Biotechnology. Biotechnology is the
use of biological organisms and processes to produce commercially valuable
products. Many of the earliest biotech
products made use of bacteria to produce recombinant proteins. That is, the bacteria produced human proteins
that could be used as therapeutics in humans.
This feat is possible because both humans and bacteria shared a common
ancestor. Remarkably, the genetic
information that human cells use to make proteins can be used by bacteria found
in human feces.
Sometimes, the use of bacteria and yeast in the production
of foods such as cheese and beer is placed under the umbrella of
biotechnology. However, for our purposes
in this class, biotechnology will be limited to the products created using
modified genetic information. For
example, the recombinant proteins mentioned above are the result of taking
genetic information from human cells and, through the use of numerous molecular
biology processes, bringing that information into bacterial cells in the
context that these organisms can understand.
Central Dogma of Biology. Despite the need to go back in time a couple billion years, humans and bacteria share a common ancestor. Evolutionary theory as well as the central dogma of biology weave together to allow bacteria to read genetic information derived from human cell to make human proteins. The central dogma of molecular biology posits that genetic information flows from DNA to RNA to protein. While the “DNA to RNA” step can be reversed, the same is not true for genetic information flowing from the form of protein back to nucleic acids such as DNA and RNA.
Last Universal Common Ancestor. Of all known living organisms, DNA serves not only as the information that is both passed down from one generation of organisms to the next but also as the source code for proteins using RNA as an intermediate, or messenger, molecule. This fact implies that the last universal common ancestor (aka LUCA) had a DNA genome. All organisms since have made use of DNA genomes as well as the genetic code used to decipher the information. With DNA genomes in all living organisms and a shared used of a genetic code, biotechnology is built on the ability of scientists to use molecular biology tools to edit and modify DNA. The possible modifications are nearly endless.
Model organisms. The
extent of biodiversity makes it impractical to study all organisms in a
laboratory setting. Instead, representative
organisms from the tree of life have been disproportionately studied. Multiple factors have guided the selection of
these “model organisms” including the ease of culturing asexual organisms and
breeding sexual organisms. Biological
principles that are determined in model organisms are usually applicable to
non-model organisms because of both the central dogma of biology and the
concept of LUCA.
In a positive feedback loop, research of a model organisms
incentivizes additional research of that model organism because tools to study
the organism are developed in one study that could be used in follow-up studies
of that organism. However, recent improvements
in molecular biology and biotechnology methods are reducing the barriers of
entry for studying new organisms and, thus, reducing our reliance on any
particular model organism.
Drosophila embryos are injected at the syncytium stage. What is syncytium? What is the advantage to transgene injection at this stage in development?
Why are two P elements often used to create transgenic flies? Why use a partially-deleted inverted repeat?
Can you envision how alcohol dehydrogenase could be used as a selectable marker in fruit flies?
Look at the number of parasites in the various stages of Plasmodium development (Fig. 16.19). What stage of the life cycle does the defensin A strategy “attack”? How about bee venom phospholipase? Single-chain antibodies? What would be the practical reason for targeting therapies to these stages of the life cycle?
The generation of sterile female transgenic salmon includes some interesting steps. What is the purpose of these steps?
Fertilize with Charr Sperm and Pressure Shock
Fertilize with Salmon Sperm and Pressure Shock
Methyl Testosterone Sex Reversal
The book states, “Using antisense RNA and ribozymes have largely been discontinued in favor of RNA interference.” Why? Is it an obsolete statement? (Stay up-to-date with CRISPR Journal.)
Why does the book use so many words to discuss “Natural Transgenics and DNA Ingestion”?
The first transgenic mice had a growth hormone, somatotropin, expressed under the regulation of a metallothionein promoter. What supplement was added to the diet to induce somatotropin expression?
In Greek mythology, what does a chimera look like? What does a chimeric mouse (usually) look like? Would you expect to see a chimeric mouse from a nuclear microinjection-based approach? How about an ESC/blastocyst injection-based approach? How about a retrovirus-based approach?
Does rBST or rTPA come from a transgenic farm animal?
How might you know whether a DNA cassette was inserted into a genome?
The LCR is full of HS. What does this tell you about the DNA in that region? What would you expect for expression levels if a gene was cloned into the beta-globin cluster?
What benefit could come from having insulator sequences on either side of your transgene?
Graff thinks figure 16.9A is drawn wrong. What do you think?
How does the Lac operon work? How is this system used in transgenic animals? Do you use lactose to induce transgene expression in mice?
Clontech sells “Tet-Off and Tet-On Gene Expression Systems”. How can it be both?
What would you do with a TRIM69-floxed mouse to knock out expression of TRIM69 in monocytic cells?
PAM stands for “protospacer adjacent motif”. What does this mean?
Compare CRISPRi and CRISPRa. Which Cas9 variant would be useful for these approaches? What Cas9 fusion protein would you create for CRISPRi? CRISPRa?
How would you set up your CRISPR-based experiment to preferentially use NHEJ DNA repair pathway? What type of mutation would you expect?
How would you set up your CRISPR-based experiment to preferentially use HR DNA repair pathway? What kind of mutation could you expect?
Why would you likely use two gRNAs in Cas9n- and RFN-based experiments? What does it mean that this would reduce your chances of causing off-target effects?
The ribozyme-gRNA-ribozyme method of creating a sgRNA sounds complex. Why wouldn’t you just clone sgRNA sequence alone in a normal PolII-based promoter? How can this RGR system produce sgRNAs in a tissue-specific manner?
Discuss the similarity between processing the CRISPR array in bacteria and processing polycistronic tRNA-gRNA (PTG) sequences.
CRISPR systems are adaptive immune systems in bacteria and archaea that provide resistance to bacteriophage. How has this been adapted to plants? How has this been adapted to animals?
Compare GMOs and GEs. Why might the latter be more acceptable?