Exam 2 (Microbes in Biotechnology)

4 steps of gene cloning

1. create recombinant plasmids with gene of interest

2. transformation

3. selection

4. gene cloning and expression

creating recombinant plasmids with gene of interest

cut open plasmid and paste gene of interest (process relies on restriction enzymes, cutting DNA at specific sites, and ligase, joining DNA)

transformation

recombinant plasmid introduced into bacteria by making bacteria competent (ex. through heat shock), altering membrane permeability, and encouraging them to take up DNA

selection

antibiotic selection identifies the bacteria that took up the plasmid (no plasmid = die, yes plasmid = colony)

gene cloning and expression

colony grown into large culture, bacteria are induced to express gene, gene is transcribed and translated into protein

PETase example of gene cloning

Ideonella skaiensis evolved an enzyme (PETase) that eats plastic; E. coli genetically modified with PETase gene so that large quantities of the enzyme can be made

gain of function research

manipulate and study pathogens in laboratory settings to help predict emerging infectious disease threats such as severe disease, drug resistance, etc) to reduce risk of disease outbreak

bioethanol from starch

microbial enzymes convert starch into sugar, yeast use the sugar for fermentation; problem is that it requires land and would-be food sources, hard to justify

bioethanol from cellulose

microbes with cellulase break down non-edible cellulose (from stalks, sawdust, woodchips, etc); problem is that microbes with cellulase are rare

bioethanol pros

less greenhouse gas emissions that regular ethanol used in gasoline, renewable

AMES test

screens for mutagenic (likely carcinogenic) chemicals by using his- bacteria that has a mutation in histidine gene, rendering it unable to synthesize histadine (essential amino acid); mutagenic chemicals could revert his- gene to his+ gene through reversion mutation, allowing it to survive on a plate

why add liver extract metabolic enzymes to AMES test?

some chemicals turn mutagenic only after being consumed