microbio chapter 9

what is biotechnology

using microbial genetics for advancements in technology

since bacteria is simple how do we modify genes

transcribed genes to modify new dna in bacteria

bacteria is transcribed and translated from what

foreign source

what is a natural example of biotechnology

recombination ; exchanging genes in form of natural recombination

lab setting

artificially manipulated ; combine genes from different sources

methods of recombinant DNA technology

inserting genes

deleting genes

modify genes

example of recombinant dna technology

manufacturing insulin

genes are inserted into a ___ during recombinant DNA

vector

vectors use a

plasmid

vectors are used as a source of getting DNA to ___ (vehicle, transportation)

destination

once dna is in destination what is made

a clone

clones are the ___ (where new bacteria cell and new dna that was inserted)

recombinant

gene product is the

harvest

tools used for recombinant dna

selection

mutation

restriction enzymes

vector

PCR

describe selection tool

looking for microbes that have a desirable trait

what cells are selected

those that contain recombinant DNA

methods for selection

antibiotic resistance and blue white screening

describe mutation

allows us to study how different genes work

describe mutagens

less specific, sees if mutation lead to a desirable trait

describe site directed mutagenesis

involves making a specific change to specific codon in gene

site directed mutagenesis happens during

PCR

restriction enzymes recognize what

a particular sequence in DNA and cut the specific sequence

the sequence being cut is usually

palindromic

describe palindromic

reads the same way forward and backward between complimentary dna strand

restriction enzymes create what ends

sticky and blunt

describe sticky ends

single stranded overhangs of dna ; 2 pieces match each other like a puzzle

describe blunt ends

cuts straight through double strands leaving no overhangs

what organism can also be used as a vector

viruses

PCR mimics what cellular process

DNA replication

PCR is what type of process

semiconservative

steps of PCR

denaturation

anneal primers

extension

describe denaturation 95°

unzips/pulls apart strands of dna using heat to break hydrogen bonds

every component needs to be heat stable after this

describe anneal primers 45°

cooling step where short single stranded dna sequences bind to matching complementary strands; added to the separate strands

mutagenesis can occur at which step

anneal primers ; manipulate to add sequence to primer

describe extension 72°

dna polyermase adds to the primer that have been annealed. adding nucleotides to be extended

all components after denaturation in PCR need to be

heat stable

purpose of PCR

amplify DNA

steps are amplified by how much

2^x ; x = number of cycles

PCR can detect what virus

COVID 19

steps for gene recombination of insulin

amplify

use primers

digest

ligate

transformation

not all bacteria

grow colonies

harvest

describe amplify

doubles number of cycles

use primers that contain ___

restriction sites that are found in plasmids

what should be digested

DNA containing restriction sites and plasmids with restriction enzymes

what is ligated

DNA and plasmid

after ligation is complete what is formed

recombinant DNA

what is contained in the recombinant DNA

gene and antibiotic gene from plasmid

describe transformation

transform component cells (bacteria) with recombinant plasmid

bacterial cell

not all bacteria are ___

transformed

describe the select step for bacteria

selects for bacteria that are clones, able to survive on antibiotics

after colonies are picked on agar plate what happens

grow on selective media

why are bacteria selected to grow on media plate

make more clone copies

the last step of recombinant DNA technology

harvest gene product

describe harvest

cell selection

ways to get DNA for processes

use genomic libraries and complementary DNA (cDNA)

describe genomic libraries

creates a library of DNA sequences

cDNA is made from

mRNA

eukaryotes contain what that prokaryotes dont

introns

what are introns

pieces of DNA not expressed

introns can be cut out using

slicing during RNA processing

since bacteria do not contain introns that dont

recognize them as something that needs to be expressed

to get cDNA, mRNA has to be

reversed transcribed

describe reversed transcribed

reversed back into DNA

what is more representative of the gene after reversed transcribed

DNA

using antibiotic resistors on plasmids differentiates between

whether or not bacteria has grew

w/ antibiotic resistors if bacteria grows it contains

the plasmid

w/ antibiotic resistors if bacteria doesn’t grow it didn’t get

transformed and the antibiotic media killed it

blue white screening contains

amp and B-galactosidase genes

with B-galactose gene is inserted into

B-galactosidase —> gets destroyed

growth on an amp plate means

vector/plasmid was taken up

if vector contains the gene the color is

white

if the vector does not contain the gene the color is

blue

what will be taken up regardless of color in amp growth

vector