Tuesday, March 26, 2013

Grow Your Own

The following is an excerpt from The Path: Origins, available April 31st.

A quiet revolution has been underway within the last four decades. Biochemist Herbert Boyer and medical doctor Stanley Cohen were awarded a half-million-dollar Lemelson-MIT Prize for their breakthrough 1973 transplantation of genes from one organism to another. Since then, recombinant DNA has become the basis of the biotechnology industry, used to create everything from food to medicines, fuel and materials.

Biotechnology applies engineering principles to nature. To use the analogy of a desktop computer, if living cells are the hardware of life, then DNA is the software which runs it. That software can now be customized to produce entirely new forms of life.

Custom DNA can be virtually assembled and tested on a computer, then assembled in the real world from swappable gene modules, just as a computer program can be assembled from subroutines using a programming language like visual C++. And, just as authorship of a computer game or word processor can be protected through copyright laws, genetic software - i.e. genetically-engineered life forms - can be patented.

Individual genes express proteins which perform specific subroutines like cutting, binding, detection, etc. These modules can be chemically synthesized by a commercial lab or extracted from living organisms, then reassembled into a completely new DNA "program" which can perform complex tasks, such as producing a custom protein like insulin, or altering a living organism to produce nutritionally-enhanced vegetables or even goats that excrete spider-silk in their milk.

Because DNA is a common code constructed from four nucleotide sequences and shared by all life forms, it can be recombined - the DNA from one organism mixed with that of another. Here's how the process works:

A bacterial cell typically contains at least one plasmid, a small loop of DNA separate from the cell's main cluster of nucleoid DNA. In the process called transformation, this plasmid is treated with an enzyme to allow the insertion of a customized gene. Human DNA is often inserted, and bacteria can be induced to absorb this modified DNA, turning them into custom DNA or protein factories.

STEP 1: Growing the culture
A bacterial colony - often E. coli - is grown in a shallow glass petri dish within a growth medium. This medium can be either a liquid broth or solid gel agar made from pre-digested milk or meat, algae gelatin, beef or yeast extract, and salt. The growth medium is incubated at 37 degrees, so large numbers of bacteria can be rapidly produced.

E. coli are selectively bred with a resistance to an antibiotic like ampicillin. This immunity can quickly spread through conjugation. During conjugation, one bacterial cell creates a pilus, a transferral tube of protein that it inserts into another bacterial cell. It then sends across a plasmid DNA loop, giving the recipient bacterium a "software upgrade" to its DNA. This is one way in which antibiotic resistance - the ability for bacteria to manufacture antibiotic-destroying enzymes, alter their membranes, or other defenses - can spread. They can also scavenge DNA remnants from dead bacteria, or be themselves infected by jumping genes, small pieces of DNA called transposons, which hop from DNA molecule to DNA molecule and become permanently integrated into the host's genome.

STEP 2: Isolating the DNA
First, the cells of the bacteria colony are lysed - split open. There are three methods commonly used. Commercially-purchased enzymes such as cellulase or lysozyme (created from snail gut yeast or chicken eggs) can break down cell membranes.

Alternatively, sonoporation uses sound wave vibrations to break apart cell membranes and release cellular components. In this process, the bacterial culture is placed in a test tube filled with water, and an ultrasonic probe is placed inside which vibrates at high speed. If the mixture is cooled with ice, this process doesn't cause denaturation, the structural breakdown of proteins.

Beadbeaters are machines which resemble blenders, used to disrupt cells through mechanical force. Chilled cultures in test tubes containing tiny steel beads are placed in an ice-filled chamber and agitated at high speed. The vigorous vibration causes cells in the solution to physically break apart.

The resulting liquid can then be strained through a filter with a hand-held plunger, or placed in a gel box, where electrical charges are used to separate DNA molecules according to their natural charges.

STEP 3: Extracting the Desired Genes
Specific genes are extracted from DNA using a restriction enzyme. Restriction enzymes - naturally-occurring proteins in microorganisms - function as a defense mechanism against foreign DNA borne by invading viruses. These protective enzymes cut up foreign DNA in the process called restriction, while the host's DNA is protected by a modification enzyme that "marks" it through methylation - the adding of tiny methyl group molecules to the DNA molecule.

There are over 600 restriction enzymes for sale commercially, but over 3000 have been studied extensively, and are regularly used as tools in laboratories.

Each restriction enzyme recognizes a specific nucleotide sequence known as a restriction site, where it severs both strands of the DNA. The enzyme EcoRI, for example, has a precise shape which allows it to travel along the DNA double helix, scanning for the nucleotide base sequence GAATTC, where it then cuts the plasmid, opening the molecule and allowing a customized sequence of DNA to be inserted.

Exposing DNA to the enzyme Ecor1 separates DNA with a staggered cut, called "sticky" because the overhangs of the open ends easily bind to other Ecor1-cut DNA fragments.

As a simpler alternative, customized lengths of single-stranded DNA (ssDNA) can be chemically synthesized in laboratories.

STEP 4: Splicing the DNA
The customized DNA is spliced into the plasmid vector (host) using another enzyme called lygase, which acts as genetic glue. In a solution, the customized DNA sequence and the open ends of the cut plasmids naturally recombine by matched base-pairing. Thus, the engineered gene naturally inserts itself into the open plasmid ring. The plasmid is then resealed by adding the enzyme DNA ligase to the solution.

Live E. coli and the pool of recombinant plasmids are mixed in a suspension of calcium chloride at freezing temperatures. Rapidly raising and lowering the temperature creates heat shock, so the cell membranes become temporarily permeable to DNA, and some of the E. coli cells absorb the recombinant plasmids.

STEP 5: Isolation and Growth
The E. coli are then placed upon a petri dish filled with growth medium, into which an antibiotic such as ampicillin has been added. The customized plasmids contain genes which make them immune to the antibiotic's effects, so all non-immune E. coli are destroyed, leaving only the engineered bacteria.

The colony of surviving E. coli is then incubated at 37 degrees. When these cells replicate, they create identical copies of the customized DNA, producing whichever proteins the customized genes express.

Plasmid-based genetic cloning is limited to accommodating external DNA of about 10,000 base pairs, but a typical human gene is on average about 27 kilobase pairs. Because of this, bioengineers have begun to use a variety of vectors in place of plasmids, including viruses; phages are one variety of virus which only infect bacteria, though they're notoriously difficult to use.

Cosmids - a mixture between plasmids and phages - can hold up to 30 kb of base pairs, enough for an average-sized gene, but for larger genes, in 1997, bioengineers created a new type of vector called a Bacterial artificial chromosome, or BAC for short. These are artificially-synthesized lengths of DNA which can hold up to 300,000 base pairs, and are more convenient to use, because they have been prepackaged with tools that simplify the process, such as antibiotic resistance genes, and convenient base-pair-matching ends with which the custom DNA can easily bond.

The plasmid for creating a BAC vector is a special F plasmid (fertility plasmid), used by bacteria to transfer DNA via conjugation during environmental stress.

If you'd like to try your hand at it, you can download free gene-designing software from www.dna20.com, and have your designs synthesized in the lab and delivered directly to your door - just like ordering a pizza.

No comments:

Post a Comment