Molecular Biotechnology Flashcards

"Any technological application that uses biological organisms, derivatives or thereof, to make of processes for specific use"
aka biotech is basically the procedures using live organisms

Traditional biotechnology refers back to when there was no knowledge of microorganisms or that genes were units of inheritance.-not scientifically based-unable to insert vectors into other organisms DNA-recently developed biotechnologies incorporate knowledge of metabolic pathways, genetics, and cell physiology for rational manipulation

This discipline did not arise on its own, which is common for many fields of study. Molecular Biotechnology draws on knowledge from different disciplines to create commercial products that are useful in a wide range of applications
Pulls from MicroBio, Biochem, genetics, chemical engineering, and cell bio

Since biotechnology takes from a wide range of disciplines (it's multidisciplinary). Universities have expertise in many fields, so expansions of that will be close to colleges/universities. Experts in various fields move frequently between universities and and the community.

-most emerge from large populations, economic stability, universities and a academic population-science based established by quality research at universities and institutions-access to market-open-minded public-seed capital from investors-need investment/funding (critical to avoid bottle-neck)

-centrofuge sample to creatte pellet of cellular material (including DNA)-Resuspend pellet in EDTA to chelate divalent metal cations-add strong alkaline solution (has SDS and NaOH) to denature the chromosomal DNA-SDS disrupts cell membrane allowing the alkali to reach the genetic material-centrofuge again to isolate plasmid in supernatant-phenol:chloroform extraction to isolate DNA andRNA into the upper aqueos layer, protein in the lower organic chloroform layer

-pelleted bacterial cells are resuspended in Buffer P1 (contains RNase A)-vortex-Add buffer P2 (denatures chromosomal DNA)-don't proceed more than 5 minutes, will denature plasmid DNA-Add Buffer N3, mix, centrofuge to form pellet-put supernatent in to QIAprep spin column, centrofuge-Remove trace nuclease activity with Buffer PB-Elute DNA with Buffer EB in microfuge tube

-have alkaline lysis step-use SDS for membrane disruption

-MBD needs EtOH precipitation-elution buffer similar to TE buffer-B&D method requires reagents to be made fresh-QIA doesn't need Ph/OH/Chloroform extraction-QIA more costly, MBD more economic

-subculturing may lead to loss of plasmid-use fresh plates (plasmids may mutate or be lost over time)-desired clone should be streaked from a glycerol stock onto fresh agar plate

-let medium cool bellow 50C before adding antibiotics-spread antibiotic on agar plates after it's solid-avoid freezing and thawing

-16H point of E. Coli growth - stationary phase where all nutrients are used up-past then, enter phase of decline, cells start to lyse, DNA becomes partly degraded

-plasmids antiobiotic resistance will be lost when no antibiotics are present-nucleotide synthesis expensive process, don't wanna do again-will have a low copy number is plasmid can't replicate, a problem because cells with low plasmid copy number will out compete high copy number cells at low antibiotic concentrations

-strains with medium-high levels of REN activity generate low quality DNA-strains that produce lots of carbohydrates produce low quality DNA (which is released during lysis and inhibit enzyme activity)-methylation and growth characteristics of strain need to be taken into account

-rich broth produces more cultures (if culture volume too high, alkaline lysis will be inefficient making a lower yield than expected)