lab 7 Flashcards
what is competency?
Bacterial cells must be in a particular physiological state before they can be transformed. This state is referred to as competency. Competency can occur naturally in some species under certain conditions.
In nature, what is the source of external DNA for bacteria?
In nature, the source of the external DNA is from other cells that have died and lysed.
what do Bacillus subtilis do to acquire foreign DNA?
take up DNA molecules present in the soil surroundings as a result of the death and breakdown of other bacteria, and they pull the DNA through their cell membrane to the inside of the cell
what is the difference between bacteria acquiring foreign DNA in nature and in lab
nature: source of external DNA is from other cells that have died and lysed
lab: source of DNA is plasmid
why cant all eukaryotic proteins be expressed in bacteria? what is a solution to this?
due to the lack of post-transcription and translation modification in prokaryotic cells, not all eukaryotic proteins can be expressed in bacteria.
solution: many eukaryotic proteins are made in bacteria, which have been transformed with recombinant plasmids carrying eukaryotic genes.
true or false, e coli enters a state of competency naturally.
false, it must be induced
how can we induce competency for e-coli?
when treated with chloride salts of the metal cations calcium, magnesium, and rubidium. Calcium chloride is the most commonly used salt. In addition, sudden cycles of heat and cold help to bring about competency.
what is the most commonly used salt to induce competency for e-coli?
calcium chloride
how does treating e-coli with chloride salts and making it go through hot-cold cycles induce competency?
affect the structure and permeability of the cell wall and membrane so that DNA molecules can pass through.
why is the method by which e-coli uptakes DNA unknown?
-Unlike salts and small organic molecules such as glucose, DNA molecules are too large to diffuse or be easily transported through the cell membrane.
- Some bacteria possess membrane proteins that recognise DNA and facilitate the uptake of small pieces, but E. coli does not have this property.
how do we suspect that e-coli takes in DNA?
It is suspected that in E. coli DNA molecules pass through any of several hundred of adhesion zones where the outer and inner cell membranes of E. coli are fused to pores in the bacterial cell wall
what are adhesion zones?
where the outer and inner cell membranes of E. coli are fused to pores in the bacterial cell wall
what is the main factor preventing DNA from diffusing through the cell wall of e-coli?
presence of negatively charged phospholipids in the cell membranes, since DNA is also negatively charged
how can we solve the problem of the DNA and the cell wall having the same charge, which prevents DNA from entering?
It is suspected that treatment of the cells at 0 degrees C crystallises the fluid cell membrane, stabilising the distribution of charged phosphates.
Cations form complexes with exposed phosphate groups, shielding the negative charges, making passage of DNA possible.
Heat shock appears to facilitate the movement by creating a thermal imbalance on either side of the membrane, physically helping to move the DNA through the adhesion zone
what is transformation efficiency?
The amount of cells transformed per 1 μg of DNA
what is the procedure of selection?
plated on agar media that would allow only the cells that acquired the DNA to grow
what’s a colony?
a colony is a group of identical cells derived from a single cell which underwent many divisions
each colony originally grew from ONE transformed cell
true or false. transformation is ways 100 percent effective
false
in this lab, which plasmid is getting introduced into e-coli?
pGLO plasmid.
what strain of the pGLO plasmid is used?
HB101
what are the interesting features of the pGLO plasmid?
-origin of replication
-the Bla or ampicillin resistance gene which codes for β-lactamase. This is a
secreted enzyme that inactivates ampicillin and allows transformed bacteria, carrying the plasmid, to grow into colonies on ampicillin containing medium.
-the gene coding for the Green Fluorescent Protein (GFP) from the jelly fish Aequoria victoria. GFP is a protein that emits green light in the range of 500nm (509nm) when excited in the ultraviolet range (395nm). Emission is fluorescent because it has a different (higher) wavelength than excitation.
-an AraC gene which codes for the AraC arabinose repressor.
-an AraC promoter, pBAD, controlling transcription of the GFP gene. this promoter functions in the L-arabinose bacterial operon for arabinose sugar catabolism
-arabinose breakdown operon
what is pBAD?
araC promoter, which is the promoter for the L-arabinose bacterial operon for arabinose sugar catabolism.
what does the expression of the GFP gene depend on? how?
on the amount of arabinose. if L-arabinose is present, it binds to the araC arabinose repressor and inactivates it, which allows for transcription of the metabolic pathway to occur. since GFP is a part of this pathway, if arabinose is present, GFP will be transcribed
why is the coding for antibacterial resistance useful for bacteria?
-In nature: can give them a selective advantage in an environment containing antibiotics
-in experiments: provides an easy screening technique to distinguish between transformed bacteria (carrying plasmids) and non-transformed bacteria
how can we use antibiotic resistance in our favour in this lab?
we can see which bacteria took up the plasmid, cause the ones that took up the plasmid will be the only ones to grow
what’s the antibiotic used in this lab?
ampicillin
what should the colonies look like if arabinose if present on the plate?
After transformation, bacteria should be forming ampicillin resistant colonies that emit green fluorescent light when exposed to UV light
What are the steps to transformation?
which substance specifically inactivates ampicillin?
β-lactamase
what is the bla gene?
ampicillin resistant gene
what are our two controls in the experiment?
positive control: just our normal cells that grow to make sure our cells are viable (no plasmid, no ampicillin)
negative control: our cells, but no plasmid so when its put into ampicillin, it doesn’t grow
for the lac operon of E-coli, what are the three genes?
lacZ, lacY and lacA
which gene codes for ß-galactosidase
lac z
what is ß-galactosidase
enzyme involved in lactose breakdown. cleaves lactose into the monosaccharides glucose and galactose
is the lac operon by default on or off?
off (allolactose turns it on)
what is IPTG?
ß-galactosidase can also be induced by the addition of IPTG (isopropyl- B-D-thiogalactoside) to a growing culture. IPTG is a non-metabolizable analog of the normal substrate, lactose
what is the inducer of ß-galactosidase?
allolactose, or IPTG
What are the three bacterial strains we are using (mutants). what do they each do?
-Lac Z+ (wild type)
-Lac Z- (cannot make the enzyme ß-galactosidase, the protein that
cleaves lactose into the monosaccharides glucose and galactose)
-Lac Zoc (has an altered operator region that does not bind lac repressor very well, thus the lac operon is active even in the absence of lactose)
what are the 2 indicator plates used?
-Maconkey lactose plate
-xgal plate
why does the gal plate have 2 subcategories of plates? what are they called?
-X-gal / glucose plates
-X-gal / IPTG / glycerol plates
-we need these 2 extra plates cause
Xgal plates are extremely sensitive. One molecule of ß-galactosidase is all
that is needed to cleave enough Xgal to turn the colony blue. For this reason you will use two types of Xgal plates when you assay the strains.
what do u know ab maconkey plates?
These plates contain lactose and a pH indicator.
Colonies able to metabolize lactose (lac Z+) generate lactic acid, acidifying
the medium and maintaining a red color.
Colonies that are unable to metabolize lactose (lac Z-) turn yellowish-white.
MacConkey plates are not very sensitive. Approximately 1000 molecules
must be metabolized to generate enough lactic acid to turn the colony red.
what do u know ab x-gal plates?
The compound Xgal (5-bromo-4-chloro-3-indoyl- ß-D-galactoside) is cleaved by the enzyme ß-galactosidase (product of the lacZ + gene) to give galactose and a blue dye, figure 6.
Therefore lacZ + colonies turn blue, while lacZ - colonies remain white.
Xgal plates are extremely sensitive. One molecule of ß-galactosidase is all
that is needed to cleave enough Xgal to turn the colony blue. For this reason you will use two types of Xgal plates when you assay the strains.
what is the Xgal/glucose repressor plates?
By adding glucose, cAMP levels will be low and the lactose operon should not be expressed provided that the operator region is also functional.
What is LB?
Lysogeny broth. It’s all the stuff that the bacteria needs to ensure proper growth (sugar, nutrients, etc.)
Wha type of Operon is the arabinose bacterial operon?
Inducible
What are the 4 plates used after our transformation experiment?
Why is IPTG useful?
Because we have have an unlimited supply of it, since b-galactisodase does not cleave it
How do Xgal/glycerol IPTG inducer plates lead to maximal transcription?
-Glycerol makes CAMP levels very high
-IPTG will never be cleaved by b-galactosidase so we have an excess
What two questions does the x-gal plate answer?
Question 1:Does the strain make ß-galactosidase?
Question 2 - If the strain does make ß-galactosidase, is it constitutively expressed, or is it inducible via IPTG induction? Careful analysis of the color of the colonies on Xgal/glucose repressor plates versus Xgal/glycerol IPTG inducer plates will tell you whether the lac operon is constitutive or inducible in that strain.
What plate is this
MacConkey plate
What plate is this
X gal
True or false, Lac Z- doesn’t show up in any of the plates
True
Identify
how many micrograms in 1 gram?
1000000
which plates have LB loaded onto them?
all of them
