Re: [DIYbio] Beginner questions!

WELCOME TO DIYBio!!!!

I'd strongly suggest starting with bacteria first since plants involves a decent amount of lab overhead to get started. Carolina.com sells kits of all kinds. A simple GFP bacteria kit should get you on the way. After familiarizing yourself with the kit, see if you can get your hands on a plasmid like pUC19 which gives you space to add genes using fairly affordable molecular scissors (restriction endonucleases) and glue (ligases). 

The core of beginner molecular biology is the cutting and pasting of DNA into a self-replicating circle of DNA that acts like compiled code in bacteria. Isolated from the rest of the genome, these circles replicate themselves via hijacking bacterial DNA machinery to make many* copies to the point where one could extract them from a liquid broth containing millions of bacterial cells. This extraction is called a miniprep. Once miniprepped, the sample of purified DNA can be physically cut with various scissors (restriction enzymes) that home in on specific sequences and cut the DNA at that location in a jagged or blunt kind of way. Pieces cut by the same scissor can later be glued together using an enzyme called Ligase. You will need a DNA text editor to visualize this better. SnapGene offers a trail version which is really simple to use and visual. Genome Compiler is another app that is totally free and even more colorful and visual. Lots of tutorials out there but at the heart of it all is molecular scissors and glue that allow the researcher to stitch together sequences to form genetic phrases which confer (hopefully and ideally) a function that can be detected. GFP (green fluorescent protein) expressing plasmids like the ones in Carolina or Edvotek Kits give you a very clear idea of whether or not your experiment worked.

Through the process of cold-hot-cold, better known as heat shock, one forces the engineered plasmid into EColi by, allegedly, making pores due to rapid temp change on the surface of the bacteria. At that point the plasmid DNA goes along its concentration gradient and enters the bacteria via said pores. Thats the hot phase. The cold phase rapidly closes the pores and traps the plasmid inside. Once inside, the plasmid does its thing. You take the sample of bacteria that was just shocked and plate it on solid agar medium containing an antibiotic that normally kills the bacteria. If the bacteria took up the plasmid, the plasmid by default also contains the genetic circuit for producing a protein which destroys or interferes with the antibiotic. This system is in place so cells that are not containing the plasmid are killed. 

The plasmid in the GFP kit also contains a circuit for expressing a protein which gives off a green glow when excited (exposed to) UV light. Cells that have this plasmid should be resistant to ampicillin (see plasmid map for specific antibiotic resistance conferred but usually ampicillin) AND glow under UV light. The documentation that comes with these kits will do a decent job at explaining the process and should come with everything needed aside from a 42C heat source, incubator, microwave or hotplate, etc.

A great resource for all things DNA is the Dolan DNA Learning Center found at DNALC.org

Once you get comfortable with bacterial editing, let me know and I'll introduce you to working with plants. Best of luck and don't hesitate to ask questions once you do some homework and googling! 

Sebastian S. Cocioba

CEO & Founder

New York Botanics, LLC

Blog: ATinyGreenCell.com

On Mar 25, 2016, at 1:25 AM, Justin S <jjs...@gmail.com> wrote:

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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