Team:TrinityCollegeDublin/Results
Results
Trinity College 2015 iGEM team constructed the required "Alpha" plasmids for the introduction of the alternative pathway of artemisinin precursor biosynthesis in E. coli B569.
During the 3A assembly of the device, after each ligation the product was PCR amplified to verify correct length for a successful ligation as well as, after certain ligations, sequenced and compared to Geneious R8 software simulated ligation products. This ensured that the final constructs contained the desired sequences without any mistakes. Time constraints arising from a delay in receiving the B569 cell line from Amyris due to legal issues concerning a materials transfer made it impossible to verify the activity of the "Alpha" constructs when inserted into the cells (which was the only means to validate our constructs for their purpose of producing dihydroartemisinic acid). The validation would require running a bioreactor for at least one week, collecting and lysing samples, functionalising (siliation) the desired intermediates and running GC-MS analysis. As such, an alternative assay was developed for the validation of enzyme activity.
Plasmids expressing just ALDH1 were constructed and DH5-alpha cells were transformed with these. The aldehyde dehydrogenase1 enzyme is not specific to artemisinic aldehyde/dihydroartemisinic aldehyde but instead acts as a dehydrogenase on a large spectrum of aldehydes including acetaldehyde.
The DH5-alpha cells expressing ALDH1 were lysed (freeze-thaw), the lysate spun down at 10K RPM for 10min and 40µl of the supernatant reacted with an acetaldehyde and cofactor solution. If ALDH1 enzymatic activity was present, acetaldehyde would be converted to acetic acid, which could subsequently be identified by running a GC-MS analysis.
While DH5-alpha E. coli cells have been documented to contain a native acetaldehyde dehydrogenase enzyme, it is expressed in low levels generally and is only upregulated as part of a stress response if the cells are grown in ethanol containing media. For our assay, the cells were never exposed to ethanol and were lysed prior to addition to acetaldehyde solution, thus never allowing this upregulation to occur. In addition to this fact, the ALDH1 generator was in a high copy number plasmid and under the control of a very strong constitutive promoter (BBa_J23101) - both factors signifying that ALDH1 would be strongly overexpressed in the transformed cell lines. As such, any native dehydrogenase activity could be negated by using the gentle freeze-thaw lysis and protein isolation techniques, only useful for working with overexpressed recombinant proteins as endogenous protein content is too low to be extracted by this means.
The experiment was carried out in quadruplicate and GC-MS data showed a peak characteristic of the presence of acetic acid (RMatch values of ~820) in each case, confirming the activity of the dehydrogenase (see graphs 1-4). No acetic acid was present in the reference standard meaning that the peak seen in the graphs is the result of recombinant dehydrogenase activity. It is therefore possible to extrapolate, based on these results that if artemisinic aldehyde/dihydroartemisinic aldehyde is present in the cell, aldehyde dehydrogenase1 would act on them converting them to artemisinic acid/dihydroartemisinic acid, as expected.
References:
Johnson B. H., Hecht M. H.; (1994)
Recombinant Proteins Can Be Isolated From E. Coli Cells Using Repeated Cycles Of Freezing And Thawing; Bio/Technology, 12, 1357-1360.
Ho K. K., Wiener H.; (2005)
Isolation and Characterization of an Aldehyde Dehydrogenase Encoded by the aldB Gene of Escherichia coli ; Journal of Bacteriology, 187(3), 1067–1073.
Teoh, K, Polichuk, D, Reed, D, & Covello, P 2009,
'Molecular cloning of an aldehyde dehydrogenase implicated in artemisinin biosynthesis in Artemisia annua', Botany, 87, 6, pp. 635-642
During the 3A assembly of the device, after each ligation the product was PCR amplified to verify correct length for a successful ligation as well as, after certain ligations, sequenced and compared to Geneious R8 software simulated ligation products. This ensured that the final constructs contained the desired sequences without any mistakes. Time constraints arising from a delay in receiving the B569 cell line from Amyris due to legal issues concerning a materials transfer made it impossible to verify the activity of the "Alpha" constructs when inserted into the cells (which was the only means to validate our constructs for their purpose of producing dihydroartemisinic acid). The validation would require running a bioreactor for at least one week, collecting and lysing samples, functionalising (siliation) the desired intermediates and running GC-MS analysis. As such, an alternative assay was developed for the validation of enzyme activity.
Plasmids expressing just ALDH1 were constructed and DH5-alpha cells were transformed with these. The aldehyde dehydrogenase1 enzyme is not specific to artemisinic aldehyde/dihydroartemisinic aldehyde but instead acts as a dehydrogenase on a large spectrum of aldehydes including acetaldehyde.
The DH5-alpha cells expressing ALDH1 were lysed (freeze-thaw), the lysate spun down at 10K RPM for 10min and 40µl of the supernatant reacted with an acetaldehyde and cofactor solution. If ALDH1 enzymatic activity was present, acetaldehyde would be converted to acetic acid, which could subsequently be identified by running a GC-MS analysis.
While DH5-alpha E. coli cells have been documented to contain a native acetaldehyde dehydrogenase enzyme, it is expressed in low levels generally and is only upregulated as part of a stress response if the cells are grown in ethanol containing media. For our assay, the cells were never exposed to ethanol and were lysed prior to addition to acetaldehyde solution, thus never allowing this upregulation to occur. In addition to this fact, the ALDH1 generator was in a high copy number plasmid and under the control of a very strong constitutive promoter (BBa_J23101) - both factors signifying that ALDH1 would be strongly overexpressed in the transformed cell lines. As such, any native dehydrogenase activity could be negated by using the gentle freeze-thaw lysis and protein isolation techniques, only useful for working with overexpressed recombinant proteins as endogenous protein content is too low to be extracted by this means.
The experiment was carried out in quadruplicate and GC-MS data showed a peak characteristic of the presence of acetic acid (RMatch values of ~820) in each case, confirming the activity of the dehydrogenase (see graphs 1-4). No acetic acid was present in the reference standard meaning that the peak seen in the graphs is the result of recombinant dehydrogenase activity. It is therefore possible to extrapolate, based on these results that if artemisinic aldehyde/dihydroartemisinic aldehyde is present in the cell, aldehyde dehydrogenase1 would act on them converting them to artemisinic acid/dihydroartemisinic acid, as expected.
References: