notebook/Protocols/carbazole”>carbazole degradation assay</a> using the GC-MS to quantitatively measure the loss of carbazole at biological sciences. This process will take 2 weeks to demonstrate LD2's ability to degrade carbazole. We are forcing it to use carbazole as a carbon and a nitrogen source in one sample and as only a nitrogen source in another sample (this sample has had glucose added to it).</p><br />
<br />
</html>[[File:AmdA colonyPCR calgary12.JPG|500px|thumb|Amplification is seen in lane 7 of this PCR of top 10 colonies into which AmdA/PSB1C3 had been transformed. This indicates that <i>AmdA</i> has been successfully inserted into a biobrick vector.|center]]<br />
[[File:NewcarAagenomePCR calgary12.jpg|500px|thumb|<i>carAa</i> amplified from pCAR1.2 plasmid of <i>Pseudomonas putida</i>.|center]] <html><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<p>The new stock of <i>carAa</i> that we had PCRed from LD2 was sent for sequencing this week and came back as a match, so we have begun constructing a RBS site and a TetR promoter (<a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>) in front of it. At this point all the <i>car</i> genes have been successfully amplified, inserted into a biobrick plasmid, and sequence verified. We also continued to attempt to construct <i>carAc</i> with an RBS site (<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>) in front of it, but had no success. On a better note, we confirmed this week that<i> carAd</i> was mutated properly and we were also able to insert an RBS site in front of it. The next step for this gene will be to attempt a plasmid switch as it is currently in <a href="http://partsregistry.org/Part:pSB1A3">pSB1A3</a> and needs to be in <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> for submission to the parts registry. After sending a new batch of the ant operon for sequencing we got the same confusing results as we did last week. We therefore hypothesized that our primers were annealing to a homologous region that is similar to the<i> ant</i> genes and very close to <i>carAa</i>. This means that we need to restart PCR on this gene as all of our current product is incorrect. The sequencing results for <i>AmdA</i> that came back this week were also somewhat confusing as they did not quite match the sequence we expected. However, they were a 100% match for the <i>AmdA</i> gene in a different strain of <i>Rhodococcus erythropolis</i>. This means that DSMZ probably sent us the wrong strain of bacteria and that we were very lucky that the primers we designed based off of the expected template actually worked to amplify a slightly different gene in another strain. The good news is that this new sequence contains no illegal cut sites, which explains why mutagenesis was not working (there was nothing to mutate) and allows us to immediately begin construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> in front of <i>AmdA</i> this week. </p><br />
<br />
</html>[[File:Caraa colonyPCR calgary12.JPG|500px|thumb|Colony PCR amplification from top 10 <i>E. coli</i> cells with a PSB1C3/<i>carAa</i> construct transformed into them. Lanes 1-3, 5, and 7 were sent for sequencing with lane 5 coming back positive.|center]]<html><br />
<br />
<h2>Week 19 (September 3- September 7)</h2><br />
<p><i>carAd</i>/<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> was successfully plasmid switched into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, making it ready for submission. Construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> and <i>carAa</i> may have been successful as the digest of the miniprep looked promising. It will be sent for sequencing next week. Unfortunately there was no progress made on the constructions of <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> or <i>carAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. These will be redigested and religated for next week. We could not amplify the <i>ant</i> operon despite trying many variations of PCR using high fidelity KAPA polymerase. This is probably due to the multiple, large regions of homology on the operon that the reverse primer binds to making amplification difficult. Because the function of this gene can theoretically be replaced by <i>AmdA</i>, we may scale back efforts dedicated to it, as the chances of successful amplification appear slim. </p><br />
<br />
<h2>Weeks 20-22 (September 10 - September 30)</h2><br />
<p>Constructs that we continued to work on this week included <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, <i>carAa</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, and <i>carAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. Some sequencing was sent, but no positive results were obtained. A new approach to biobrick the <i>ant</i> operon was implemented this week. A new forward primer was designed that would anneal about 1500 base pairs upstream of the <i>ant</i> operon, avoiding the region of homology that is repeated at various points on the plasmid. Once this region is successfully amplified we could use this amplified DNA as a template for another PCR reaction which would use the normal <i>ant</i> primers. Since the template would no longer include regions of homology these primers should be free to work properly and amplify the <i>ant</i> operon. The PCR using the new primers was started this week, and although amplification at 4500 base pairs was observed as expected, there was also a lot of non-specific amplification. This means that to purify the amplified DNA that we want and avoid the other products a dreaded gel extraction had to be performed which, as usual, did not work. At this point it was decided that further attempts to amplify the <i>ant</i> operon would be futile and our efforts would be better served working on constructing the parts we do have. Unfortunately no progress was made in these weeks on construction. </p><br />
<br />
<br />
<br />
<br />
</html><br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/carbazoleTeam:Calgary/Notebook/Protocols/carbazole2012-10-03T23:34:29Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=Carbazole Degradation Time-course Assay|<br />
CONTENT=<html><br />
<h2>Media Preparation</h2><br />
<p><b>B-N Medium for Culturing <i>Pseudomonas</i> LD2</b></p><br />
<p>The B-N (basic with no nitrogen) medium with trace metals is specifically designed for <i>Pseudomonas</i> LD2 growth when analyzing biodegradation of a nitrogen-containing compound.</p><br />
<p>Weigh out appropriate amounts of all compounds except FeSO<font style="text-transform: lowercase;">4</font>.7H<font style="text-transform: lowercase;">2</font>O and dissolve, with stirring, in 1L of milliQ H<font style="text-transform: lowercase;">2</font>O. </p><br />
<br />
<br />
<center><br />
<table border="0"><br />
<br />
<tr><br />
<td> K<font style="text-transform: lowercase;">2</font>HPO<font style="text-transform: lowercase;">4</font>:</td><br />
<td>0.5g</td><br />
</tr><br />
<br />
<tr><br />
<td> Na<font style="text-transform: lowercase;">2</font>SO<font style="text-transform: lowercase;">4</font>:</td><br />
<td>2.0g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>MgSO<font style="text-transform: lowercase;">4</font> 7H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>0.2g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>FeSO<font style="text-transform: lowercase;">4</font> 7H<font style="text-transform: lowercase;">2</font>O </td><br />
<td>Trace</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> Trace metals:</td><br />
<td>1.0mL</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>Up to 1.0L</td><br />
</tr><br />
<br />
</table><br />
</center><br />
<br />
<p>When all the salts are dissolved, add FeSO<font style="text-transform: lowercase;">4</font>.7H<font style="text-transform: lowercase;">2</font>O and trace metals and stir for at least 5min to ensure that all compounds are dissolved. <br />
<p>The trace metal solution is prepared as follows:</p><br />
<br />
<center><br />
<table border="0"><br />
<br />
<tr><br />
<td>CaCl<font style="text-transform: lowercase;">2</font> 2H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>3.7g</td><br />
</tr><br />
<br />
<tr><br />
<td>H<font style="text-transform: lowercase;">3</font>BO<font style="text-transform: lowercase;">3</font>:</td><br />
<td>2.5g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> MnCl<font style="text-transform: lowercase;">2</font>:</td><br />
<td>0.87g</td><br />
</tr><br />
<br />
<tr><br />
<td> FeCl<font style="text-transform: lowercase;">3</font>:</td><br />
<td>0.44g </td><br />
</tr><br />
<br />
<tr><br />
<td>ZnCl<font style="text-transform: lowercase;">3</font>:</td><br />
<td>0.44g</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td> Na<font style="text-transform: lowercase;">2</font >MoO<font style="text-transform: lowercase;">2</font > 2H<font style="text-transform: lowercase;">2</font >O:</td><br />
<td>0.29g</td><br />
</tr><br />
<br />
<tr><br />
<td>CoCl<font style="text-transform: lowercase;">2</font >:</td><br />
<td>0.01g </td><br />
</tr><br />
<br />
<tr><br />
<td>CuCl<font style="text-transform: lowercase;">2</font >:</td><br />
<td>0.0001g </td><br />
</tr><br />
<br />
<tr><br />
<td> H<font style="text-transform: lowercase;">2</font>O:</td><br />
<td>Up to 1.0L</td><br />
</tr><br />
<br />
</table><br />
</center><br />
<br />
<p>Dispense appropriate amounts of B-N media into glass culture flasks and autoclave.</p><br />
<br />
<h2>Carbazole Degradation Time-course Experiment</h2><br />
<p>This is a time course experiment designed to monitor carbazole loss by collecting bacterial culture samples over time, followed by <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oextraction"> organic extraction</a> and GC-MS analysis.</p><br />
<ol><br />
<li>Grow up overnight cultures of <i>Pseudomonas</i> LD2.</li><br />
<li>Make B-N media in 250 mL Erlenmeyer flasks. Add 50mL to each Erlenmeyer flask and autoclave.</li><br />
<li>Prepare 2 Erlenmeyer flasks as blanks (B-N media only) at day 0 and day 14 as negative controls.</li><br />
<li>Add 15 mg carbazole in its crystalline form, 2.5mg glucose (glucose can be dissolved in a small amount of water and filter sterilized), and 100uL of cells to one set of flasks. This serves as the positive control (2 samples at each time point).</li><br />
<li>Add 15 mg carbazole in its crystalline form and 100uL of cells to another set of flasks. This serves as the experimental sample (2 samples at each time point).</li><br />
<li>Place stopper on the flasks and incubate in the shaker at 28oC.</li><br />
<li>At the appropriate time points, take out the flasks from the shaker and add enough acid to bring the pH of the solutions to ~2.</li><br />
<li>Each sample is extracted, and analyzed using a GC-MS.</li><br />
</ol><br />
<br />
</html>}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/dmszfreezedryTeam:Calgary/Notebook/Protocols/dmszfreezedry2012-10-03T23:30:13Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
<br />
TITLE=Revival of Freeze-dried Bacterial Cultures from DSMZ|<br />
CONTENT=<html><br />
<p><br />
The following protocol is used to reactivate and culture bacterial strains received from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH).<br />
</p><br />
<ol><br />
<li>Remove the glass ampoule from plastic tube.</li><br />
<li>Check the indicator to ensure an intact seal (red or blue – good seal, pink or orange – humidity is present in the sample).</li><br />
<li>Heat the tip (pointy tip up) of glass ampoule over a Bunsen burner flame thoroughly.</li><br />
<li>Drop some water onto the tip to crack glass all around.</li><br />
<li>Carefully strike the glass tip with forceps (or a similar tool) to break off the tip.</li><br />
<li>Remove the insulation material and extract the smaller vial with forceps. If the bacteria in the vial is an anaerobic strain, ensure anaerobic conditions are used during the rest of the procedure.</li><br />
<li>Remove the cotton plug from vial and keep it sterile.</li><br />
<li>Add 0.5 mL recommended medium as indicated by the accompanying instructions to the dehydrated culture and insert cotton plug back into the vial. Allow the culture to rehydrate for about 30 min.</li><br />
<li>Remove the cotton plug again. Mix very gently with an inoculation loop and transfer to 5mL of a recommended liquid medium in a 15mL tube. Mix well.</li><br />
<li>Pipette 100uL of liquid culture onto plate as a spot and make a streak plate.</li><br />
</ol><br />
<ul><li>Additional incubation directions are provided on the <a href="http://www.dsmz.de/home.html">DSMZ website</a>.</li></ul><br />
</html><br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/DenitrogenationTeam:Calgary/Notebook/Denitrogenation2012-10-03T23:24:00Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Denitrogenation Journal|<br />
CONTENT=<br />
<html><br />
<br />
<h2>Week 1 (May 1-4)</h2><br />
<p>The following section covers wetlab aspect of our overall project focusing on microbial conversion of naphthenic acids into economically-valuable hydrocarbons. The approach taken in this endeavour will be from four strategic starting points - ring cleavage, decarboxylation, denitrogenation, and desulfurization. Overall, the 'hydrocarbons' aspect of the project is a critical one to our overall design and construction of a bioreactor capable of not only detecting but also converting naphthenic acids and other toxins to useful or at least innocuous compounds. </p><br />
<br />
<h2>Week 2-3 (May 7-18)</h2><br />
<p>In the first two weeks of iGEM our group has focused on reviewing literature regarding the bioremediation of nitrogen groups attached to naphthenic acids. The most prevalent N heterocycle is carbazole, representing 75% of total nitrogen by mass. The upper pathway of carbazole biodegradation is catalyzed by the enzymes coded for by the <i>car</i> operon, <i>CarA</i> (<i>CarAaAcAd</i>), <i>CarB</i> (<i>CarBaBb</i>), and <i>CarC</i>. These enzymes convert carbazole to anthralinic acid. The lower pathway is catalyzed by the enzymes of the <i>ant</i> operon, <i>antA, B, and C</i>, yielding catechol while releasing CO2 and NH3. The <i>car</i> and <i>ant</i> operons are both regulated by the P<sup>ant</sup> promoter which is induced by the protein, antR. <i>carAa</i> also has its own promoter which is not induced by <i>antR</i> (Diaz & Garcia, 2010). We have also investigated an alternative pathway using <i>CarA</i> combined with an amidase (<i>amdA</i>) that selectively cleaves NH2 from an intermediate of the car pathway (Xu et al, 2006). This could bypass much of the car/ant pathway and is possibly more efficient.</p><br />
<p>We have decided to use <i>Pseudomonas resinovorans</i> and <i>Rhodococcus erythropolis</i> to derive these genes from. <i>carABC</i> and <i>antABC</i> from<i> P. resinovorans</i> has been shown to have a wide range of nitrogen containing substrate specificity. <i>R. erythropolis</i> contains the <i>amdA</i> gene that we wish to use, and some evidence suggests that it may also be able to degrade sulfur rings through its <i>carABC</i> pathway.</p><br />
<br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p>This week we reviewed the primers listed in the database and also designed some new ones. Primers for <i>carAa</i>,<i> carAc</i>, and <i>carAd</i> were designed individually, while primers for <i>CarBaBbC</i> and <i>antABC</i> were designed to encompass multiple genes in a sequence. These primers were designed to be used on <i>Pseudomonas putida</i> which we decided to use as our gene source since it was available to us as opposed to ordering <i>Pseudomonas resinovorans</i>. We also designed a primer for the<i> AmdA</i> gene from <i>Rhodococcus erthyroplois</i>. In addition to ordering these primers we also ordered the nitrogen containing compounds that we will need to test these enzymes on. We decided on using carbazole to make sure the enzymes can perform their natural function as well as pyrrolidine to test them on a similar ring structure. We also ordered cyclohexamine in order to independently test the function of the alternative <i>AmdA</i> pathway. Finally, we decided to eventually order 4-Piperidine butyric acid hydrochloride to test how the enzymes will work on nitrogen containing naphthenic acids. However, we decided since it is a very expensive compound we would wait to make sure the enzyme's work on more simple compounds before ordering it.</p><br />
<p>We also started our work in the wet lab by plating colonies and making an overnight culture of -80 freezer stock <i>Psuedomonas putida</i> on Thursday. We also resuspended the primers for <i>carAa</i>, <i>carAc</i>, <i>carAd</i>,<i> antA</i>, <i>antB</i>, and <i>ant C</i> that were already in the database. We were able to use the colonies that grew on the streak plates to start a colony PCR to attempt to isolate each of these genes on Friday.</p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2><br />
<p>On Monday and Tuesday we used the database primers to attempt to isolate <i>carAa</i>, <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i>antB</i>, and <i>antC</i> from the <i>Psuedomonas putida</i> we plated last week. <i>carAd</i>, <i>antB</i>, and <i>antC</i> were all put in the gradient PCR machine to account for the wide range of their primers’ melting points, while the others were done via regular PCR. Unfortunately, no positive results were obtained from these reactions. The PCR on <i>carAc</i> and <i>antA</i> was repeated on Wednesday, still not giving positive results. Finally, we attempted to use a salt concentration gradient on the PCR reaction for <i>carAc</i> and <i>antA</i>, using concentrations that ranged from 1.0 microlitres/tube to 2.0 microlitres/tube in increments of 0.2 microlitres/tube. This helped as <i>carAc</i> showed bands in samples that had concentrations of 1.2 and 1.4 microlitres/tube. <i> antA</i> also showed bands, however they were not the correct size, indicating contamination and/or non-specific annealing of the primer. The positive control also showed bands of the correct size. Earlier in the week we also made overnight cultures of 3 environmental strains (28, 29, 30) of <i>Psuedomonas putida</i> from -80 glycerol stock. On Friday we performed a genomic prep on these cultures, and plan on attempting to amplify genes from the isolated DNA next week. </p><br />
<br />
</html>[[File:CarAa and carAc Ucalgary12.png|500px|thumb|Gel of PCR using database primers for <i>carAa</i> and <i>carAc</i> on <i>Pseudomonas putida</i>|center]]<html><br />
<br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> We started off this week by determining the DNA concentration of our genomic prep samples from last week using the nanodrop. DNA concentration for all three<i> P. putida</i> strains was at least 1000 ng/microlitre, well above what was needed for PCR. 1/2 and 1/3 dilutions were prepared for all three strains so as not to have an excess of template DNA in PCR reactions. PCR was performed on all 3 strains using primers for <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i> antB</i>, and <i>antC</i> using 6 replicates per gene. The only successful amplification appeared to be <i>antB</i> and <i>carAc</i>, both from strain 28 (with weaker bands in strain 29). We then performed another PCR, just on those two genes with an increased amount of Taq polymerase to hopefully get enough amplified DNA to move forward with. This resulted in strong bands for both at the expected size. We then performed PCR purification using the Qiaquick kit and obtained samples containing 33.5 ng/microlitre of <i>antB</i> DNA and 129 ng/microlitre of <i>carAc </i>DNA. These concentrations were both sufficient to begin a restriction digest and ligation of these parts into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. Next week we hope to verify the results of the restriction digest, continue to amplify <i>carAc</i> and <i>antB</i> from strain 28, and hopefully submit a biobrick for sequencing.</p><br />
<br />
</html>[[File:AntB and carAc Ucalgary.png|500px|thumb|Gel of PCR ran on genomic DNA of <i>Pseudomonas putida</i> environmental strain 28 using primers for <i>carAc</i> and <i>antB</i>. |center]]<html><br />
<br />
<br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p>The purified PCR products for <i>carAc</i> and <i>antB</i> were digested with restriction enzymes EcoRI+SpeI, EcoRI+PstI, and XbaI+PstI. However, only <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmids that had been digested with EcoRI+SpeI and EcoRI+PstI were available to attempt ligation. Gel results were inconclusive on the restriction digest product as the plasmid size appeared much larger than expected. However, transformation was still attempted on the ligation products for both genes (both using EcoRI+SpeI ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid). The transformation products were plated onto chloroamphenicol (chlor) plates to select for colonies that had the chloro resistance gene on the <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid. Also this week another round of PCR was performed for the <i> car</i> and <i> ant</i> genes, however all, but <i>carAc</i> showed bands in the negative control lane, possibly indicating contamination or the formation of primer dimers. The <i>carAc</i> bands were fairly weak and a PCR purification resulted in very low DNA concentration, insufficient to move onto restriction digest. Next week's plan hinges heavily on the result of the transformation from this Friday. </p><br />
<br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>The results of our transformation on <i>carAc</i> and <i>antB</i> from last week showed mostly red colonies indicating that the ligation was unsuccessful and that the PSB1C3 plasmid simply closed on itself with no insert. However, there were three colonies for <i>carAc</i> that were white (cut with both X+P and E+P). Colony PCR was performed on these three colonies using biobrick primer sets to verify that the part had been inserted in these colonies, but unfortunately all PCR results were negative indicating an unsuccessful ligation.</p><br />
<p>We also performed a miniprep on <i>Pseudomonas putida</i> strain #28 this week using a home-made protocol rather than the Qiagen kit. Miniprep A had a DNA concentration of 1539.8 ng/μL and Miniprep B had DNA concentration of 1001.2 ng/μL possibly indicating that there may have been a lot of genomic DNA contamination. However miniprep A product was still used as a PCR template for a reaction attempting to isolate <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i>antB</i>, and <i>antC</i>. Special conditions for this PCR included replacing 60 μL of water with 60 μL of betaine heated to 37 degrees Celsius and running a Mg gradient ranging from 0.5 μL - 2.5 μL per tube for each gene. Of these only <i>antB</i> had bands of the right size in 3 of the lanes (Mg concentrations of 1.5, 2, and 2.5 μL). However, all of the <i>antB</i> bands (including the negative control) contained a contamination band at around 100 bases, probably due to the formation of primer dimers. This forced us to do a gel extraction rather than a PCR purification. This gel extraction did not work as the nanodrop results indicated high amounts of agarose contamination. Another PCR was performed using the same conditions, but using miniprep B instead as there was no more miniprep A product remaining. Unfortunately there were no bands for <i>antB</i>, only the 100 base pair contamination bands. </p><br />
<br />
</html>[[File:AntA and antC Ucalgary.png|500px|thumb|Gel of PCR ran on miniprep A of <i>Pseudomonas putida</i> attempting to isolate <i>antA</i> and <i>antC</i> genes.|center]]<br />
[[File:CarAc and carAd Ucalgary.png|500px|thumb|Gel of PCR ran on miniprep A of <i>Pseudomonas putida</i> attempting to isolate <i>carAc</i> and <i>carAd</i> genes.|center]]<br />
[[File:Minipret A antB and carAc Ucalgary.png|500px|thumb|Gel of PCR ran on miniprep A showing bands 3-5 of the correct size corresponding to antB genes and colony PCR ran using biobrick primers attempting to determine if <i>carAc</i> genes were successfully transformed into PSB1C3 plasmid.|center]] <html><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>This week we modified our PCR protocol to adjust for the homemade Taq polymerase we were using. Template DNA was boiled for 10 minutes on its own to replace the initialization step. This is because this polymerase could not tolerate the 95 degree heat during the regular initialization step. Another modification we made was to boil our primers for 5 minutes prior to adding them to the master mix. This was done to discourage the formation of primer-dimers. Finally we also attempted a touch-down PCR which started at an annealing temperature greater than the Tm of our primers and gradually worked its way down to below the Tm of the primers. This was done to increase the specificity of our reaction and also eliminate the formation of primer-dimers. Unfortunately these techniques still saw the formation of dimers, preventing us from performing a PCR purification. Again, the gel purification procedure did not lead to successful purification of our target gene. Due to the problems we have had so far our goal for next week is to perform an assay experiment to try and determine which of the <i>Pseudomonas putida</i> strains we have access to can actually degrade carbazole and therefore contain the pCAR1 plasmid. </p><br />
<br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The goal for this week was to grow different strains of<i> Pseudomonas</i> in media containing B-N media + glucose with only carbazole as a source of nitrogen. In theory only those strains which contained the pCAR1 plasmid would be able to use carbazole as a nitrogen source and thrive in this media. The strains we tested from glycerol stock included <i>P. putida</i> LD1, <i>P.putida</i> environmental strains 28-30, and <i>P. fluorescens</i> PF5. Each of these strains had two cultures grown; one with carbazole and one without it. <i>E. coli</i> grown with carbazole was used as a negative control as it does not contain the pCAR plasmid. At timepoints of 22, 46, and 117 hours of growth samples were taken to do an OD600 reading to measure bacterial growth and had an ammonia assay performed on it. The detection of ammonia acted as a proxy for detecting carbazole degradation as it is an end-product of the pathway. With this in mind, an additional control strain of LD1 was grown in media containing glucose and ammonia. This is to detect the possibility that <i>Pseudomonas</i> can use ammonia as a source of nitrogen after it has degraded carbazole which would be problematic as far as using ammonia levels to determine carbazole degradation. So far we have been having difficulties reading the results of the ammonia assay with a plate reader and may resort to testing our samples using a different assay protocol next week. The OD600 readings were initially very high at 22 hours and then dropped at 46 hours. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>This week we took our last readings from the experiment that we started last week. The OD600 readings taken at 117 hours showed minimal growth over the weekend for all strains except for environmental strain #29 (with carbazole) which increased from 0.086 to 0.313. No other strain showed an increase of 0.100 or greater. This shows that only strain #29 was able to thrive in the carbazole media, possibly indicating that it contains the pCAR plasmid enabling it to use carbazole as a nitrogen source. To verify this we used a new ammonia assay protocol to measure the production of ammonia in the different cultures and theoretically how much carbazole was degraded. Unlike the previous assay we were able to produce a standard curve with a consistent slope based on samples with known amounts of ammonia. However, the only samples that showed any elevation of ammonia levels above the blank were the LD1 samples that were cultured with ammonia chloride to control for the possibility of pseudomonas using ammonia as a nitrogen source. The ammonia levels in this sample were relatively constant which would indicate that the LD1 was not using ammonia as a nitrogen source. Unfortunately the fact that none of the test samples had elevated ammonia levels did not give us any evidence that these strains contain the pCAR plasmid. </p><br />
<br />
</html>[[File: Standard_curve_for_ammonia_assay_UCalgary2012_denitrification_group.png|500px|thumb|Standard curve for ammonia assay using OD635 absorbance.|center|]]<html><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>We received a new strain of Pseudomonas, LD2, this week that had previously been used to study the pCAR1 plasmid. It was grown up in PCA media and had its colonies used for colony PCR. We also received our new primers this week, further increasing the chances of successful PCR. Using the new strain and the new primers <i>carAa</i>, <i>carAc</i>, and <i>carAd</i> all showed at least some bands of close to the correct size. After PCR purification we obtained DNA concentrations of 21.3 ng/μL for <i>carAa</i>, 41.9 ng/ μL for <i>carAc</i>, and 32.8 ng/μL for<i> carAd</i>. These products were then restriction digested using EcoRI + PstI sites. </p><br />
<br />
</html>[[File:07.20.2012 CarAa and CarAc.jpg|500px|thumb|Gel of colony PCR from <i>Pseudomonas sp. LD2</i> for <i>carAa</i> and <i>carAc</i> genes. <i>carAa</i> expected size is 1155 bp and <i>carAc</i> is 323 bp.|center]]<br />
[[File:07.20.2012 CarAd colony PCR from LD2.jpg|500px|thumb|Gel of colony PCR from <i>Pseudomonas sp. LD2</i> for <i>carAd</i> gene. <i>carAd</i> expected size is 989 bp.|center]] <html><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The restriction digested <i>car</i> genes were ligated into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> cells. White colonies grew for<i> carAa</i> and <i>carAc</i> suggesting that ligation and transformation were successful, however no colonies grew for <i>carAd</i> suggesting that the transformation did not work. Colony PCR was performed to confirm the presence of these genes within the vectors, however only <i>carAc</i> was positive. The colonies containing the <i>carAc</i> plasmid were then mini prepped and restriction digested to isolate the plasmid and send for sequencing. Further attempts at transforming<i> carAa</i> and <i>carAd</i> were not successful. Another goal was to perform colony PCR on the <i>P. putida</i> to isolate the<i> antABC</i> genes using both the primers for the entire operon and those for the individual genes. No attempt showed any bands, although salt and temperature gradients were used as well as different types of polymerase. LD2 cultures were grown up to be plasmid prepped and genomic prepped in order to have a higher concentration of template DNA in the PCR master mix and more PCR will be attempted on these genes next week. Mutagenesis primers were also designed this week to remove the illegal restriction cut sites in the<i> carAd</i> and <i>antB</i> genes (NotI and 2xEcoRI respectively). Only 1 mutagenesis primer was designed for <i>antB</i> as one of its EcoRI sites was very close to the beginning of the gene and could be removed using a new primer. During this process it was also discovered that the original ant primers were designed for the wrong genes and therefore do not match the intended sequence. Although the new <i>antABC</i> primers were designed using the correct gene the PCR may not be working due to the length of the operon so the design of new individual primers may be necessary. Finally, we also aimed to grow up the freeze dried culture of <i>Rhodococcus erythropolis</i> (source of the<i> AmdA</i> gene) that we received from DSMZ. The reactivation media was produced and autoclaved to begin the process next week.</p><br />
<br />
</html>[[File:07.23.2012.Restriction Digest of Car genes.jpg|500px|thumb|Gel of restriction digest of car genes. A refers to <i>carAa</i>, C to <i>carAc</i>, and D to <i>carAd</i>. Ladder shows that after purification and digestion DNA is still the correct size.|center]]<br />
[[File:CarAc biobrick confirmation PCR.jpg|500px|thumb|Gel of confirmation PCR done on competent Top 10 <i>E. coli</i> cells in which a PSB1C3 vector containing the <i>carAc</i> gene had been inserted. Red X marks the 500 bp mark on the weak ladder showing the product to be the expected size (323 bp gene + 2*100 bp biobrick sites on each end).|center]] <html><br />
<br />
<h2>Week 14 (July 30 - August 3)</h2><br />
<p>We received the sequencing results this week for <i>carAc</i> which came back as a 100% match to the expected nucleotide sequence. We also successfully ligated both<i> carAa</i> and<i> carAd</i> into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into top 10 <i>E. coli</i> cells. Colony PCR verified the presence of the biobrick plasmid in these colonies. These were both miniprepped, digested and sent for sequencing by the end of the week. Using a salt gradient, addition of DMSO, and the KAPA buffer specifically designed for GC-rich amplicons we were able to amplify the ant operon from a genomic prep of LD2. However, all attempts at ligating and transforming this sequence were met with the unfortunate presence of red colonies (indicating failed insertion into the plasmid). Next week we will recut a <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> vector with Xba and Pst to attempt to mitigate this problem. We also reactivated the <i>R. erythropolis</i> following the DSMZ protocol for reviving freeze-dried cultures that can be found <a href=https://2012.igem.org/Team:Calgary/Notebook/Protocols/dmszfreezedry>here</a> using DSMZ media #1 and grew it on plates made from DSMZ media #220 at 28 degrees. Colonies grew very quickly and were used for colony PCR and to make overnight cultures for downstream applications. The colony PCR using the amidase primers was unsuccessful, so a genomic prep was performed on the overnight culture. Unfortunately we lacked some of the required equipment and the genomic prep did not work, but with a few procedural modifications and the right equipment we are confident it will work next week. A glycerol stock of the<i> R. erythropoplis</i> was also made from another overnight culture. We also started construction on the <i>carAc </i>part this week by inserting a ribosome binding site (B0034) in front of the gene. Finally, since the<i> ant</i> operon was successfully amplified by PCR we designed mutagenesis primers for the 2nd EcoRI site in the operon rather than making new individual primers. We also designed a mutagenesis primer to remove the SpeI site in <i>AmdA</i>. Now that we have conclusively proven that the LD2 strain contains the pCAR1 plasmid we aim to demonstrate its ability to degrade carbazole and other nitrogen containing heterocycles using an assay experiment similar to the one performed in Week 10.</p><br />
<br />
</html>[[File:07.30.2012. CarAa, CarAd.jpg|500px|thumb|<i>carAa</i> and <i>carAd</i> amplified from genomic prep of <i>Pseudomonas sp. LD2</i>. Amplicons are larger than expected, potentially the gel ran diagonally. The 6th lane for each gene was a negative control.|center]]<br />
[[File:07.31.2012.Ant operon.jpg|500px|thumb|<i>ant</i> operon amplified from genomic prep of <i>Pseudomonas sp. LD2</i> at the expected size of 3000 bp.|center]]<br />
[[File:08.01.2012 CarAdBB.jpg|500px|thumb|Lanes 2,4,5, and 11 contain <i>carAd</i> genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent top 10 <i>E.Coli</i>.|center]]<br />
[[File:08.01.2012 CarAaBB.jpg|500px|thumb|Lane 9 contains <i>carAa</i> genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent top 10 <i>E.Coli</i>. Although larger than expected, the positive control RFP generator in lane 10 is also larger than its expected size (~1200 bp including biobrick bookends) indicating that the gel may have a slant in it.|center]]<html><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p>This week we received the sequencing results for <i>carAa</i> and <i>carAd</i>. <i>carAd</i> was a match, however <i>carAa</i> did not match at all and had misplaced biobrick restriction enzyme sites. Due to this we decided to restart the biobricking process for <i>carAa</i> all the way back to PCR from the plasmid. By the end of the week we had progressed to the point of transforming it into competent <i>E. coli</i> cells, however a thick lawn of bacteria had grown overnight making it impossible to pick colonies. A streak plate was made from this plate to attempt to get less dense growth. Since the sequencing came back positive for <i>carAd</i> the next step was to perform site directed mutagenesis on its NotI restriction enzyme site using the primers that were designed previously (for site-directed mutagenesis optimization, please refer to Desulfurization Journal Week 14). The PCR was successful, showing very bright bands at 3000 bp, and the PCR product was digested with DpnI to remove parental DNA. Transformation of this product will indicate whether the mutagenesis was successful. We were also able to successfully perform a genomic prep of <i>Rhodococcus erythropolis</i> and amplify the <i>AmdA</i> gene this week. It was ligated into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> by Friday, and a confirmation colony PCR was run on Friday evening to verify that the gene was inserted. These results will be available by Monday. After many attempts the <i>ant</i> operon was finally ligated and transformed as well this week. Confirmation PCR showed bands of about 3000 bp which is consistent with the size of the operon. By Friday the successful colonies had been grown up and miniprepped, ready to send for sequencing early next week. The <i>carAc</i>/B0034 construct was also successfully transformed this week and verified by running a PCR using the B0034 forward primer. It was also miniprepped and is ready to send for sequencing next week. </p><br />
<p>In addition to this work constructing biobricks we also started an experiment to demonstrate the function of the pCAR plasmid in LD2. Strains of LD2 were grown up in LB and then washed and placed in B-N media + glucose and various experimental additions. These additions included carbazole, 4-PBAH (a nitrogen containing naphthenic acid), ammonia chloride, and a negative control with no nitrogen source. Strains of <i>E. coli</i> were also grown under the same conditions to compare growth and ammonia production of LD2 to a strain that does not contain the pCAR plasmid. These results should act as a preview of what we expect our final biobrick containing <i>E. col</i>i species to produce as well as allowing us to establish LD2 as a positive control for this experiment. </p><br />
<br />
<br />
</html>[[File:08.07.2012 amdA pcr from genomic prep.jpg|500px|thumb|<i>AmdA</i> amplified from the genomic prep of <i>R. Erythropolis</i> shows bands just greater than 1500 base pairs in lanes 2,4-6,9, and 12-13. Lane 14 was a negative control. The expected size of <i>AmdA</i> is 1566 base pairs.|center]]<br />
[[File:08.09.2012 confirmation pcr of ant operon.jpg|500px|thumb|The two labelled lanes show amplification at 3000 base pairs from a confirmation colony PCR using the biobrick primers. The expected size of the <i>ant</i> operon with the biobrick bookends is about 3100 base pairs.|center]]<br />
[[File:08.10.2012 carad notI mutagenesis.jpg|500px|thumb|Strong amplification at just above 3000 base pairs from the <i>carAd</i> PCR using mutagenic primers. Since PSB1C3 is 2070 base pairs and <i>carAd</i> is 989 base pairs the amplification should run at about 3059 base pairs. 3 different amounts of template DNA were tested in lanes 2-7 as indicated and a positive control showed weaker amplification in lanes 8-9 at about 4000 base pairs.|center]]<br />
<html><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>The sequencing for the <i>carAc</i>/<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> construct came back with only <i>carAc</i> and no evidence of a <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> site preceding it. The confirmation PCR that was run last week using the <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> forward primer seemed to indicate that it was inserted, however since <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> is only 12 base pairs the primer may not have been specific enough. The construction on this part was restarted this week. <i>carAa</i> colony PCR on last week's transformation showed two positive colonies, however upon culturing, miniprepping, and digesting these colonies the plasmid appeared to be too large (3000 base pairs as opposed to the expected 2000). This may be due to a problem of a mislabeled plasmid tube that has been experienced by other team members, causing the gene to be inserted into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a> rather than <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. To test this, a streak plate was made from these colonies and grown on an ampicillin resistant plate. Mutagenized <i>carAd</i> was successfully transformed into Top 10 <i>E. coli</i> cells, cultured, and miniprepped. The miniprep product was digested with NotI to test whether the NotI site was successfully removed. These results were good, as the plasmid ran at 2000 base pairs and the insert at just under 1000 base pairs in the mutagenized <i>carAd</i> whereas the non-mutagenized <i>carAd</i> had its insert cut into 2 bands. This will be sent for sequencing next week to confirm the mutation. Unfortunately the sequencing results came back negative for the <i>ant</i> operon meaning we will need to restart genomic PCR on it next week. Multiple transformations and confirmation PCRs were run on <i>AmdA</i> this week, however none displayed a band size of 1700-1800 base pairs as expected. We have continued to try different digested plasmids as other team members also had difficulties this week with ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, possibly indicating a problem with our Antarctic Phosphatase stock.</p><br />
<p>The other news from this week was the final results from our carbazole degradation experiment attempting to demonstrate LD2's ability to degrade carbazole and hopefully other nitrogen containing compounds. Unfortunately, these results were very difficult to make sense of as the LD2 that were cultured with carbazole grew, but did not show elevated ammonia levels which would be indicative of carbazole degradation. One of our controls was LD2 cultured with ammonium chloride to test whether LD2 was capable of using ammonia as a nitrogen source. This would pose a problem for us as we were planning to use an increase in ammonia levels as a proxy for carbazole degradation rather than measuring carbazole degradation directly using GC-MS. However, the LD2 showed significantly less ammonia after 44 hours when compared to 26 hours indicating that it may be degraded, either by the LD2 or by an unaccounted for abiotic process. This has lead us to decide to use GC-MS and directly measure carbazole levels in our sample the next time we do this experiment.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<p>The <i>carAa</i> was re-streaked onto ampicillin plates and grew, probably showing that it was ligated into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a>, not <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, which explained the irregularity of our confirmation digest last week. It was sent for sequencing, but in case it was incorrect we also started a new <i>carAa</i> pipeline by PCRing it from the <i>Pseudomonas</i> genome again and redigesting and ligating. It was transformed into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> over the weekend. <i>carAc</i> did not see a lot of progress this week as we spent much of the time attempting to construct a RBS (B0034) in front of it, but were unsuccessful. <i>carAd</i> was also subjected to this construction with B0034 and we were able to send it for sequencing by the end of the week. Several new constructs were also started for <i>carAd</i> in case the sequencing is negative. The <i>ant</i> operon was successfully transformed and confirmed by colony PCR this week, after which it was grown up and sent for sequencing. We also began to attempt mutagenesis on one of the EcoRI sites in the operon, but had no amplification of the plasmid. We also re-digested some PCR purified product to start another ligation in case sequencing failed. After many attempts we were also able to finally successfully transform an <i>AmdA</i>/PSB1C3 plasmid into Top 10 cells this week. After a miniprep they showed bands of about 1800 base pairs on the confirmation digest gel, so they were also sent for sequencing this week and had mutagenesis started on the illegal SpeI site. Unfortunately, the sequencing sent early in the week came back with ambiguous results. The forward reaction for<i> antABC</i> was a match, but its reverse reaction matched to <i>carAa</i>. Both <i>carAa</i> reactions matched nothing. Since these two genes are over 20 000 base pairs apart on the template plasmid it is virtually impossible that they were actually combined into one plasmid (PCR extension time would not have been long enough and even if it was the product would have run much above the 3000 base pair mark). This is possibly a mistake on Eurofins end, so we are not 100% sure if we have 1 good gene, both of them, or neither. Also the sequence for mutagenized <i>carAd</i> showed only about the first 100 base pairs, which were correct however not long enough meaning it was a poor reaction and inconclusive. Finally, we also re-started our carbazole degradation assay using the GC-MS at biological sciences. This process will take 2 weeks to demonstrate LD2's ability to degrade carbazole. We are forcing it to use carbazole as a carbon and a nitrogen source in one sample and as only a nitrogen source in another sample (this sample has had glucose added to it).</p><br />
<br />
</html>[[File:AmdA colonyPCR calgary12.JPG|500px|thumb|Amplification is seen in lane 7 of this PCR of top 10 colonies into which AmdA/PSB1C3 had been transformed. This indicates that <i>AmdA</i> has been successfully inserted into a biobrick vector.|center]]<br />
[[File:NewcarAagenomePCR calgary12.jpg|500px|thumb|<i>carAa</i> amplified from pCAR1.2 plasmid of <i>Pseudomonas putida</i>.|center]] <html><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<p>The new stock of <i>carAa</i> that we had PCRed from LD2 was sent for sequencing this week and came back as a match, so we have begun constructing a RBS site and a TetR promoter (<a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>) in front of it. At this point all the <i>car</i> genes have been successfully amplified, inserted into a biobrick plasmid, and sequence verified. We also continued to attempt to construct <i>carAc</i> with an RBS site (<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>) in front of it, but had no success. On a better note, we confirmed this week that<i> carAd</i> was mutated properly and we were also able to insert an RBS site in front of it. The next step for this gene will be to attempt a plasmid switch as it is currently in <a href="http://partsregistry.org/Part:pSB1A3">pSB1A3</a> and needs to be in <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> for submission to the parts registry. After sending a new batch of the ant operon for sequencing we got the same confusing results as we did last week. We therefore hypothesized that our primers were annealing to a homologous region that is similar to the<i> ant</i> genes and very close to <i>carAa</i>. This means that we need to restart PCR on this gene as all of our current product is incorrect. The sequencing results for <i>AmdA</i> that came back this week were also somewhat confusing as they did not quite match the sequence we expected. However, they were a 100% match for the <i>AmdA</i> gene in a different strain of <i>Rhodococcus erythropolis</i>. This means that DSMZ probably sent us the wrong strain of bacteria and that we were very lucky that the primers we designed based off of the expected template actually worked to amplify a slightly different gene in another strain. The good news is that this new sequence contains no illegal cut sites, which explains why mutagenesis was not working (there was nothing to mutate) and allows us to immediately begin construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> in front of <i>AmdA</i> this week. </p><br />
<br />
</html>[[File:Caraa colonyPCR calgary12.JPG|500px|thumb|Colony PCR amplification from top 10 <i>E. coli</i> cells with a PSB1C3/<i>carAa</i> construct transformed into them. Lanes 1-3, 5, and 7 were sent for sequencing with lane 5 coming back positive.|center]]<html><br />
<br />
<h2>Week 19 (September 3- September 7)</h2><br />
<p><i>carAd</i>/<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> was successfully plasmid switched into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, making it ready for submission. Construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> and <i>carAa</i> may have been successful as the digest of the miniprep looked promising. It will be sent for sequencing next week. Unfortunately there was no progress made on the constructions of <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> or <i>carAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. These will be redigested and religated for next week. We could not amplify the <i>ant</i> operon despite trying many variations of PCR using high fidelity KAPA polymerase. This is probably due to the multiple, large regions of homology on the operon that the reverse primer binds to making amplification difficult. Because the function of this gene can theoretically be replaced by <i>AmdA</i>, we may scale back efforts dedicated to it, as the chances of successful amplification appear slim. </p><br />
<br />
<h2>Weeks 20-22 (September 10 - September 30)</h2><br />
<p>Constructs that we continued to work on this week included <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, <i>carAa</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, and <i>carAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. Some sequencing was sent, but no positive results were obtained. A new approach to biobrick the <i>ant</i> operon was implemented this week. A new forward primer was designed that would anneal about 1500 base pairs upstream of the <i>ant</i> operon, avoiding the region of homology that is repeated at various points on the plasmid. Once this region is successfully amplified we could use this amplified DNA as a template for another PCR reaction which would use the normal <i>ant</i> primers. Since the template would no longer include regions of homology these primers should be free to work properly and amplify the <i>ant</i> operon. The PCR using the new primers was started this week, and although amplification at 4500 base pairs was observed as expected, there was also a lot of non-specific amplification. This means that to purify the amplified DNA that we want and avoid the other products a dreaded gel extraction had to be performed which, as usual, did not work. At this point it was decided that further attempts to amplify the <i>ant</i> operon would be futile and our efforts would be better served working on constructing the parts we do have. Unfortunately no progress was made in these weeks on construction. </p><br />
<br />
<br />
<br />
<br />
</html><br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/ProtocolsTeam:Calgary/Notebook/Protocols2012-10-03T23:18:44Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
<br />
TITLE=Protocols|<br />
CONTENT = <html><br />
<p>Here is a list of all the procedures we used this summer. Each contains a description and list of materials required.</p><br />
<ul><br />
<br />
<h2>General Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/agarosegel">Agarose Gel Electrophresis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/gemomicprep">Bacterial Genomic DNA Purification</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/transformation">Bacterial Transformation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/construction">Construction Techniques</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Carbazole GC-MS Analysis">GC-MS Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/gelextraction">Gel Extraction</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/lbagar">LB Agar Plates</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oextraction">Organic Extraction</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/onculture">Overnight Cultures</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/pcrpurification">PCR Purification</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/plasmidminiprep">Plasmid Purification (from <i>E. coli</i>)</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/compcells">Preparing Chemically Competent Cells (<i>E. coli</i>)</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/glycerolstock">Preparing Glycerol Stocks (<i>E. coli</i>)<br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/dnarehydration">Rehydration of Registry DNA</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/dmszfreezedry">Reviving Freeze-dried Bacterial Cultures from DSMZ</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">Site-Directed Mutagenesis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/taqpcr">Taq PCR Protocol</a></li><br />
<br />
<h2>Electrochemistry Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/cvs">Cyclic Voltammetry</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/potstd">Potentiostatic Standard Curve Generation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/potentiostatic">Reporter Expression Detection</a></li><br />
<br />
<br />
<h2>Desulfurization Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/catalase">Catalase assay</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/desulfur">Desulfurization Assay</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/hpac">HpaC assay</a></li><br />
<br />
<h2>Decarboxylation Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/PetroBrick Validation Assay">PetroBrick Validation Assay </a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oleT in Validation Assay">oleT Validation Assay </a></li><br />
<br />
<h2>Denitrogenation Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/carbazole">Carbazole Degradation Assay</a></li><br />
<br />
<h2>Decatecholization Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/decatecholization">Decatecholization Assay</a></li><br />
<h2>Transposon Mutant Library for Toxin Detection</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">Transposon-Mediated Mutant Library Generation</a></li><br />
<br />
<h2>Kill Switch Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgcircuit">Characterization of mgtA regulation with GFP </a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgtacircuit">Characterization of mgtA regulation with S7 killgene </a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/nucleaseassay">Nuclease assay</a></li><br />
<br />
<h2>Bioreactor Assays Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/bacgrowth">Bacterial Growth Experiments</a></li><br />
<p>Belt Selection Tests:</p><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/bactest">Bacteria Test</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/hcseptest">Hydrocarbon Separation Test</a></li><li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/hctest">Hydrocarbon Test</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/hdbeltskim">NA and Hexadecane Belt Skim Test</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tailingtest">Tailings Test</a></li><br />
</ul><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<h2>Modeling Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Modelvalidation">Modelling validation experiments</a></li><br />
<br />
<br />
<br />
</ul><br />
<br />
</html><br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/dmszfreezedryTeam:Calgary/Notebook/Protocols/dmszfreezedry2012-10-03T23:17:11Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
<br />
TITLE=Revival of Freeze-dried Bacterial Cultures from DSMZ|<br />
CONTENT=<html><br />
<p><br />
The following protocol is used to reactivate and culture bacterial strains received from DMSZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH).<br />
</p><br />
<ol><br />
<li>Remove the glass ampoule from plastic tube.</li><br />
<li>Check the indicator to ensure an intact seal (red or blue – good seal, pink or orange – humidity is present in the sample).</li><br />
<li>Heat the tip (pointy tip up) of glass ampoule over a Bunsen burner flame thoroughly.</li><br />
<li>Drop some water onto the tip to crack glass all around.</li><br />
<li>Carefully strike the glass tip with forceps (or a similar tool) to break off the tip.</li><br />
<li>Remove the insulation material and extract the smaller vial with forceps. If the bacteria in the vial is an anaerobic strain, ensure anaerobic conditions are used during the rest of the procedure.</li><br />
<li>Remove the cotton plug from vial and keep it sterile.</li><br />
<li>Add 0.5 mL recommended medium as indicated by the accompanying instructions to the dehydrated culture and insert cotton plug back into the vial. Allow the culture to rehydrate for about 30 min.</li><br />
<li>Remove the cotton plug again. Mix very gently with an inoculation loop and transfer to 5mL of a recommended liquid medium in a 15mL tube. Mix well.</li><br />
<li>Pipette 100uL of liquid culture onto plate as a spot and make a streak plate.</li><br />
</ol><br />
<ul><li>Additional incubation directions are provided on the <a href="http://www.dsmz.de/home.html">DSMZ website</a>.</li></ul><br />
</html><br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/DenitrogenationTeam:Calgary/Notebook/Denitrogenation2012-10-03T23:11:12Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Denitrogenation Journal|<br />
CONTENT=<br />
<html><br />
<br />
<h2>Week 1 (May 1-4)</h2><br />
<p>The following section covers wetlab aspect of our overall project focusing on microbial conversion of naphthenic acids into economically-valuable hydrocarbons. The approach taken in this endeavour will be from four strategic starting points - ring cleavage, decarboxylation, denitrogenation, and desulfurization. Overall, the 'hydrocarbons' aspect of the project is a critical one to our overall design and construction of a bioreactor capable of not only detecting but also converting naphthenic acids and other toxins to useful or at least innocuous compounds. </p><br />
<br />
<h2>Week 2-3 (May 7-18)</h2><br />
<p>In the first two weeks of iGEM our group has focused on reviewing literature regarding the bioremediation of nitrogen groups attached to naphthenic acids. The most prevalent N heterocycle is carbazole, representing 75% of total nitrogen by mass. The upper pathway of carbazole biodegradation is catalyzed by the enzymes coded for by the <i>car</i> operon, <i>CarA</i> (<i>CarAaAcAd</i>), <i>CarB</i> (<i>CarBaBb</i>), and <i>CarC</i>. These enzymes convert carbazole to anthralinic acid. The lower pathway is catalyzed by the enzymes of the <i>ant</i> operon, <i>antA, B, and C</i>, yielding catechol while releasing CO2 and NH3. The <i>car</i> and <i>ant</i> operons are both regulated by the P<sup>ant</sup> promoter which is induced by the protein, antR. <i>carAa</i> also has its own promoter which is not induced by <i>antR</i> (Diaz & Garcia, 2010). We have also investigated an alternative pathway using <i>CarA</i> combined with an amidase (<i>amdA</i>) that selectively cleaves NH2 from an intermediate of the car pathway (Xu et al, 2006). This could bypass much of the car/ant pathway and is possibly more efficient.</p><br />
<p>We have decided to use <i>Pseudomonas resinovorans</i> and <i>Rhodococcus erythropolis</i> to derive these genes from. <i>carABC</i> and <i>antABC</i> from<i> P. resinovorans</i> has been shown to have a wide range of nitrogen containing substrate specificity. <i>R. erythropolis</i> contains the <i>amdA</i> gene that we wish to use, and some evidence suggests that it may also be able to degrade sulfur rings through its <i>carABC</i> pathway.</p><br />
<br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p>This week we reviewed the primers listed in the database and also designed some new ones. Primers for <i>carAa</i>,<i> carAc</i>, and <i>carAd</i> were designed individually, while primers for <i>CarBaBbC</i> and <i>antABC</i> were designed to encompass multiple genes in a sequence. These primers were designed to be used on <i>Pseudomonas putida</i> which we decided to use as our gene source since it was available to us as opposed to ordering <i>Pseudomonas resinovorans</i>. We also designed a primer for the<i> AmdA</i> gene from <i>Rhodococcus erthyroplois</i>. In addition to ordering these primers we also ordered the nitrogen containing compounds that we will need to test these enzymes on. We decided on using carbazole to make sure the enzymes can perform their natural function as well as pyrrolidine to test them on a similar ring structure. We also ordered cyclohexamine in order to independently test the function of the alternative <i>AmdA</i> pathway. Finally, we decided to eventually order 4-Piperidine butyric acid hydrochloride to test how the enzymes will work on nitrogen containing naphthenic acids. However, we decided since it is a very expensive compound we would wait to make sure the enzyme's work on more simple compounds before ordering it.</p><br />
<p>We also started our work in the wet lab by plating colonies and making an overnight culture of -80 freezer stock <i>Psuedomonas putida</i> on Thursday. We also resuspended the primers for <i>carAa</i>, <i>carAc</i>, <i>carAd</i>,<i> antA</i>, <i>antB</i>, and <i>ant C</i> that were already in the database. We were able to use the colonies that grew on the streak plates to start a colony PCR to attempt to isolate each of these genes on Friday.</p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2><br />
<p>On Monday and Tuesday we used the database primers to attempt to isolate <i>carAa</i>, <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i>antB</i>, and <i>antC</i> from the <i>Psuedomonas putida</i> we plated last week. <i>carAd</i>, <i>antB</i>, and <i>antC</i> were all put in the gradient PCR machine to account for the wide range of their primers’ melting points, while the others were done via regular PCR. Unfortunately, no positive results were obtained from these reactions. The PCR on <i>carAc</i> and <i>antA</i> was repeated on Wednesday, still not giving positive results. Finally, we attempted to use a salt concentration gradient on the PCR reaction for <i>carAc</i> and <i>antA</i>, using concentrations that ranged from 1.0 microlitres/tube to 2.0 microlitres/tube in increments of 0.2 microlitres/tube. This helped as <i>carAc</i> showed bands in samples that had concentrations of 1.2 and 1.4 microlitres/tube. <i> antA</i> also showed bands, however they were not the correct size, indicating contamination and/or non-specific annealing of the primer. The positive control also showed bands of the correct size. Earlier in the week we also made overnight cultures of 3 environmental strains (28, 29, 30) of <i>Psuedomonas putida</i> from -80 glycerol stock. On Friday we performed a genomic prep on these cultures, and plan on attempting to amplify genes from the isolated DNA next week. </p><br />
<br />
</html>[[File:CarAa and carAc Ucalgary12.png|500px|thumb|Gel of PCR using database primers for <i>carAa</i> and <i>carAc</i> on <i>Pseudomonas putida</i>|center]]<html><br />
<br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> We started off this week by determining the DNA concentration of our genomic prep samples from last week using the nanodrop. DNA concentration for all three<i> P. putida</i> strains was at least 1000 ng/microlitre, well above what was needed for PCR. 1/2 and 1/3 dilutions were prepared for all three strains so as not to have an excess of template DNA in PCR reactions. PCR was performed on all 3 strains using primers for <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i> antB</i>, and <i>antC</i> using 6 replicates per gene. The only successful amplification appeared to be <i>antB</i> and <i>carAc</i>, both from strain 28 (with weaker bands in strain 29). We then performed another PCR, just on those two genes with an increased amount of Taq polymerase to hopefully get enough amplified DNA to move forward with. This resulted in strong bands for both at the expected size. We then performed PCR purification using the Qiaquick kit and obtained samples containing 33.5 ng/microlitre of <i>antB</i> DNA and 129 ng/microlitre of <i>carAc </i>DNA. These concentrations were both sufficient to begin a restriction digest and ligation of these parts into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. Next week we hope to verify the results of the restriction digest, continue to amplify <i>carAc</i> and <i>antB</i> from strain 28, and hopefully submit a biobrick for sequencing.</p><br />
<br />
</html>[[File:AntB and carAc Ucalgary.png|500px|thumb|Gel of PCR ran on genomic DNA of <i>Pseudomonas putida</i> environmental strain 28 using primers for <i>carAc</i> and <i>antB</i>. |center]]<html><br />
<br />
<br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p>The purified PCR products for <i>carAc</i> and <i>antB</i> were digested with restriction enzymes EcoRI+SpeI, EcoRI+PstI, and XbaI+PstI. However, only <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmids that had been digested with EcoRI+SpeI and EcoRI+PstI were available to attempt ligation. Gel results were inconclusive on the restriction digest product as the plasmid size appeared much larger than expected. However, transformation was still attempted on the ligation products for both genes (both using EcoRI+SpeI ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid). The transformation products were plated onto chloroamphenicol (chlor) plates to select for colonies that had the chloro resistance gene on the <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid. Also this week another round of PCR was performed for the <i> car</i> and <i> ant</i> genes, however all, but <i>carAc</i> showed bands in the negative control lane, possibly indicating contamination or the formation of primer dimers. The <i>carAc</i> bands were fairly weak and a PCR purification resulted in very low DNA concentration, insufficient to move onto restriction digest. Next week's plan hinges heavily on the result of the transformation from this Friday. </p><br />
<br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>The results of our transformation on <i>carAc</i> and <i>antB</i> from last week showed mostly red colonies indicating that the ligation was unsuccessful and that the PSB1C3 plasmid simply closed on itself with no insert. However, there were three colonies for <i>carAc</i> that were white (cut with both X+P and E+P). Colony PCR was performed on these three colonies using biobrick primer sets to verify that the part had been inserted in these colonies, but unfortunately all PCR results were negative indicating an unsuccessful ligation.</p><br />
<p>We also performed a miniprep on <i>Pseudomonas putida</i> strain #28 this week using a home-made protocol rather than the Qiagen kit. Miniprep A had a DNA concentration of 1539.8 ng/μL and Miniprep B had DNA concentration of 1001.2 ng/μL possibly indicating that there may have been a lot of genomic DNA contamination. However miniprep A product was still used as a PCR template for a reaction attempting to isolate <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i>antB</i>, and <i>antC</i>. Special conditions for this PCR included replacing 60 μL of water with 60 μL of betaine heated to 37 degrees Celsius and running a Mg gradient ranging from 0.5 μL - 2.5 μL per tube for each gene. Of these only <i>antB</i> had bands of the right size in 3 of the lanes (Mg concentrations of 1.5, 2, and 2.5 μL). However, all of the <i>antB</i> bands (including the negative control) contained a contamination band at around 100 bases, probably due to the formation of primer dimers. This forced us to do a gel extraction rather than a PCR purification. This gel extraction did not work as the nanodrop results indicated high amounts of agarose contamination. Another PCR was performed using the same conditions, but using miniprep B instead as there was no more miniprep A product remaining. Unfortunately there were no bands for <i>antB</i>, only the 100 base pair contamination bands. </p><br />
<br />
</html>[[File:AntA and antC Ucalgary.png|500px|thumb|Gel of PCR ran on miniprep A of <i>Pseudomonas putida</i> attempting to isolate <i>antA</i> and <i>antC</i> genes.|center]]<br />
[[File:CarAc and carAd Ucalgary.png|500px|thumb|Gel of PCR ran on miniprep A of <i>Pseudomonas putida</i> attempting to isolate <i>carAc</i> and <i>carAd</i> genes.|center]]<br />
[[File:Minipret A antB and carAc Ucalgary.png|500px|thumb|Gel of PCR ran on miniprep A showing bands 3-5 of the correct size corresponding to antB genes and colony PCR ran using biobrick primers attempting to determine if <i>carAc</i> genes were successfully transformed into PSB1C3 plasmid.|center]] <html><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>This week we modified our PCR protocol to adjust for the homemade Taq polymerase we were using. Template DNA was boiled for 10 minutes on its own to replace the initialization step. This is because this polymerase could not tolerate the 95 degree heat during the regular initialization step. Another modification we made was to boil our primers for 5 minutes prior to adding them to the master mix. This was done to discourage the formation of primer-dimers. Finally we also attempted a touch-down PCR which started at an annealing temperature greater than the Tm of our primers and gradually worked its way down to below the Tm of the primers. This was done to increase the specificity of our reaction and also eliminate the formation of primer-dimers. Unfortunately these techniques still saw the formation of dimers, preventing us from performing a PCR purification. Again, the gel purification procedure did not lead to successful purification of our target gene. Due to the problems we have had so far our goal for next week is to perform an assay experiment to try and determine which of the <i>Pseudomonas putida</i> strains we have access to can actually degrade carbazole and therefore contain the pCAR1 plasmid. </p><br />
<br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The goal for this week was to grow different strains of<i> Pseudomonas</i> in media containing B-N media + glucose with only carbazole as a source of nitrogen. In theory only those strains which contained the pCAR1 plasmid would be able to use carbazole as a nitrogen source and thrive in this media. The strains we tested from glycerol stock included <i>P. putida</i> LD1, <i>P.putida</i> environmental strains 28-30, and <i>P. fluorescens</i> PF5. Each of these strains had two cultures grown; one with carbazole and one without it. <i>E. coli</i> grown with carbazole was used as a negative control as it does not contain the pCAR plasmid. At timepoints of 22, 46, and 117 hours of growth samples were taken to do an OD600 reading to measure bacterial growth and had an ammonia assay performed on it. The detection of ammonia acted as a proxy for detecting carbazole degradation as it is an end-product of the pathway. With this in mind, an additional control strain of LD1 was grown in media containing glucose and ammonia. This is to detect the possibility that <i>Pseudomonas</i> can use ammonia as a source of nitrogen after it has degraded carbazole which would be problematic as far as using ammonia levels to determine carbazole degradation. So far we have been having difficulties reading the results of the ammonia assay with a plate reader and may resort to testing our samples using a different assay protocol next week. The OD600 readings were initially very high at 22 hours and then dropped at 46 hours. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>This week we took our last readings from the experiment that we started last week. The OD600 readings taken at 117 hours showed minimal growth over the weekend for all strains except for environmental strain #29 (with carbazole) which increased from 0.086 to 0.313. No other strain showed an increase of 0.100 or greater. This shows that only strain #29 was able to thrive in the carbazole media, possibly indicating that it contains the pCAR plasmid enabling it to use carbazole as a nitrogen source. To verify this we used a new ammonia assay protocol to measure the production of ammonia in the different cultures and theoretically how much carbazole was degraded. Unlike the previous assay we were able to produce a standard curve with a consistent slope based on samples with known amounts of ammonia. However, the only samples that showed any elevation of ammonia levels above the blank were the LD1 samples that were cultured with ammonia chloride to control for the possibility of pseudomonas using ammonia as a nitrogen source. The ammonia levels in this sample were relatively constant which would indicate that the LD1 was not using ammonia as a nitrogen source. Unfortunately the fact that none of the test samples had elevated ammonia levels did not give us any evidence that these strains contain the pCAR plasmid. </p><br />
<br />
</html>[[File: Standard_curve_for_ammonia_assay_UCalgary2012_denitrification_group.png|500px|thumb|Standard curve for ammonia assay using OD635 absorbance.|center|]]<html><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>We received a new strain of Pseudomonas, LD2, this week that had previously been used to study the pCAR1 plasmid. It was grown up in PCA media and had its colonies used for colony PCR. We also received our new primers this week, further increasing the chances of successful PCR. Using the new strain and the new primers <i>carAa</i>, <i>carAc</i>, and <i>carAd</i> all showed at least some bands of close to the correct size. After PCR purification we obtained DNA concentrations of 21.3 ng/μL for <i>carAa</i>, 41.9 ng/ μL for <i>carAc</i>, and 32.8 ng/μL for<i> carAd</i>. These products were then restriction digested using EcoRI + PstI sites. </p><br />
<br />
</html>[[File:07.20.2012 CarAa and CarAc.jpg|500px|thumb|Gel of colony PCR from <i>Pseudomonas sp. LD2</i> for <i>carAa</i> and <i>carAc</i> genes. <i>carAa</i> expected size is 1155 bp and <i>carAc</i> is 323 bp.|center]]<br />
[[File:07.20.2012 CarAd colony PCR from LD2.jpg|500px|thumb|Gel of colony PCR from <i>Pseudomonas sp. LD2</i> for <i>carAd</i> gene. <i>carAd</i> expected size is 989 bp.|center]] <html><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The restriction digested <i>car</i> genes were ligated into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> cells. White colonies grew for<i> carAa</i> and <i>carAc</i> suggesting that ligation and transformation were successful, however no colonies grew for <i>carAd</i> suggesting that the transformation did not work. Colony PCR was performed to confirm the presence of these genes within the vectors, however only <i>carAc</i> was positive. The colonies containing the <i>carAc</i> plasmid were then mini prepped and restriction digested to isolate the plasmid and send for sequencing. Further attempts at transforming<i> carAa</i> and <i>carAd</i> were not successful. Another goal was to perform colony PCR on the <i>P. putida</i> to isolate the<i> antABC</i> genes using both the primers for the entire operon and those for the individual genes. No attempt showed any bands, although salt and temperature gradients were used as well as different types of polymerase. LD2 cultures were grown up to be plasmid prepped and genomic prepped in order to have a higher concentration of template DNA in the PCR master mix and more PCR will be attempted on these genes next week. Mutagenesis primers were also designed this week to remove the illegal restriction cut sites in the<i> carAd</i> and <i>antB</i> genes (NotI and 2xEcoRI respectively). Only 1 mutagenesis primer was designed for <i>antB</i> as one of its EcoRI sites was very close to the beginning of the gene and could be removed using a new primer. During this process it was also discovered that the original ant primers were designed for the wrong genes and therefore do not match the intended sequence. Although the new <i>antABC</i> primers were designed using the correct gene the PCR may not be working due to the length of the operon so the design of new individual primers may be necessary. Finally, we also aimed to grow up the freeze dried culture of <i>Rhodococcus erythropolis</i> (source of the<i> AmdA</i> gene) that we received from DSMZ. The reactivation media was produced and autoclaved to begin the process next week.</p><br />
<br />
</html>[[File:07.23.2012.Restriction Digest of Car genes.jpg|500px|thumb|Gel of restriction digest of car genes. A refers to <i>carAa</i>, C to <i>carAc</i>, and D to <i>carAd</i>. Ladder shows that after purification and digestion DNA is still the correct size.|center]]<br />
[[File:CarAc biobrick confirmation PCR.jpg|500px|thumb|Gel of confirmation PCR done on competent Top 10 <i>E. coli</i> cells in which a PSB1C3 vector containing the <i>carAc</i> gene had been inserted. Red X marks the 500 bp mark on the weak ladder showing the product to be the expected size (323 bp gene + 2*100 bp biobrick sites on each end).|center]] <html><br />
<br />
<h2>Week 14 (July 30 - August 3)</h2><br />
<p>We received the sequencing results this week for <i>carAc</i> which came back as a 100% match to the expected nucleotide sequence. We also successfully ligated both<i> carAa</i> and<i> carAd</i> into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into top 10 <i>E. coli</i> cells. Colony PCR verified the presence of the biobrick plasmid in these colonies. These were both miniprepped, digested and sent for sequencing by the end of the week. Using a salt gradient, addition of DMSO, and the KAPA buffer specifically designed for GC-rich amplicons we were able to amplify the ant operon from a genomic prep of LD2. However, all attempts at ligating and transforming this sequence were met with the unfortunate presence of red colonies (indicating failed insertion into the plasmid). Next week we will recut a <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> vector with Xba and Pst to attempt to mitigate this problem. We also reactivated the <i>R. erythropolis</i> following the DSMZ protocol for reviving freeze-dried cultures that can be found <a href=https://2012.igem.org/Team:Calgary/Notebook/Protocols/dmszfreezedry>here</a> using DSMZ media #1 and grew it on plates made from DSMZ media #220 at 28 degrees. Colonies grew very quickly and were used for colony PCR and to make overnight cultures for downstream applications. The colony PCR using the amidase primers was unsuccessful, so a genomic prep was performed on the overnight culture. Unfortunately we lacked some of the required equipment and the genomic prep did not work, but with a few procedural modifications and the right equipment we are confident it will work next week. A glycerol stock of the<i> R. erythropoplis</i> was also made from another overnight culture. We also started construction on the <i>carAc </i>part this week by inserting a ribosome binding site (B0034) in front of the gene. Finally, since the<i> ant</i> operon was successfully amplified by PCR we designed mutagenesis primers for the 2nd EcoRI site in the operon rather than making new individual primers. We also designed a mutagenesis primer to remove the SpeI site in <i>AmdA</i>. Now that we have conclusively proven that the LD2 strain contains the pCAR1 plasmid we aim to demonstrate its ability to degrade carbazole and other nitrogen containing heterocycles using an assay experiment similar to the one performed in Week 10.</p><br />
<br />
</html>[[File:07.30.2012. CarAa, CarAd.jpg|500px|thumb|<i>carAa</i> and <i>carAd</i> amplified from genomic prep of <i>Pseudomonas sp. LD2</i>. Amplicons are larger than expected, potentially the gel ran diagonally. The 6th lane for each gene was a negative control.|center]]<br />
[[File:07.31.2012.Ant operon.jpg|500px|thumb|<i>ant</i> operon amplified from genomic prep of <i>Pseudomonas sp. LD2</i> at the expected size of 3000 bp.|center]]<br />
[[File:08.01.2012 CarAdBB.jpg|500px|thumb|Lanes 2,4,5, and 11 contain <i>carAd</i> genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent top 10 <i>E.Coli</i>.|center]]<br />
[[File:08.01.2012 CarAaBB.jpg|500px|thumb|Lane 9 contains <i>carAa</i> genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent top 10 <i>E.Coli</i>. Although larger than expected, the positive control RFP generator in lane 10 is also larger than its expected size (~1200 bp including biobrick bookends) indicating that the gel may have a slant in it.|center]]<html><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p>This week we received the sequencing results for <i>carAa</i> and <i>carAd</i>. <i>carAd</i> was a match, however <i>carAa</i> did not match at all and had misplaced biobrick restriction enzyme sites. Due to this we decided to restart the biobricking process for <i>carAa</i> all the way back to PCR from the plasmid. By the end of the week we had progressed to the point of transforming it into competent <i>E. coli</i> cells, however a thick lawn of bacteria had grown overnight making it impossible to pick colonies. A streak plate was made from this plate to attempt to get less dense growth. Since the sequencing came back positive for <i>carAd</i> the next step was to perform site directed mutagenesis on its NotI restriction enzyme site using the primers that were designed previously (for site-directed mutagenesis optimization, please refer to Desulfurization Journal Week 14). The PCR was successful, showing very bright bands at 3000 bp, and the PCR product was digested with DpnI to remove parental DNA. Transformation of this product will indicate whether the mutagenesis was successful. We were also able to successfully perform a genomic prep of <i>Rhodococcus erythropolis</i> and amplify the <i>AmdA</i> gene this week. It was ligated into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> by Friday, and a confirmation colony PCR was run on Friday evening to verify that the gene was inserted. These results will be available by Monday. After many attempts the <i>ant</i> operon was finally ligated and transformed as well this week. Confirmation PCR showed bands of about 3000 bp which is consistent with the size of the operon. By Friday the successful colonies had been grown up and miniprepped, ready to send for sequencing early next week. The <i>carAc</i>/B0034 construct was also successfully transformed this week and verified by running a PCR using the B0034 forward primer. It was also miniprepped and is ready to send for sequencing next week. </p><br />
<p>In addition to this work constructing biobricks we also started an experiment to demonstrate the function of the pCAR plasmid in LD2. Strains of LD2 were grown up in LB and then washed and placed in B-N media + glucose and various experimental additions. These additions included carbazole, 4-PBAH (a nitrogen containing naphthenic acid), ammonia chloride, and a negative control with no nitrogen source. Strains of <i>E. coli</i> were also grown under the same conditions to compare growth and ammonia production of LD2 to a strain that does not contain the pCAR plasmid. These results should act as a preview of what we expect our final biobrick containing <i>E. col</i>i species to produce as well as allowing us to establish LD2 as a positive control for this experiment. </p><br />
<br />
<br />
</html>[[File:08.07.2012 amdA pcr from genomic prep.jpg|500px|thumb|<i>AmdA</i> amplified from the genomic prep of <i>R. Erythropolis</i> shows bands just greater than 1500 base pairs in lanes 2,4-6,9, and 12-13. Lane 14 was a negative control. The expected size of <i>AmdA</i> is 1566 base pairs.|center]]<br />
[[File:08.09.2012 confirmation pcr of ant operon.jpg|500px|thumb|The two labelled lanes show amplification at 3000 base pairs from a confirmation colony PCR using the biobrick primers. The expected size of the <i>ant</i> operon with the biobrick bookends is about 3100 base pairs.|center]]<br />
[[File:08.10.2012 carad notI mutagenesis.jpg|500px|thumb|Strong amplification at just above 3000 base pairs from the <i>carAd</i> PCR using mutagenic primers. Since PSB1C3 is 2070 base pairs and <i>carAd</i> is 989 base pairs the amplification should run at about 3059 base pairs. 3 different amounts of template DNA were tested in lanes 2-7 as indicated and a positive control showed weaker amplification in lanes 8-9 at about 4000 base pairs.|center]]<br />
<html><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>The sequencing for the <i>carAc</i>/<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> construct came back with only <i>carAc</i> and no evidence of a <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> site preceding it. The confirmation PCR that was run last week using the <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> forward primer seemed to indicate that it was inserted, however since <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> is only 12 base pairs the primer may not have been specific enough. The construction on this part was restarted this week. <i>carAa</i> colony PCR on last week's transformation showed two positive colonies, however upon culturing, miniprepping, and digesting these colonies the plasmid appeared to be too large (3000 base pairs as opposed to the expected 2000). This may be due to a problem of a mislabeled plasmid tube that has been experienced by other team members, causing the gene to be inserted into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a> rather than <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. To test this, a streak plate was made from these colonies and grown on an ampicillin resistant plate. Mutagenized <i>carAd</i> was successfully transformed into Top 10 <i>E. coli</i> cells, cultured, and miniprepped. The miniprep product was digested with NotI to test whether the NotI site was successfully removed. These results were good, as the plasmid ran at 2000 base pairs and the insert at just under 1000 base pairs in the mutagenized <i>carAd</i> whereas the non-mutagenized <i>carAd</i> had its insert cut into 2 bands. This will be sent for sequencing next week to confirm the mutation. Unfortunately the sequencing results came back negative for the <i>ant</i> operon meaning we will need to restart genomic PCR on it next week. Multiple transformations and confirmation PCRs were run on <i>AmdA</i> this week, however none displayed a band size of 1700-1800 base pairs as expected. We have continued to try different digested plasmids as other team members also had difficulties this week with ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, possibly indicating a problem with our Antarctic Phosphatase stock.</p><br />
<p>The other news from this week was the final results from our carbazole degradation experiment attempting to demonstrate LD2's ability to degrade carbazole and hopefully other nitrogen containing compounds. Unfortunately, these results were very difficult to make sense of as the LD2 that were cultured with carbazole grew, but did not show elevated ammonia levels which would be indicative of carbazole degradation. One of our controls was LD2 cultured with ammonium chloride to test whether LD2 was capable of using ammonia as a nitrogen source. This would pose a problem for us as we were planning to use an increase in ammonia levels as a proxy for carbazole degradation rather than measuring carbazole degradation directly using GC-MS. However, the LD2 showed significantly less ammonia after 44 hours when compared to 26 hours indicating that it may be degraded, either by the LD2 or by an unaccounted for abiotic process. This has lead us to decide to use GC-MS and directly measure carbazole levels in our sample the next time we do this experiment.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<p>The <i>carAa</i> was re-streaked onto ampicillin plates and grew, probably showing that it was ligated into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a>, not <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, which explained the irregularity of our confirmation digest last week. It was sent for sequencing, but in case it was incorrect we also started a new <i>carAa</i> pipeline by PCRing it from the <i>Pseudomonas</i> genome again and redigesting and ligating. It was transformed into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> over the weekend. <i>carAc</i> did not see a lot of progress this week as we spent much of the time attempting to construct a RBS (B0034) in front of it, but were unsuccessful. <i>carAd</i> was also subjected to this construction with B0034 and we were able to send it for sequencing by the end of the week. Several new constructs were also started for <i>carAd</i> in case the sequencing is negative. The <i>ant</i> operon was successfully transformed and confirmed by colony PCR this week, after which it was grown up and sent for sequencing. We also began to attempt mutagenesis on one of the EcoRI sites in the operon, but had no amplification of the plasmid. We also re-digested some PCR purified product to start another ligation in case sequencing failed. After many attempts we were also able to finally successfully transform an <i>AmdA</i>/PSB1C3 plasmid into Top 10 cells this week. After a miniprep they showed bands of about 1800 base pairs on the confirmation digest gel, so they were also sent for sequencing this week and had mutagenesis started on the illegal SpeI site. Unfortunately, the sequencing sent early in the week came back with ambiguous results. The forward reaction for<i> antABC</i> was a match, but its reverse reaction matched to <i>carAa</i>. Both <i>carAa</i> reactions matched nothing. Since these two genes are over 20 000 base pairs apart on the template plasmid it is virtually impossible that they were actually combined into one plasmid (PCR extension time would not have been long enough and even if it was the product would have run much above the 3000 base pair mark). This is possibly a mistake on Eurofins end, so we are not 100% sure if we have 1 good gene, both of them, or neither. Also the sequence for mutagenized <i>carAd</i> showed only about the first 100 base pairs, which were correct however not long enough meaning it was a poor reaction and inconclusive. Finally, we also re-started our carbazole degradation assay using the GC-MS at biological sciences. This process will take 2 weeks to demonstrate LD2's ability to degrade carbazole. We are forcing it to use carbazole as a carbon and a nitrogen source in one sample and as only a nitrogen source in another sample (this sample has had glucose added to it).</p><br />
<br />
</html>[[File:AmdA colonyPCR calgary12.JPG|500px|thumb|Amplification is seen in lane 7 of this PCR of top 10 colonies into which AmdA/PSB1C3 had been transformed. This indicates that <i>AmdA</i> has been successfully inserted into a biobrick vector.|center]]<br />
[[File:NewcarAagenomePCR calgary12.jpg|500px|thumb|<i>carAa</i> amplified from pCAR1.2 plasmid of <i>Pseudomonas putida</i>.|center]] <html><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<p>The new stock of <i>carAa</i> that we had PCRed from LD2 was sent for sequencing this week and came back as a match, so we have begun constructing a RBS site and a TetR promoter (<a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>) in front of it. At this point all the <i>car</i> genes have been successfully amplified, inserted into a biobrick plasmid, and sequence verified. We also continued to attempt to construct <i>carAc</i> with an RBS site (<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>) in front of it, but had no success. On a better note, we confirmed this week that<i> carAd</i> was mutated properly and we were also able to insert an RBS site in front of it. The next step for this gene will be to attempt a plasmid switch as it is currently in <a href="http://partsregistry.org/Part:pSB1A3">pSB1A3</a> and needs to be in <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> for submission to the parts registry. After sending a new batch of the ant operon for sequencing we got the same confusing results as we did last week. We therefore hypothesized that our primers were annealing to a homologous region that is similar to the<i> ant</i> genes and very close to <i>carAa</i>. This means that we need to restart PCR on this gene as all of our current product is incorrect. The sequencing results for <i>AmdA</i> that came back this week were also somewhat confusing as they did not quite match the sequence we expected. However, they were a 100% match for the <i>AmdA</i> gene in a different strain of <i>Rhodococcus erythropolis</i>. This means that DSMZ probably sent us the wrong strain of bacteria and that we were very lucky that the primers we designed based off of the expected template actually worked to amplify a slightly different gene in another strain. The good news is that this new sequence contains no illegal cut sites, which explains why mutagenesis was not working (there was nothing to mutate) and allows us to immediately begin construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> in front of <i>AmdA</i> this week. </p><br />
<br />
</html>[[File:Caraa colonyPCR calgary12.JPG|500px|thumb|Colony PCR amplification from top 10 <i>E. coli</i> cells with a PSB1C3/<i>carAa</i> construct transformed into them. Lanes 1-3, 5, and 7 were sent for sequencing with lane 5 coming back positive.|center]]<html><br />
<br />
<h2>Week 19 (September 3- September 7)</h2><br />
<p><i>carAd</i>/<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> was successfully plasmid switched into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, making it ready for submission. Construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> and <i>carAa</i> may have been successful as the digest of the miniprep looked promising. It will be sent for sequencing next week. Unfortunately there was no progress made on the constructions of <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> or <i>carAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. These will be redigested and religated for next week. We could not amplify the <i>ant</i> operon despite trying many variations of PCR using high fidelity KAPA polymerase. This is probably due to the multiple, large regions of homology on the operon that the reverse primer binds to making amplification difficult. Because the function of this gene can theoretically be replaced by <i>AmdA</i>, we may scale back efforts dedicated to it, as the chances of successful amplification appear slim. </p><br />
<br />
<h2>Weeks 20-22 (September 10 - September 30)</h2><br />
<p>Constructs that continued this week included <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, <i>carAa</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, and <i>carAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. Some sequencing was sent, but no positive results were obtained. A new approach to biobrick the <i>ant</i> operon was implemented this week. A new forward primer was designed that would anneal about 1500 base pairs upstream of the <i>ant</i> operon, avoiding the region of homology that is repeated at various points on the plasmid. Once this region is successfully amplified we could use this amplified DNA as a template for another PCR reaction which would use the normal <i>ant</i> primers. Since the template would no longer include regions of homology these primers should be free to work properly and amplify the <i>ant</i> operon. The PCR using the new primers was started this week, and although amplification at 4500 base pairs was observed as expected, there was also a lot of non-specific amplification. This means that to purify the amplified DNA that we want and avoid the other products a dreaded gel extraction had to be performed which, as usual, did not work. At this point it was decided that further attempts to amplify the <i>ant</i> operon would be futile and our efforts would be better served working on constructing the parts we do have. Unfortunately no progress was made in these weeks on construction. </p><br />
<br />
<br />
<br />
<br />
</html><br />
}}</div>Anyakornilohttp://2012.igem.org/Team:Calgary/Notebook/DenitrogenationTeam:Calgary/Notebook/Denitrogenation2012-10-03T23:09:19Z<p>Anyakornilo: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Denitrogenation Journal|<br />
CONTENT=<br />
<html><br />
<br />
<h2>Week 1 (May 1-4)</h2><br />
<p>The following section covers wetlab aspect of our overall project focusing on microbial conversion of naphthenic acids into economically-valuable hydrocarbons. The approach taken in this endeavour will be from four strategic starting points - ring cleavage, decarboxylation, denitrogenation, and desulfurization. Overall, the 'hydrocarbons' aspect of the project is a critical one to our overall design and construction of a bioreactor capable of not only detecting but also converting naphthenic acids and other toxins to useful or at least innocuous compounds. </p><br />
<br />
<h2>Week 2-3 (May 7-18)</h2><br />
<p>In the first two weeks of iGEM our group has focused on reviewing literature regarding the bioremediation of nitrogen groups attached to naphthenic acids. The most prevalent N heterocycle is carbazole, representing 75% of total nitrogen by mass. The upper pathway of carbazole biodegradation is catalyzed by the enzymes coded for by the <i>car</i> operon, <i>CarA</i> (<i>CarAaAcAd</i>), <i>CarB</i> (<i>CarBaBb</i>), and <i>CarC</i>. These enzymes convert carbazole to anthralinic acid. The lower pathway is catalyzed by the enzymes of the <i>ant</i> operon, <i>antA, B, and C</i>, yielding catechol while releasing CO2 and NH3. The <i>car</i> and <i>ant</i> operons are both regulated by the P<sup>ant</sup> promoter which is induced by the protein, antR. <i>carAa</i> also has its own promoter which is not induced by <i>antR</i> (Diaz & Garcia, 2010). We have also investigated an alternative pathway using <i>CarA</i> combined with an amidase (<i>amdA</i>) that selectively cleaves NH2 from an intermediate of the car pathway (Xu et al, 2006). This could bypass much of the car/ant pathway and is possibly more efficient.</p><br />
<p>We have decided to use <i>Pseudomonas resinovorans</i> and <i>Rhodococcus erythropolis</i> to derive these genes from. <i>carABC</i> and <i>antABC</i> from<i> P. resinovorans</i> has been shown to have a wide range of nitrogen containing substrate specificity. <i>R. erythropolis</i> contains the <i>amdA</i> gene that we wish to use, and some evidence suggests that it may also be able to degrade sulfur rings through its <i>carABC</i> pathway.</p><br />
<br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p>This week we reviewed the primers listed in the database and also designed some new ones. Primers for <i>carAa</i>,<i> carAc</i>, and <i>carAd</i> were designed individually, while primers for <i>CarBaBbC</i> and <i>antABC</i> were designed to encompass multiple genes in a sequence. These primers were designed to be used on <i>Pseudomonas putida</i> which we decided to use as our gene source since it was available to us as opposed to ordering <i>Pseudomonas resinovorans</i>. We also designed a primer for the<i> AmdA</i> gene from <i>Rhodococcus erthyroplois</i>. In addition to ordering these primers we also ordered the nitrogen containing compounds that we will need to test these enzymes on. We decided on using carbazole to make sure the enzymes can perform their natural function as well as pyrrolidine to test them on a similar ring structure. We also ordered cyclohexamine in order to independently test the function of the alternative <i>AmdA</i> pathway. Finally, we decided to eventually order 4-Piperidine butyric acid hydrochloride to test how the enzymes will work on nitrogen containing naphthenic acids. However, we decided since it is a very expensive compound we would wait to make sure the enzyme's work on more simple compounds before ordering it.</p><br />
<p>We also started our work in the wet lab by plating colonies and making an overnight culture of -80 freezer stock <i>Psuedomonas putida</i> on Thursday. We also resuspended the primers for <i>carAa</i>, <i>carAc</i>, <i>carAd</i>,<i> antA</i>, <i>antB</i>, and <i>ant C</i> that were already in the database. We were able to use the colonies that grew on the streak plates to start a colony PCR to attempt to isolate each of these genes on Friday.</p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2><br />
<p>On Monday and Tuesday we used the database primers to attempt to isolate <i>carAa</i>, <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i>antB</i>, and <i>antC</i> from the <i>Psuedomonas putida</i> we plated last week. <i>carAd</i>, <i>antB</i>, and <i>antC</i> were all put in the gradient PCR machine to account for the wide range of their primers’ melting points, while the others were done via regular PCR. Unfortunately, no positive results were obtained from these reactions. The PCR on <i>carAc</i> and <i>antA</i> was repeated on Wednesday, still not giving positive results. Finally, we attempted to use a salt concentration gradient on the PCR reaction for <i>carAc</i> and <i>antA</i>, using concentrations that ranged from 1.0 microlitres/tube to 2.0 microlitres/tube in increments of 0.2 microlitres/tube. This helped as <i>carAc</i> showed bands in samples that had concentrations of 1.2 and 1.4 microlitres/tube. <i> antA</i> also showed bands, however they were not the correct size, indicating contamination and/or non-specific annealing of the primer. The positive control also showed bands of the correct size. Earlier in the week we also made overnight cultures of 3 environmental strains (28, 29, 30) of <i>Psuedomonas putida</i> from -80 glycerol stock. On Friday we performed a genomic prep on these cultures, and plan on attempting to amplify genes from the isolated DNA next week. </p><br />
<br />
</html>[[File:CarAa and carAc Ucalgary12.png|500px|thumb|Gel of PCR using database primers for <i>carAa</i> and <i>carAc</i> on <i>Pseudomonas putida</i>|center]]<html><br />
<br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> We started off this week by determining the DNA concentration of our genomic prep samples from last week using the nanodrop. DNA concentration for all three<i> P. putida</i> strains was at least 1000 ng/microlitre, well above what was needed for PCR. 1/2 and 1/3 dilutions were prepared for all three strains so as not to have an excess of template DNA in PCR reactions. PCR was performed on all 3 strains using primers for <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i> antB</i>, and <i>antC</i> using 6 replicates per gene. The only successful amplification appeared to be <i>antB</i> and <i>carAc</i>, both from strain 28 (with weaker bands in strain 29). We then performed another PCR, just on those two genes with an increased amount of Taq polymerase to hopefully get enough amplified DNA to move forward with. This resulted in strong bands for both at the expected size. We then performed PCR purification using the Qiaquick kit and obtained samples containing 33.5 ng/microlitre of <i>antB</i> DNA and 129 ng/microlitre of <i>carAc </i>DNA. These concentrations were both sufficient to begin a restriction digest and ligation of these parts into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. Next week we hope to verify the results of the restriction digest, continue to amplify <i>carAc</i> and <i>antB</i> from strain 28, and hopefully submit a biobrick for sequencing.</p><br />
<br />
</html>[[File:AntB and carAc Ucalgary.png|500px|thumb|Gel of PCR ran on genomic DNA of <i>Pseudomonas putida</i> environmental strain 28 using primers for <i>carAc</i> and <i>antB</i>. |center]]<html><br />
<br />
<br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p>The purified PCR products for <i>carAc</i> and <i>antB</i> were digested with restriction enzymes EcoRI+SpeI, EcoRI+PstI, and XbaI+PstI. However, only <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmids that had been digested with EcoRI+SpeI and EcoRI+PstI were available to attempt ligation. Gel results were inconclusive on the restriction digest product as the plasmid size appeared much larger than expected. However, transformation was still attempted on the ligation products for both genes (both using EcoRI+SpeI ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid). The transformation products were plated onto chloroamphenicol (chlor) plates to select for colonies that had the chloro resistance gene on the <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid. Also this week another round of PCR was performed for the <i> car</i> and <i> ant</i> genes, however all, but <i>carAc</i> showed bands in the negative control lane, possibly indicating contamination or the formation of primer dimers. The <i>carAc</i> bands were fairly weak and a PCR purification resulted in very low DNA concentration, insufficient to move onto restriction digest. Next week's plan hinges heavily on the result of the transformation from this Friday. </p><br />
<br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>The results of our transformation on <i>carAc</i> and <i>antB</i> from last week showed mostly red colonies indicating that the ligation was unsuccessful and that the PSB1C3 plasmid simply closed on itself with no insert. However, there were three colonies for <i>carAc</i> that were white (cut with both X+P and E+P). Colony PCR was performed on these three colonies using biobrick primer sets to verify that the part had been inserted in these colonies, but unfortunately all PCR results were negative indicating an unsuccessful ligation.</p><br />
<p>We also performed a miniprep on <i>Pseudomonas putida</i> strain #28 this week using a home-made protocol rather than the Qiagen kit. Miniprep A had a DNA concentration of 1539.8 ng/μL and Miniprep B had DNA concentration of 1001.2 ng/μL possibly indicating that there may have been a lot of genomic DNA contamination. However miniprep A product was still used as a PCR template for a reaction attempting to isolate <i>carAc</i>, <i>carAd</i>, <i>antA</i>, <i>antB</i>, and <i>antC</i>. Special conditions for this PCR included replacing 60 μL of water with 60 μL of betaine heated to 37 degrees Celsius and running a Mg gradient ranging from 0.5 μL - 2.5 μL per tube for each gene. Of these only <i>antB</i> had bands of the right size in 3 of the lanes (Mg concentrations of 1.5, 2, and 2.5 μL). However, all of the <i>antB</i> bands (including the negative control) contained a contamination band at around 100 bases, probably due to the formation of primer dimers. This forced us to do a gel extraction rather than a PCR purification. This gel extraction did not work as the nanodrop results indicated high amounts of agarose contamination. Another PCR was performed using the same conditions, but using miniprep B instead as there was no more miniprep A product remaining. Unfortunately there were no bands for <i>antB</i>, only the 100 base pair contamination bands. </p><br />
<br />
</html>[[File:AntA and antC Ucalgary.png|500px|thumb|Gel of PCR ran on miniprep A of <i>Pseudomonas putida</i> attempting to isolate <i>antA</i> and <i>antC</i> genes.|center]]<br />
[[File:CarAc and carAd Ucalgary.png|500px|thumb|Gel of PCR ran on miniprep A of <i>Pseudomonas putida</i> attempting to isolate <i>carAc</i> and <i>carAd</i> genes.|center]]<br />
[[File:Minipret A antB and carAc Ucalgary.png|500px|thumb|Gel of PCR ran on miniprep A showing bands 3-5 of the correct size corresponding to antB genes and colony PCR ran using biobrick primers attempting to determine if <i>carAc</i> genes were successfully transformed into PSB1C3 plasmid.|center]] <html><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>This week we modified our PCR protocol to adjust for the homemade Taq polymerase we were using. Template DNA was boiled for 10 minutes on its own to replace the initialization step. This is because this polymerase could not tolerate the 95 degree heat during the regular initialization step. Another modification we made was to boil our primers for 5 minutes prior to adding them to the master mix. This was done to discourage the formation of primer-dimers. Finally we also attempted a touch-down PCR which started at an annealing temperature greater than the Tm of our primers and gradually worked its way down to below the Tm of the primers. This was done to increase the specificity of our reaction and also eliminate the formation of primer-dimers. Unfortunately these techniques still saw the formation of dimers, preventing us from performing a PCR purification. Again, the gel purification procedure did not lead to successful purification of our target gene. Due to the problems we have had so far our goal for next week is to perform an assay experiment to try and determine which of the <i>Pseudomonas putida</i> strains we have access to can actually degrade carbazole and therefore contain the pCAR1 plasmid. </p><br />
<br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The goal for this week was to grow different strains of<i> Pseudomonas</i> in media containing B-N media + glucose with only carbazole as a source of nitrogen. In theory only those strains which contained the pCAR1 plasmid would be able to use carbazole as a nitrogen source and thrive in this media. The strains we tested from glycerol stock included <i>P. putida</i> LD1, <i>P.putida</i> environmental strains 28-30, and <i>P. fluorescens</i> PF5. Each of these strains had two cultures grown; one with carbazole and one without it. <i>E. coli</i> grown with carbazole was used as a negative control as it does not contain the pCAR plasmid. At timepoints of 22, 46, and 117 hours of growth samples were taken to do an OD600 reading to measure bacterial growth and had an ammonia assay performed on it. The detection of ammonia acted as a proxy for detecting carbazole degradation as it is an end-product of the pathway. With this in mind, an additional control strain of LD1 was grown in media containing glucose and ammonia. This is to detect the possibility that <i>Pseudomonas</i> can use ammonia as a source of nitrogen after it has degraded carbazole which would be problematic as far as using ammonia levels to determine carbazole degradation. So far we have been having difficulties reading the results of the ammonia assay with a plate reader and may resort to testing our samples using a different assay protocol next week. The OD600 readings were initially very high at 22 hours and then dropped at 46 hours. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>This week we took our last readings from the experiment that we started last week. The OD600 readings taken at 117 hours showed minimal growth over the weekend for all strains except for environmental strain #29 (with carbazole) which increased from 0.086 to 0.313. No other strain showed an increase of 0.100 or greater. This shows that only strain #29 was able to thrive in the carbazole media, possibly indicating that it contains the pCAR plasmid enabling it to use carbazole as a nitrogen source. To verify this we used a new ammonia assay protocol to measure the production of ammonia in the different cultures and theoretically how much carbazole was degraded. Unlike the previous assay we were able to produce a standard curve with a consistent slope based on samples with known amounts of ammonia. However, the only samples that showed any elevation of ammonia levels above the blank were the LD1 samples that were cultured with ammonia chloride to control for the possibility of pseudomonas using ammonia as a nitrogen source. The ammonia levels in this sample were relatively constant which would indicate that the LD1 was not using ammonia as a nitrogen source. Unfortunately the fact that none of the test samples had elevated ammonia levels did not give us any evidence that these strains contain the pCAR plasmid. </p><br />
<br />
</html>[[File: Standard_curve_for_ammonia_assay_UCalgary2012_denitrification_group.png|500px|thumb|Standard curve for ammonia assay using OD635 absorbance.|center|]]<html><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>We received a new strain of Pseudomonas, LD2, this week that had previously been used to study the pCAR1 plasmid. It was grown up in PCA media and had its colonies used for colony PCR. We also received our new primers this week, further increasing the chances of successful PCR. Using the new strain and the new primers <i>carAa</i>, <i>carAc</i>, and <i>carAd</i> all showed at least some bands of close to the correct size. After PCR purification we obtained DNA concentrations of 21.3 ng/μL for <i>carAa</i>, 41.9 ng/ μL for <i>carAc</i>, and 32.8 ng/μL for<i> carAd</i>. These products were then restriction digested using EcoRI + PstI sites. </p><br />
<br />
</html>[[File:07.20.2012 CarAa and CarAc.jpg|500px|thumb|Gel of colony PCR from <i>Pseudomonas sp. LD2</i> for <i>carAa</i> and <i>carAc</i> genes. <i>carAa</i> expected size is 1155 bp and <i>carAc</i> is 323 bp.|center]]<br />
[[File:07.20.2012 CarAd colony PCR from LD2.jpg|500px|thumb|Gel of colony PCR from <i>Pseudomonas sp. LD2</i> for <i>carAd</i> gene. <i>carAd</i> expected size is 989 bp.|center]] <html><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The restriction digested <i>car</i> genes were ligated into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> cells. White colonies grew for<i> carAa</i> and <i>carAc</i> suggesting that ligation and transformation were successful, however no colonies grew for <i>carAd</i> suggesting that the transformation did not work. Colony PCR was performed to confirm the presence of these genes within the vectors, however only <i>carAc</i> was positive. The colonies containing the <i>carAc</i> plasmid were then mini prepped and restriction digested to isolate the plasmid and send for sequencing. Further attempts at transforming<i> carAa</i> and <i>carAd</i> were not successful. Another goal was to perform colony PCR on the <i>P. putida</i> to isolate the<i> antABC</i> genes using both the primers for the entire operon and those for the individual genes. No attempt showed any bands, although salt and temperature gradients were used as well as different types of polymerase. LD2 cultures were grown up to be plasmid prepped and genomic prepped in order to have a higher concentration of template DNA in the PCR master mix and more PCR will be attempted on these genes next week. Mutagenesis primers were also designed this week to remove the illegal restriction cut sites in the<i> carAd</i> and <i>antB</i> genes (NotI and 2xEcoRI respectively). Only 1 mutagenesis primer was designed for <i>antB</i> as one of its EcoRI sites was very close to the beginning of the gene and could be removed using a new primer. During this process it was also discovered that the original ant primers were designed for the wrong genes and therefore do not match the intended sequence. Although the new <i>antABC</i> primers were designed using the correct gene the PCR may not be working due to the length of the operon so the design of new individual primers may be necessary. Finally, we also aimed to grow up the freeze dried culture of <i>Rhodococcus erythropolis</i> (source of the<i> AmdA</i> gene) that we received from DSMZ. The reactivation media was produced and autoclaved to begin the process next week.</p><br />
<br />
</html>[[File:07.23.2012.Restriction Digest of Car genes.jpg|500px|thumb|Gel of restriction digest of car genes. A refers to <i>carAa</i>, C to <i>carAc</i>, and D to <i>carAd</i>. Ladder shows that after purification and digestion DNA is still the correct size.|center]]<br />
[[File:CarAc biobrick confirmation PCR.jpg|500px|thumb|Gel of confirmation PCR done on competent Top 10 <i>E. coli</i> cells in which a PSB1C3 vector containing the <i>carAc</i> gene had been inserted. Red X marks the 500 bp mark on the weak ladder showing the product to be the expected size (323 bp gene + 2*100 bp biobrick sites on each end).|center]] <html><br />
<br />
<h2>Week 14 (July 30 - August 3)</h2><br />
<p>We received the sequencing results this week for <i>carAc</i> which came back as a 100% match to the expected nucleotide sequence. We also successfully ligated both<i> carAa</i> and<i> carAd</i> into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into top 10 <i>E. coli</i> cells. Colony PCR verified the presence of the biobrick plasmid in these colonies. These were both miniprepped, digested and sent for sequencing by the end of the week. Using a salt gradient, addition of DMSO, and the KAPA buffer specifically designed for GC-rich amplicons we were able to amplify the ant operon from a genomic prep of LD2. However, all attempts at ligating and transforming this sequence were met with the unfortunate presence of red colonies (indicating failed insertion into the plasmid). Next week we will recut a <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> vector with Xba and Pst to attempt to mitigate this problem. We also reactivated the <i>R. erythropolis</i> following the DSMZ protocol for reviving freeze-dried cultures that can be found <a href=”https://2012.igem.org/Team:Calgary/Notebook/Protocols/dmszfreezedry”>here</a> using DSMZ media #1 and grew it on plates made from DSMZ media #220 at 28 degrees. Colonies grew very quickly and were used for colony PCR and to make overnight cultures for downstream applications. The colony PCR using the amidase primers was unsuccessful, so a genomic prep was performed on the overnight culture. Unfortunately we lacked some of the required equipment and the genomic prep did not work, but with a few procedural modifications and the right equipment we are confident it will work next week. A glycerol stock of the<i> R. erythropoplis</i> was also made from another overnight culture. We also started construction on the <i>carAc </i>part this week by inserting a ribosome binding site (B0034) in front of the gene. Finally, since the<i> ant</i> operon was successfully amplified by PCR we designed mutagenesis primers for the 2nd EcoRI site in the operon rather than making new individual primers. We also designed a mutagenesis primer to remove the SpeI site in <i>AmdA</i>. Now that we have conclusively proven that the LD2 strain contains the pCAR1 plasmid we aim to demonstrate its ability to degrade carbazole and other nitrogen containing heterocycles using an assay experiment similar to the one performed in Week 10.</p><br />
<br />
</html>[[File:07.30.2012. CarAa, CarAd.jpg|500px|thumb|<i>carAa</i> and <i>carAd</i> amplified from genomic prep of <i>Pseudomonas sp. LD2</i>. Amplicons are larger than expected, potentially the gel ran diagonally. The 6th lane for each gene was a negative control.|center]]<br />
[[File:07.31.2012.Ant operon.jpg|500px|thumb|<i>ant</i> operon amplified from genomic prep of <i>Pseudomonas sp. LD2</i> at the expected size of 3000 bp.|center]]<br />
[[File:08.01.2012 CarAdBB.jpg|500px|thumb|Lanes 2,4,5, and 11 contain <i>carAd</i> genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent top 10 <i>E.Coli</i>.|center]]<br />
[[File:08.01.2012 CarAaBB.jpg|500px|thumb|Lane 9 contains <i>carAa</i> genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent top 10 <i>E.Coli</i>. Although larger than expected, the positive control RFP generator in lane 10 is also larger than its expected size (~1200 bp including biobrick bookends) indicating that the gel may have a slant in it.|center]]<html><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p>This week we received the sequencing results for <i>carAa</i> and <i>carAd</i>. <i>carAd</i> was a match, however <i>carAa</i> did not match at all and had misplaced biobrick restriction enzyme sites. Due to this we decided to restart the biobricking process for <i>carAa</i> all the way back to PCR from the plasmid. By the end of the week we had progressed to the point of transforming it into competent <i>E. coli</i> cells, however a thick lawn of bacteria had grown overnight making it impossible to pick colonies. A streak plate was made from this plate to attempt to get less dense growth. Since the sequencing came back positive for <i>carAd</i> the next step was to perform site directed mutagenesis on its NotI restriction enzyme site using the primers that were designed previously (for site-directed mutagenesis optimization, please refer to Desulfurization Journal Week 14). The PCR was successful, showing very bright bands at 3000 bp, and the PCR product was digested with DpnI to remove parental DNA. Transformation of this product will indicate whether the mutagenesis was successful. We were also able to successfully perform a genomic prep of <i>Rhodococcus erythropolis</i> and amplify the <i>AmdA</i> gene this week. It was ligated into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> by Friday, and a confirmation colony PCR was run on Friday evening to verify that the gene was inserted. These results will be available by Monday. After many attempts the <i>ant</i> operon was finally ligated and transformed as well this week. Confirmation PCR showed bands of about 3000 bp which is consistent with the size of the operon. By Friday the successful colonies had been grown up and miniprepped, ready to send for sequencing early next week. The <i>carAc</i>/B0034 construct was also successfully transformed this week and verified by running a PCR using the B0034 forward primer. It was also miniprepped and is ready to send for sequencing next week. </p><br />
<p>In addition to this work constructing biobricks we also started an experiment to demonstrate the function of the pCAR plasmid in LD2. Strains of LD2 were grown up in LB and then washed and placed in B-N media + glucose and various experimental additions. These additions included carbazole, 4-PBAH (a nitrogen containing naphthenic acid), ammonia chloride, and a negative control with no nitrogen source. Strains of <i>E. coli</i> were also grown under the same conditions to compare growth and ammonia production of LD2 to a strain that does not contain the pCAR plasmid. These results should act as a preview of what we expect our final biobrick containing <i>E. col</i>i species to produce as well as allowing us to establish LD2 as a positive control for this experiment. </p><br />
<br />
<br />
</html>[[File:08.07.2012 amdA pcr from genomic prep.jpg|500px|thumb|<i>AmdA</i> amplified from the genomic prep of <i>R. Erythropolis</i> shows bands just greater than 1500 base pairs in lanes 2,4-6,9, and 12-13. Lane 14 was a negative control. The expected size of <i>AmdA</i> is 1566 base pairs.|center]]<br />
[[File:08.09.2012 confirmation pcr of ant operon.jpg|500px|thumb|The two labelled lanes show amplification at 3000 base pairs from a confirmation colony PCR using the biobrick primers. The expected size of the <i>ant</i> operon with the biobrick bookends is about 3100 base pairs.|center]]<br />
[[File:08.10.2012 carad notI mutagenesis.jpg|500px|thumb|Strong amplification at just above 3000 base pairs from the <i>carAd</i> PCR using mutagenic primers. Since PSB1C3 is 2070 base pairs and <i>carAd</i> is 989 base pairs the amplification should run at about 3059 base pairs. 3 different amounts of template DNA were tested in lanes 2-7 as indicated and a positive control showed weaker amplification in lanes 8-9 at about 4000 base pairs.|center]]<br />
<html><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>The sequencing for the <i>carAc</i>/<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> construct came back with only <i>carAc</i> and no evidence of a <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> site preceding it. The confirmation PCR that was run last week using the <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> forward primer seemed to indicate that it was inserted, however since <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> is only 12 base pairs the primer may not have been specific enough. The construction on this part was restarted this week. <i>carAa</i> colony PCR on last week's transformation showed two positive colonies, however upon culturing, miniprepping, and digesting these colonies the plasmid appeared to be too large (3000 base pairs as opposed to the expected 2000). This may be due to a problem of a mislabeled plasmid tube that has been experienced by other team members, causing the gene to be inserted into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a> rather than <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. To test this, a streak plate was made from these colonies and grown on an ampicillin resistant plate. Mutagenized <i>carAd</i> was successfully transformed into Top 10 <i>E. coli</i> cells, cultured, and miniprepped. The miniprep product was digested with NotI to test whether the NotI site was successfully removed. These results were good, as the plasmid ran at 2000 base pairs and the insert at just under 1000 base pairs in the mutagenized <i>carAd</i> whereas the non-mutagenized <i>carAd</i> had its insert cut into 2 bands. This will be sent for sequencing next week to confirm the mutation. Unfortunately the sequencing results came back negative for the <i>ant</i> operon meaning we will need to restart genomic PCR on it next week. Multiple transformations and confirmation PCRs were run on <i>AmdA</i> this week, however none displayed a band size of 1700-1800 base pairs as expected. We have continued to try different digested plasmids as other team members also had difficulties this week with ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, possibly indicating a problem with our Antarctic Phosphatase stock.</p><br />
<p>The other news from this week was the final results from our carbazole degradation experiment attempting to demonstrate LD2's ability to degrade carbazole and hopefully other nitrogen containing compounds. Unfortunately, these results were very difficult to make sense of as the LD2 that were cultured with carbazole grew, but did not show elevated ammonia levels which would be indicative of carbazole degradation. One of our controls was LD2 cultured with ammonium chloride to test whether LD2 was capable of using ammonia as a nitrogen source. This would pose a problem for us as we were planning to use an increase in ammonia levels as a proxy for carbazole degradation rather than measuring carbazole degradation directly using GC-MS. However, the LD2 showed significantly less ammonia after 44 hours when compared to 26 hours indicating that it may be degraded, either by the LD2 or by an unaccounted for abiotic process. This has lead us to decide to use GC-MS and directly measure carbazole levels in our sample the next time we do this experiment.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<p>The <i>carAa</i> was re-streaked onto ampicillin plates and grew, probably showing that it was ligated into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a>, not <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, which explained the irregularity of our confirmation digest last week. It was sent for sequencing, but in case it was incorrect we also started a new <i>carAa</i> pipeline by PCRing it from the <i>Pseudomonas</i> genome again and redigesting and ligating. It was transformed into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> over the weekend. <i>carAc</i> did not see a lot of progress this week as we spent much of the time attempting to construct a RBS (B0034) in front of it, but were unsuccessful. <i>carAd</i> was also subjected to this construction with B0034 and we were able to send it for sequencing by the end of the week. Several new constructs were also started for <i>carAd</i> in case the sequencing is negative. The <i>ant</i> operon was successfully transformed and confirmed by colony PCR this week, after which it was grown up and sent for sequencing. We also began to attempt mutagenesis on one of the EcoRI sites in the operon, but had no amplification of the plasmid. We also re-digested some PCR purified product to start another ligation in case sequencing failed. After many attempts we were also able to finally successfully transform an <i>AmdA</i>/PSB1C3 plasmid into Top 10 cells this week. After a miniprep they showed bands of about 1800 base pairs on the confirmation digest gel, so they were also sent for sequencing this week and had mutagenesis started on the illegal SpeI site. Unfortunately, the sequencing sent early in the week came back with ambiguous results. The forward reaction for<i> antABC</i> was a match, but its reverse reaction matched to <i>carAa</i>. Both <i>carAa</i> reactions matched nothing. Since these two genes are over 20 000 base pairs apart on the template plasmid it is virtually impossible that they were actually combined into one plasmid (PCR extension time would not have been long enough and even if it was the product would have run much above the 3000 base pair mark). This is possibly a mistake on Eurofins end, so we are not 100% sure if we have 1 good gene, both of them, or neither. Also the sequence for mutagenized <i>carAd</i> showed only about the first 100 base pairs, which were correct however not long enough meaning it was a poor reaction and inconclusive. Finally, we also re-started our carbazole degradation assay using the GC-MS at biological sciences. This process will take 2 weeks to demonstrate LD2's ability to degrade carbazole. We are forcing it to use carbazole as a carbon and a nitrogen source in one sample and as only a nitrogen source in another sample (this sample has had glucose added to it).</p><br />
<br />
</html>[[File:AmdA colonyPCR calgary12.JPG|500px|thumb|Amplification is seen in lane 7 of this PCR of top 10 colonies into which AmdA/PSB1C3 had been transformed. This indicates that <i>AmdA</i> has been successfully inserted into a biobrick vector.|center]]<br />
[[File:NewcarAagenomePCR calgary12.jpg|500px|thumb|<i>carAa</i> amplified from pCAR1.2 plasmid of <i>Pseudomonas putida</i>.|center]] <html><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<p>The new stock of <i>carAa</i> that we had PCRed from LD2 was sent for sequencing this week and came back as a match, so we have begun constructing a RBS site and a TetR promoter (<a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>) in front of it. At this point all the <i>car</i> genes have been successfully amplified, inserted into a biobrick plasmid, and sequence verified. We also continued to attempt to construct <i>carAc</i> with an RBS site (<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>) in front of it, but had no success. On a better note, we confirmed this week that<i> carAd</i> was mutated properly and we were also able to insert an RBS site in front of it. The next step for this gene will be to attempt a plasmid switch as it is currently in <a href="http://partsregistry.org/Part:pSB1A3">pSB1A3</a> and needs to be in <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> for submission to the parts registry. After sending a new batch of the ant operon for sequencing we got the same confusing results as we did last week. We therefore hypothesized that our primers were annealing to a homologous region that is similar to the<i> ant</i> genes and very close to <i>carAa</i>. This means that we need to restart PCR on this gene as all of our current product is incorrect. The sequencing results for <i>AmdA</i> that came back this week were also somewhat confusing as they did not quite match the sequence we expected. However, they were a 100% match for the <i>AmdA</i> gene in a different strain of <i>Rhodococcus erythropolis</i>. This means that DSMZ probably sent us the wrong strain of bacteria and that we were very lucky that the primers we designed based off of the expected template actually worked to amplify a slightly different gene in another strain. The good news is that this new sequence contains no illegal cut sites, which explains why mutagenesis was not working (there was nothing to mutate) and allows us to immediately begin construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> in front of <i>AmdA</i> this week. </p><br />
<br />
</html>[[File:Caraa colonyPCR calgary12.JPG|500px|thumb|Colony PCR amplification from top 10 <i>E. coli</i> cells with a PSB1C3/<i>carAa</i> construct transformed into them. Lanes 1-3, 5, and 7 were sent for sequencing with lane 5 coming back positive.|center]]<html><br />
<br />
<h2>Week 19 (September 3- September 7)</h2><br />
<p><i>carAd</i>/<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> was successfully plasmid switched into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, making it ready for submission. Construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> and <i>carAa</i> may have been successful as the digest of the miniprep looked promising. It will be sent for sequencing next week. Unfortunately there was no progress made on the constructions of <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> or <i>carAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. These will be redigested and religated for next week. We could not amplify the <i>ant</i> operon despite trying many variations of PCR using high fidelity KAPA polymerase. This is probably due to the multiple, large regions of homology on the operon that the reverse primer binds to making amplification difficult. Because the function of this gene can theoretically be replaced by <i>AmdA</i>, we may scale back efforts dedicated to it, as the chances of successful amplification appear slim. </p><br />
<br />
<h2>Weeks 20-22 (September 10 - September 30)</h2><br />
<p>Constructs that continued this week included <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, <i>carAa</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, and <i>carAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. Some sequencing was sent, but no positive results were obtained. A new approach to biobrick the <i>ant</i> operon was implemented this week. A new forward primer was designed that would anneal about 1500 base pairs upstream of the <i>ant</i> operon, avoiding the region of homology that is repeated at various points on the plasmid. Once this region is successfully amplified we could use this amplified DNA as a template for another PCR reaction which would use the normal <i>ant</i> primers. Since the template would no longer include regions of homology these primers should be free to work properly and amplify the <i>ant</i> operon. The PCR using the new primers was started this week, and although amplification at 4500 base pairs was observed as expected, there was also a lot of non-specific amplification. This means that to purify the amplified DNA that we want and avoid the other products a dreaded gel extraction had to be performed which, as usual, did not work. At this point it was decided that further attempts to amplify the <i>ant</i> operon would be futile and our efforts would be better served working on constructing the parts we do have. Unfortunately no progress was made in these weeks on construction. </p><br />
<br />
<br />
<br />
<br />
</html><br />
}}</div>Anyakornilo