Metabolic engineering/biofuel project help
Posted: Wed Jan 17, 2018 6:06 pm
Hello,
My project is based off of producing butanol as a biofuel with Saccharomyces cerevisiae by using a different starting point in a published pathway. It is mostly based off of the research paper "Metabolic engineering of a synergistic pathway for n-butanol production in Saccharomyces cerevisiae”.
I am having trouble in a few different areas:
1. In the paper, an s. cerevisiae strain is modified in various ways to create different strains, named in the paper. I plan to use the strain the paper references as THR5*. The supplementary information for the paper shows the plasmids needed to create the strain but I am a bit confused about how to interpret this information. Essentially, for THR5*, what would I have to obtain (primers, etc) and in what form? From what I understand so far, the plasmids needed for THR5* would be pRS426 - THR* and pRS424 (If this is correct, what would be the fragments/PCR template/primer sequence for pRS424 and how do I interpret this information and know what to order?)
2. The S. cerevisiae strain used in the experiment is S. cerevisiae HZ848. The supplementary information says that THR5* uses S. cerevisiae HZ848 adh1Δ. If I want to compare the n-butanol yield with strain THR5* when using aspartame vs aspartame, would it be wise to obtain both the wild-type and adh1Δ strains (would using the wild-type strain also help to control the experiment more and how?)
3. I am having trouble locating the strains I would need (wild-type and/or adh1Δ, depending on the answer to #2 (above)). I have heard that if I can get in touch with the authors of the paper, they should be able to provide the strains I need. However, I am having trouble contacting them (no replies to multiple emails I have sent (first email was one month ago) and do not pick up the phones at their labs either). Any tips for contacting them or ways to get their attention?
4. The methods section mentions this:
S. cerevisiae strains were grown on synthetic dropout (SD) medium agar plates containing the appropriate medium composition for selection (0.17% Difco yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate and 0.083% amino acid dropout mix, 0.01% adenine hemisulfate and 2% glucose).
Where can I find the synthetic dropout medium agar plates (is the medium composition provided a composition that comes on pre-made agar plates or is this something where I would have to make the medium according to the composition provided and then create my own agar plates? If so, how would I do that?)
5. Not sure if this can be answered by anyone but the paper authors but the methods reference an amino acid dropout mix in the medium composition. I have looked online and can only find mixes that omit certain amino acids/combinations of amino acids and am confused about which mix to order/if the mix used makes a difference.
5. Finally, if there is anyone who has experience with yeast/yeast transformation, would there be any way for them to show me how to do a yeast transformation or help me in doing one? I am a bit confused about the protocol and my teacher has not done one either.
Thanks in advance,
Mitali
My project is based off of producing butanol as a biofuel with Saccharomyces cerevisiae by using a different starting point in a published pathway. It is mostly based off of the research paper "Metabolic engineering of a synergistic pathway for n-butanol production in Saccharomyces cerevisiae”.
I am having trouble in a few different areas:
1. In the paper, an s. cerevisiae strain is modified in various ways to create different strains, named in the paper. I plan to use the strain the paper references as THR5*. The supplementary information for the paper shows the plasmids needed to create the strain but I am a bit confused about how to interpret this information. Essentially, for THR5*, what would I have to obtain (primers, etc) and in what form? From what I understand so far, the plasmids needed for THR5* would be pRS426 - THR* and pRS424 (If this is correct, what would be the fragments/PCR template/primer sequence for pRS424 and how do I interpret this information and know what to order?)
2. The S. cerevisiae strain used in the experiment is S. cerevisiae HZ848. The supplementary information says that THR5* uses S. cerevisiae HZ848 adh1Δ. If I want to compare the n-butanol yield with strain THR5* when using aspartame vs aspartame, would it be wise to obtain both the wild-type and adh1Δ strains (would using the wild-type strain also help to control the experiment more and how?)
3. I am having trouble locating the strains I would need (wild-type and/or adh1Δ, depending on the answer to #2 (above)). I have heard that if I can get in touch with the authors of the paper, they should be able to provide the strains I need. However, I am having trouble contacting them (no replies to multiple emails I have sent (first email was one month ago) and do not pick up the phones at their labs either). Any tips for contacting them or ways to get their attention?
4. The methods section mentions this:
S. cerevisiae strains were grown on synthetic dropout (SD) medium agar plates containing the appropriate medium composition for selection (0.17% Difco yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate and 0.083% amino acid dropout mix, 0.01% adenine hemisulfate and 2% glucose).
Where can I find the synthetic dropout medium agar plates (is the medium composition provided a composition that comes on pre-made agar plates or is this something where I would have to make the medium according to the composition provided and then create my own agar plates? If so, how would I do that?)
5. Not sure if this can be answered by anyone but the paper authors but the methods reference an amino acid dropout mix in the medium composition. I have looked online and can only find mixes that omit certain amino acids/combinations of amino acids and am confused about which mix to order/if the mix used makes a difference.
5. Finally, if there is anyone who has experience with yeast/yeast transformation, would there be any way for them to show me how to do a yeast transformation or help me in doing one? I am a bit confused about the protocol and my teacher has not done one either.
Thanks in advance,
Mitali