3 ms·

Thanks! Could you shed some more light on how one actually takes a gene from a plant (in a lab setting I suppose) and put it into yeast?

by a_imho 8y ago

negus 8y ago
https://en.wikipedia.org/wiki/Illumina_dye_sequencing https://en.wikipedia.org/wiki/Illumina_dye_sequencing https://en.wikipedia.org/wiki/CRISPR https://en.wikipedia.org/wiki/CRISPR
jakobegger 8y ago
Step 1 is you hope that someone already identified the gene(s) that do what you want. You look it up in a database and copy-paste the DNA sequence into a word document. Step 2 you adjust the DNA sequence for the target organism. This might mean using different codons, changing the start of the gene, etc. Usually you also append a few extra genes like GFP or antibiotic resistance genes that make working with your DNA sequence easier. Step 3 upload your DNA sequence to an online store for DNA synthesis. A few days later you'll get an envelope with a small plastic vial with your synthesized DNA. Step 4 Now you need to get your DNA into the target organism. I'm not sure how you do that for yeast, but there's probably a well known protocol you can follow. You might also first put your DNA into bacteria to make more of it. Step 5 now grow your cells, and put them under the microscope, check if they glow green (that's why you put the GFP in there) and perform various other analysis to see if what you did works. It probably didn't, so you go back and change something, and try again.
Obi_Juan_Kenobi 8y ago
* Extract RNA * Reverse-transcribe this into DNA * PCR amplify target sequence using custom primers. Generally you design the primers to create restriction sites flaking the sequence of interest. * Restriction digest the amplicons * Pop that into a cloning vector, e.g. Gateway * Clone and isolate a colony * Sanger sequence to confirm you've got what you want * Move construct into expression vector * Transform yeast with expression vector construct * Sequence some colonies to find one that worked well (these vectors use resistance genes to provide an easy screening process) * Once you've confirmed it worked, you're all set. You can just use plasmids or do a stable transformation; the latter is harder but it's what you'd want for commercial production I think.

Thanks! Could you shed some more light on how one actually takes a gene from a plant (in a lab setting I suppose) and put it into yeast?

by a_imho 8y ago

Thanks! Could you shed some more light on how one actually takes a gene from a plant (in a lab setting I suppose) and put it into yeast?

negus 8y ago

https://en.wikipedia.org/wiki/Illumina_dye_sequencing https://en.wikipedia.org/wiki/Illumina_dye_sequencing https://en.wikipedia.org/wiki/CRISPR https://en.wikipedia.org/wiki/CRISPR

jakobegger 8y ago

Step 1 is you hope that someone already identified the gene(s) that do what you want. You look it up in a database and copy-paste the DNA sequence into a word document. Step 2 you adjust the DNA sequence for the target organism. This might mean using different codons, changing the start of the gene, etc. Usually you also append a few extra genes like GFP or antibiotic resistance genes that make working with your DNA sequence easier. Step 3 upload your DNA sequence to an online store for DNA synthesis. A few days later you'll get an envelope with a small plastic vial with your synthesized DNA. Step 4 Now you need to get your DNA into the target organism. I'm not sure how you do that for yeast, but there's probably a well known protocol you can follow. You might also first put your DNA into bacteria to make more of it. Step 5 now grow your cells, and put them under the microscope, check if they glow green (that's why you put the GFP in there) and perform various other analysis to see if what you did works. It probably didn't, so you go back and change something, and try again.

Obi_Juan_Kenobi 8y ago

* Extract RNA * Reverse-transcribe this into DNA * PCR amplify target sequence using custom primers. Generally you design the primers to create restriction sites flaking the sequence of interest. * Restriction digest the amplicons * Pop that into a cloning vector, e.g. Gateway * Clone and isolate a colony * Sanger sequence to confirm you've got what you want * Move construct into expression vector * Transform yeast with expression vector construct * Sequence some colonies to find one that worked well (these vectors use resistance genes to provide an easy screening process) * Once you've confirmed it worked, you're all set. You can just use plasmids or do a stable transformation; the latter is harder but it's what you'd want for commercial production I think.