Team:Washington/Alkanes/Results

From 2011.igem.org

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(GSNS Analysis Shows Alkane Production Works)
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=GSNS Analysis Shows Alkane Production Works=
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=GCMS Analysis Shows The PeroBrick Works=
We preformed GC-MS analysis on cell cultures expressing both Acyl-ACP reductase (AAR) and Aldehyde Decarbonylase (ADC). To act as controls and in order to show that the alkane production system is working as expected, we also analyzed cell cultures expressing either only AAR, or only ADC.
We preformed GC-MS analysis on cell cultures expressing both Acyl-ACP reductase (AAR) and Aldehyde Decarbonylase (ADC). To act as controls and in order to show that the alkane production system is working as expected, we also analyzed cell cultures expressing either only AAR, or only ADC.
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[[File:Washinton_AARADCGC-MS.png|center|700px|thumb|GC plot showing that alkanes are only produced when both ADC and AAR are expressed.]]
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[[Image:Washinton_AARADCGC-MS.png|center|600px|border|]]
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'''No significant hydrocarbons peaks in samples extracted from cells expressing only ADC.'''  The blue GCMS chromatagram trace on the bottom show no significant peaks are found on the ADC only control within the time range that we expect alkane signal based upon GC-MS runs on chemical standards (8-10.5 minutes). This is expected, as ''E. coli'' does not normally produce any long chain length aldehyde substrates.
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===No significant hydrocarbons peaks in samples extracted from cells expressing only AAR===
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'''C16 alcohols are observed when AAR is expressed on its own.'''  The red GCMS chromatagram trace in the middle has a significant peak at 10.2 minutes corresponding to the C16 alcohol (as confirmed by comparison of the peak's MS spectra to a reference library) is observed when AAR is expressed in MG1655 E. coli.  The production of C16 alcohols in cells expressing AAR (but not in cells expressing only ADC) is consistent with AAR reducing even chain length Acyl-ACPs into even chain length fatty aldehydes, which are further reduced by aldehyde dehydrogenases to the alcohol.
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No significant peaks are found on the AAR-Only control GC plot within the time range that we expect alkane signal based upon GC-MS runs on chemical standards (8-10.5 minutes). This is expected, as ''E. coli'' does not normally express native decarbonylase enzymes.
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===No significant hydrocarbons peaks in samples extracted from cells expressing only ADC===
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'''The PetroBrick Works!  C13, C15, and C17 alkanes are produced when both AAR and ADC are expressed.'''  The green GCMS chromatagram trace in the middle has a significant new peaks corresponding to the C13 (8.2min) and C15 (9.2min) alkanes, as well as a peak at 10.2 minutes that contains both the C16 alcohol and the C17 alkene. The fact that the C17 alkene and C16 alcohol peak overlaps with the C16 alcohol peak makes quantification of C17 akene yields impossible, but we have sufficient evidence to determine that both molecules are present in the 10.2 minute peak( the later section of the peak has an MS spectra consistent with the C16 alcohol, and the earlier section is consistent with the C17 alkene). The C13 and C15 alkane peaks showed MS spectra highly consistent with C13 and C15 alkane reference spectra. In addition, both alkane molecules eluted off of the GC at the same time as C13 and C15 alkane standards, further increasing confidence that the 8.2 min and 9.2 min peaks do correspond to the C13 and C15 alkanes. Odd chain length alkane production is what is expected from our system, as ADC removes a carbonyl group from even chain length aldehydes produced by AAR, yielding an odd chain length alkane.The mass spectra for the peaks at 8.2 and 9.3 elute and have the same MS spectra as our standards, as well as the NIST compound library.  The peak at 10.2 corresponded to a mixture of C17 alkene and C16 alcohol.  Therefore quantifying the amount of C13 and C15 alkanes was readily achieved by constructing a standard curve.  The coelution of the C17 with the significant alcohol peaks made it difficult to quantify the level of C17 alkene produced.
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No significant peaks are found on the ADC-Only control GC plot within the time range that we expect alkane signal based upon GC-MS runs on chemical standards (8-10.5 minutes). This is expected, as ''E. coli'' does not normally produce any long chain length aldehyde substrates.
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=Initial Quantization=
 
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===C16-Alcohol production by cells expressing AAR===
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The GC plot from the extraction from a cell culture expressing only AAR shows a significant peak at 10.2 minutes corresponding to the C16 alcohol (as confirmed by comparison of the peak's MS spectra to a reference library). Wild-type E. coli produces multiple aldehyde dehydrogenases that convert aldehydes to alcohols. The production of C16 alcohols in cells expressing AAR (but not in cells expressing only ADC) is consistent with AAR reducing even chain length Acyl-ACPs into even chain length fatty aldehydes, which are further reduced by aldehyde dehydrogenases to the alcohol.
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=Initial Quantization of Alkane Production=
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In order to be able to know how much alkane was being produced by our ''E. coli'', we spiked known amounts of alkane into cell cultures known to not produce alkanes. We then extracted using ethyl acetate, and analyzed extracts using GC-MS. Since peak area corresponds to the amount of each substance present, we used these GC plots to make a standard curve that allows us to convert peak area into an absolute amount. To determine how much alkane was being produced by our alkane production construct, we grew up 3 E. coli cultures expressing ADC and AAR in a standard M9 media ([http://2011.igem.org/Team:Washington/alkanebiosynthesis link]), and analyzed using GC-MS.
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===C17 Alkene production occluded by C16-Alcohols===
 
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Detecting the C17 alkenes was occluded by this C16 alcohol production, as the C16 alcohol elutes at a very similar output time. During optimization we found that the laboratory cloning strain XL1 Blue, did not produce commensurate quantities of C16 alcohols, potentially allowing for further quantification of C17 alkenes.
 
[[File:Washinton 2011 Pre-Optimization Quant.png|center|550px|thumb|Diagram showing yields of the C13 and C15 alkanes, and C17 alkenes. Note: This C17 quantification is a rough approximation.]]
[[File:Washinton 2011 Pre-Optimization Quant.png|center|550px|thumb|Diagram showing yields of the C13 and C15 alkanes, and C17 alkenes. Note: This C17 quantification is a rough approximation.]]
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=Optimized Production=
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=== Alkane production by cells expressing ADC and AAR===
 
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In cells expressing ADC and AAR, we saw peaks corresponding to the C13 (8.2min) and C15 (9.2min) alkanes, as well as a peak at 10.2 minutes that contains both the C16 alcohol and the C17 alkene. The fact that the C17 alkene and C16 alcohol peak overlaps with the C16 alcohol peak makes quantification of C17 akene yields impossible, but we have sufficient evidence to determine that both molecules are present in the 10.2 minute peak( the earlier section of the peak has an MS spectra consistent with the C16 alcohol, and the later section is consistent with the C17 alkene). The C13 and C15 alkane peaks showed MS spectra highly consistent with C13 and C15 alkane reference spectra. In addition, both alkane molecules eluted off of the GC at the same time as C13 and C15 alkane standards, further increasing confidence that the 8.2 min and 9.2 min peaks do correspond to the C13 and C15 alkanes. Odd chain length alkane production is what is expected from our system, as ADC removes a carbonyl group from even chain length aldehydes produced by AAR, yielding an odd chain length alkane.
 
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===Quantification of alkane yield===
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=Optimized Production=
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In order to be able to know how much alkane was being produced by our ''E. coli'', we spiked known amounts of alkane into cell cultures known to not produce alkanes. We then extracted using ethyl acetate, and analyzed extracts using GC-MS. Since peak area corresponds to the amount of each substance present, we used these GC plots to make a standard curve that allows us to convert peak area into an absolute amount. To determine how much alkane was being produced by our alkane production construct, we grew up 3 E. coli cultures expressing ADC and AAR in a standard M9 media ([http://2011.igem.org/Team:Washington/alkanebiosynthesis link]), and analyzed using GC-MS.
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By optimizing the method of growth, media, vectors, and cell line we were able to succesfull increase yeilds of alkane production over 50-fold. In addition, the peak at C17 no longer had any trace of alcohol and all three peaks were clearly the desired alkenes, as determined by comparrsion to our product standards and the standard NIST reference spectrum library. The paramters we adjusted for optimization are discussed in detail at [http://2011.igem.org/Team:Washington/Alkanes/Future/Vector our systems optimization] page under future directions, as this is an on going process.
[[File:Washinton 2011 Optimization Quant.png|center|550px|thumb|Diagram showing yields of the C13 and C15 alkanes, and C17 alkenes.]]
[[File:Washinton 2011 Optimization Quant.png|center|550px|thumb|Diagram showing yields of the C13 and C15 alkanes, and C17 alkenes.]]

Revision as of 06:06, 16 September 2011


GCMS Analysis Shows The PeroBrick Works

We preformed GC-MS analysis on cell cultures expressing both Acyl-ACP reductase (AAR) and Aldehyde Decarbonylase (ADC). To act as controls and in order to show that the alkane production system is working as expected, we also analyzed cell cultures expressing either only AAR, or only ADC.

Washinton AARADCGC-MS.png

No significant hydrocarbons peaks in samples extracted from cells expressing only ADC. The blue GCMS chromatagram trace on the bottom show no significant peaks are found on the ADC only control within the time range that we expect alkane signal based upon GC-MS runs on chemical standards (8-10.5 minutes). This is expected, as E. coli does not normally produce any long chain length aldehyde substrates.

C16 alcohols are observed when AAR is expressed on its own. The red GCMS chromatagram trace in the middle has a significant peak at 10.2 minutes corresponding to the C16 alcohol (as confirmed by comparison of the peak's MS spectra to a reference library) is observed when AAR is expressed in MG1655 E. coli. The production of C16 alcohols in cells expressing AAR (but not in cells expressing only ADC) is consistent with AAR reducing even chain length Acyl-ACPs into even chain length fatty aldehydes, which are further reduced by aldehyde dehydrogenases to the alcohol.

The PetroBrick Works! C13, C15, and C17 alkanes are produced when both AAR and ADC are expressed. The green GCMS chromatagram trace in the middle has a significant new peaks corresponding to the C13 (8.2min) and C15 (9.2min) alkanes, as well as a peak at 10.2 minutes that contains both the C16 alcohol and the C17 alkene. The fact that the C17 alkene and C16 alcohol peak overlaps with the C16 alcohol peak makes quantification of C17 akene yields impossible, but we have sufficient evidence to determine that both molecules are present in the 10.2 minute peak( the later section of the peak has an MS spectra consistent with the C16 alcohol, and the earlier section is consistent with the C17 alkene). The C13 and C15 alkane peaks showed MS spectra highly consistent with C13 and C15 alkane reference spectra. In addition, both alkane molecules eluted off of the GC at the same time as C13 and C15 alkane standards, further increasing confidence that the 8.2 min and 9.2 min peaks do correspond to the C13 and C15 alkanes. Odd chain length alkane production is what is expected from our system, as ADC removes a carbonyl group from even chain length aldehydes produced by AAR, yielding an odd chain length alkane.The mass spectra for the peaks at 8.2 and 9.3 elute and have the same MS spectra as our standards, as well as the NIST compound library. The peak at 10.2 corresponded to a mixture of C17 alkene and C16 alcohol. Therefore quantifying the amount of C13 and C15 alkanes was readily achieved by constructing a standard curve. The coelution of the C17 with the significant alcohol peaks made it difficult to quantify the level of C17 alkene produced.




Initial Quantization of Alkane Production

In order to be able to know how much alkane was being produced by our E. coli, we spiked known amounts of alkane into cell cultures known to not produce alkanes. We then extracted using ethyl acetate, and analyzed extracts using GC-MS. Since peak area corresponds to the amount of each substance present, we used these GC plots to make a standard curve that allows us to convert peak area into an absolute amount. To determine how much alkane was being produced by our alkane production construct, we grew up 3 E. coli cultures expressing ADC and AAR in a standard M9 media (link), and analyzed using GC-MS.


Diagram showing yields of the C13 and C15 alkanes, and C17 alkenes. Note: This C17 quantification is a rough approximation.




Optimized Production

By optimizing the method of growth, media, vectors, and cell line we were able to succesfull increase yeilds of alkane production over 50-fold. In addition, the peak at C17 no longer had any trace of alcohol and all three peaks were clearly the desired alkenes, as determined by comparrsion to our product standards and the standard NIST reference spectrum library. The paramters we adjusted for optimization are discussed in detail at our systems optimization page under future directions, as this is an on going process.

Diagram showing yields of the C13 and C15 alkanes, and C17 alkenes.