Re: [DIYbio] Beginner questions!

Much thanks for the answers. Is there a bio equivalent of "Hello World" or some simple example of the process of modifying a plant or bacteria? I would like to get familiar with the steps and the reagent vendors. What companies do people use for reagents that are hobbyist friendly? Does anyone actually get base pair sequences delivered to their house/hackerspace or is that not yet accessible to hobbyists? 

On Thursday, March 24, 2016 at 12:56:00 PM UTC-7, Nathan McCorkle wrote:

On Tue, Mar 22, 2016 at 9:53 PM, Justin S <jjs...@gmail.com> wrote:

Hi all,

I'm a total beginner, but I want to get started doing some experimental biology of my own. I'm hoping the community here can help fill in some gaps for me!

1) Suppose I want to modify the DNA of some bacteria with the goal of producing some protein. How does one go about figuring out what sequence needs to be added to the bacterial genome?

Read up on the protein (use google scholar), is the amino acid sequence known, DNA sequence, RNA sequence? Is the protein found in multiple species, or sub-species? Has anyone done experiments with any of the genes in other species (transgenic). If the protein sequence nor the DNA/RNA sequences are known, then you've got some work ahead of you (i.e. mutate the organism a little at a time, when the protein stops functioning or being present, you've found a lead... as long as the mutations can be tracked/found, i.e. a transposon sequence, so you can extract the surrounding DNA, which likely is the gene or some related regulator) (there are many approaches to this, I only gave a very generic method that is pretty old, but also quite a brute-force approach and thus intensive).

 

2) How is the sequence inserted? I'm aware of crispr, but I'm guessing there are other techniques. What's available to the layperson?

usually enzymes, but some organisms can take up DNA fragments naturally (natural competence) and integrate them... other times they can be coaxed into self-integrating (i.e. SLICE), or it can be done in stages (enzyme insert into a virus, use virus to insert into larger organism) (enzyme into plasmid, plasmid into bacteria, bacteria infect plant) (gene gun) (the list goes on...)

 

3) How does one verify the desired proteins are produced? 

western blot (attempt to bind the target-protein to antibodies that are specific to the target-protein shape/charge-distribution/etc), enzyme functional/kinetic experiments (i.e. if it glows, GFP is present... if it doesn't die when exposed to antibiotics, it has the antibiotic-resistance gene.... if provided a reactant, a product specific to the protein is produced), protein tagging (add some known protein sequence onto your target-protein, which has a standard purification method that is known to be highly specific/selective) (https://en.wikipedia.org/wiki/Protein_tag) (the list goes on with various tricks)

--

-Nathan

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