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Team:Alberta/Achievements/ProofofConcept
From 2011.igem.org
Proof of Concept
Growth
We wanted to see how well N. crassa grew on cellulosic media. We tested using grass clippings and wheat straw. Race tubes were used to determine the rate of hyphal growth of N. Crassa with different growth mediums. The following are graphs of the results:
The graphs show similar growth rates for the grass and wheat straw as compared to the minimal media control VsuTB. This demonstrates that N. crassa does grow well on cellulosic biomass which makes it a good organism to use for large scale production of biodiesel.
Genetics
The multiple assembly approach as described in the RFC were tested to determine the efficiency and fidelity of the assembly method. A key requirement of this approach is that upon ligation, ends must find partners precisely without promiscuity. To test this requirement and select ends that meet this criterion we subdivided the coding sequence of the alpha subunit of beta galactosidase into nine segments ending four base 5' overhangs that were selected to eliminate duplication Also included were two segments encoding a promoter (Bba J23001) and one segment encoding the transcriptional terminator Bba 1002 as shown below:
Segments were produced from desalted phosphorylated oligonucleotides (IDT) that had been annealed over a two-hour temperature gradient from 70 to 25 degrees. Plasmids that contained the BetaGal cassette were then assembled using the biobytes sequential assembly method on magnetic beads as described by the Alberta iGEM 2010 team. Briefly, all segments were mixed and ligated degrees. simultaneously at 50-fold molar excess to a bead-bound origin of replication derived from pSB1C3 at the restrictive temperatureof 37. Following several washes, the KanR gene was ligated and capped for recircularization. The construct was then released from the beads and to avoid bias in colony selection, transformants were plated onto media lacking XGal. Ten colonies were then selected for sequencing. Notably, the DNA from all ten colonies contained the complete alpha-BetaGal cassette attesting to the precision of the biobyte assembly method. Only one -1 frameshift was detected in the first promoter segment representing an accuracy of synthesis of 1 mistake in 3000 base pairs. The fact the no mutations were detected at the boundaries of segment ligation illustrates that any of the overhangs defined above were suitable for the N. crassa gene insertion design described above.
Esterification
N. crassa was esterified directly and the fatty acids were analyzed using GC-MS, an example of the GC plot is given below:
This shows that N. crassa can be esterified using our direct esterification method to produce our biodiesel, which are fatty acid methyl esters, especially the C16 fatty acids which are expected to accumulate as a result of our genetic manipulations.
