lab 2: bacterial transformation Flashcards
bacterial transformation
-an important technique in molecular biology
-changing the genotype & phenotype of a bacteria by uptake of foreign/new DNA
DNA
-contains genetic instructions specifying the biological development
-instructions are carried out by proteins
proteins
-made up of amino acids joined by peptide bonds
-proteins are synthesized in rough ER
-all amino acids have: acidic group (-COOH) = carboxyl group; amino group (H2N); R group that varies (determines the identity of amino acid
bacteria
-prokaryotes
-no nucleus
-bacterial DNA is organized in 1 double stranded circular molecule
-DNA is located in nucleoid
-
Escherichia coli (E. Coli)
-host organism we use for the lab
-one of the most studied organisms
-grows rapidly
-bacteria competence (the chances for successful transformation)
genetic transformations
-inserting new DNA so the bacteria contains whatever products we want to mass produce
-ex: proteins like insulin (made from bacteria & yeast), genetically modified foods (GMOs)
bioremediation
the use of either naturally occurring or deliberately introduced microorganisms or other forms of life to consume & break down environmental pollutants, in order to clean up a polluted site
plasmids
-small circular pieces of DNA that replicate independently from the host’s chromosomal DNA
-can be engineered to carry genes from one organism into a bacterial cell
-contain a gene that confers some selective advantage (AMP & Beta-galactosidase)
-can be used to carry genes into a cell
AMP
-ampicillin
-resistance to an antibiotic
Beta-galactosidase
produces an enzyme that breaks down lactose & a chemical like lactose, X-Gal -> Indo Blu
bacterial conjugation
-bacteria can pass their plasmid on to other bacteria by bacterial conjugation
plasmid conditions for successful transformation
- a host into which DNA can be inserted
- a means of carrying the DNA into the host
- a method for selecting & isolating the successfully transformed organisms
what’s carried on the plasmid?
-amp= ampicillin resistance gene
-betagalactosidase gene= turns bacteria blue
result 1: no change = control
bacteria on the control plate (no antibiotic & no X-gal) will grow beige “lawn”
result 2: antibiotic resistance
-amp
-this gene codes for a protein that confers antibiotic resistance
-it will survive if exposed to antibiotics
result 3: turning bacteria blue
-betagalactosidase gene
-this gene codes for an enzyme that breaks down the X-Gal substance to make Indo Blu pigment
calcium chloride
neutralizes negative charge of DNA
ampicillin
antibiotic designed to kill any bacteria that lack the plasmid
X-gal
the sugar that is present in order for the bacteria to express the blue color
luria broth
-a nutritionally rich medium given to E.Coli after they have been stressed with temperature changes
-used as a food source
transformation
bacteria take up plasmid DNA from their environment
LB/AMP/X+ plate
-the purpose is to grow only transformed colonies which express the blue color (protein)
-colony count should be greatly reduced since only a few colonies contain the plasmid
-plasmid in presence of XGAL turned transformed (AMP resistance) bacteria blue
LB+ plate
-the purpose is to show that the E.Coli are viable & healthy after the transformed procedures
-all bacteria should grow & express white color
-this is a positive control for LB/A/X+ plate
-the bacteria will contain the plasmid
-have a lawn of bacteria; this proves that the bacteria are capable of growing on the agar & there was nothing preventing growth besides the ampicilllin
LB- plate
-the purpose is to show that the E.Coli are viable & healthy after the transformation procedures
-all bacteria should grow & express white color
-this is the positive control for the LB/A/X- plate
-the bacteria will NOT contain plasmid
-also proves that bacteria is capable of growing on agar & nothing preventing growth beside ampicillin
LB/AMP/X- plate
-the purpose is to show that without the plasmid, the bacteria can not grow in the presence of ampicillin
-bacteria killed by AMP so no transformed bacteria to turn blue
LB/AMP-
-no growth
LB/AMP+
-small growth
-this shows that the bacteria was transformed & developed a resistance to ampicillin
