Biology Lab Practical 2
types of point mutations
Substitutions Missense mutations Deletions Frameshift mutations
Which plates could transformed bacteria grow on? Why?
+pGLO plates because contains the amp resistant gene, bla
Which plates contain transformed bacteria? How do you know?
+pGLO plates because had the amp resistant gene otherwise bacteria would not be able to survive due to amp in LB; bla inactivates the amp present in the LB nutrient agar to allow bacterial growth; only transformed bacteria that contains the plasmid and express bla can grow on plates that contain amp (small % take up plasmid and can transform)
Which plates produce bacteria which glow? Why?
+pGLO/amp/ara; bacteria on this plate are the only ones that should glow when exposed to long-wave UV light. the pGLO gene is controlled by using a promoter that is only active in the presence of the sugar arabinose
How would your results change if you were missing a component of the protocol?
- arabinose molecule induces araC to promote transcription and activate the promoter to turn "on" the GFP gene (glow) - - CaCl buffer neutralized DNA and allowed for uptake of new DNA followed - heat shock increased the uptake of foreign DNA significantly - plasmid allows for amp resistance (bla) and replication (ori)
How are they similar?
- defect in the LPS cell wall due to rfa gene causing them to be more permeable to chemicals in solution (vulnerable to exogenous mutagens) - defective DNA excision-repair mechanisms due to uvrB gene, which prevents DNA repair
Which plates could non-transformed bacteria grow on? Why?
-pGLO LB (just nutrient agar with E. coli)
Which plate was the negative control for growth?
-pGLO LB/amp; No bacteria should grow on this plate. If it does, it means the bacterial culture has acquired resistance and is no longer suitable for use in this experiment
Which plate was the positive control for growth?
-pGLO LB; This control is to be sure the bacteria are viable after the chemical and heat transformation procedure. This plate should be covered with a bacterial "lawn"
How do you calculate transformation efficiency?
1) total quantity of pGLO plasmid used (µg) 2) amt of pGLO used per plate (µg) concentration: total pGLO/final volume µl 3) count total # of transformed cells 4) calculate: total # of cells growing/ amt of DNA (amt of pGLO used per plate)
What is the dilution factor?
10^-1, 10^-2, 10^-3
What is the positive control in the Ames test experiment?
1535: sodium azide NaN3 is a white solid that is highly soluble in water. Because it easily kills bacteria in high enough concentration, it is used as a preservative in some chemical solutions 1538: 4NOP is red dye; highly insoluble in water, produced positive results and considered a potential mutagen which confirms the exp. method works
5 genetic regions of pGLO plasmid
Arabinose operon (PBAD) araC gene GFP gene Beta-lactamase gene (bla) Ori region
How much do you need to dilute an overnight culture that contains 2.8 x 10^9 CFU/ml if you want to have a plate with approx 30 colonies?
CFU/ml = # of colonies per ml plated / total Dilution factor 30/ 2.8 x 10^9
transformation solution (CaCl2)
Ca2+ cation neutralizes the negative charges of the DNA phosphate backbone and phospholipids of the cell membrane, allowing DNA to enter the cells; as Cl ions enter the cell, water molecules accompany the charged particles and causes the cells to swell and is necessary for the uptake of DNA; must be followed by heat
What 2 main health risks we're we exposed to in this lab?
E. coli and samonella
What 2 regions of the pGLO plasmid are modified arabinose operon?
PBAD promoter and araC gene
spontaneous reversion
Reversion of a mutation occurs when a second mutation restores the function that was lost as a result of the first mutation, can either be original or second site reversion
How do we expose the bacteria to the mutagen?
S. typhimurium added onto media with trace amounts histidine along w/ mutagen; The number of colonies growing on the plate indicates the number of revertants
What bacteria does it use?
Salmonella typhimurium (TA1535/ TA1538)
What is the media of the plates we used for the Ames test? Why is this important?
Since the bacteria require histidine to make many of their proteins, these mutant bacteria will die unless the media in which they are grown contains histidine
How are the two bacterial strains different?
TA1535: T to C missense in the hisG leading to a leucine to poline amino acid substitution TA1538: deletion of 1 base pair C in the hisD gene, which causes a -1 frameshift mutation; changes 2 amino acids and brings a stop codon into reading frame133
Understand the regulation and expression of the ara operon
Which sequences, proteins, and chemicals act as promoters, activators, repressors, operators, inducers
auxotroph
a mutant organism that cannot synthesize an an essential molecule (i.e. amino acid); Can only grow in complete media
arabinose
a simple sugar molecule that "turns on" the gene
metabolites
a substance formed during the metabolism of a compound; compound might not be mutagenic, but metabolite could be (need further testing after Ames)
conjugation
a type of genetic recombination: DNA is transferred from one bacteria to another with the aid of a bacterial structure (pilus) know what they are, how they occur, and how they differ
transformation
a type of genetic recombination: occurs when a bacteria receives a small amt of DNA from the environment
transduction
a type of genetic recombination: occurs when a bacteria-specific virus (bacteriophage) xfers a small amt of DNA from a lysed bacteria to another cell of the same species
reversions
ability of a chemical to induce back mutations of an auxotroph to its original prototrophic state
bla (beta lactamose gene)
allows the bacteria to be ampicillin resistant; under the control of a constituitive promoter, meaning it is always expressed - Ampicillin is an antibiotic, normally E. Coli cannot survive in its presence, and bla allows the bacteria to survive
ampicillin
antibiotic, member of the penicillin family; bla inactivates the amp present in the LB nutrient agar to allow bacterial growth; only transformed bacteria that contains the plasmid and express bla can grow on plates that contain amp (small % take up plasmid and can transform); untransformed cells cannot grow on amp selection plates
components of the ara operon
araB, araA, araD, araC, araI, araO1, araO2
PBAD
arabinose operon promoter; when araC binds to araI and araO2, DNA is looped and blocks access to the promoter, repressing transcription
prototroph
bacteria that is able to synthesize all of the biochemicals needed for its own growth Wild-type; Capable of growing on a minimal medium
competent cells
bacteria which are able to uptake DNA and are made so by treatment with calcium chloride in the early phase of growth
The bacterial cell membrane is permeable to ______ ions, but not to _____ ions.
chloride; calcium
arabinose operon
codes for 3 proteins required to metabolize arabinose, under the control of a single promoter; demonstrates both negative and positive control, diff for CRP; also shows how protein can act as a switch upon binding of a small molecule
operon
combination of promoter (regulatory DNA sequence) + transcriptions genes
second site reversion
complementary mutation; Reversion by removal of another base
Both _____ and ______ are commonly implicated in the cell to cell xfer abilities to resist certain drugs and toxins
conjugation and transduction
araA
converts arabinose to ribulose
araD
converts ribulose to xylulose and can then be metabolized via the pentose phosphate pathway
What were the experimental substances being tested for mutagenicity?
crystal violet, ethyl methanesulfonate, soy sauce
agar
derived from seaweed; melts when heated, forms a solid gel when cooled and functions to provide a solid support on which to culture bacteria
genotoxicity
describes the property of chemical agents that damages the genetic information within a cell causing mutations, which may lead to cancer; all mutagens are genotoxic, whereas not all genotoxic substances are mutagenic
CRP
doesn't directly help RNA bind to promoter, in the presence of arabinose, promotes rearrangement of araC from a state of repressing to activating transcription of PBAD promoter
genotype
genetic makeup of an organism
pGLO plasmid
genetically engineered DNA plasmid, that includes the jellyfish gene that codes for GFP, bla, and ori; structural genes (araBAD) were replaced for the GFP gene, which leaves GFP under control of araC
steps of heat shocking
ice to water bath @ 42C for 90 seconds to ice
araI
inducer site; initiator region containing operator as well as promoter; araC bound @ this site can simultaneously bind to araO2 site to repress transcription from PBAD promoter; in the absence of arabinose, araC helps activate expression of PBAD
What was the role of arabinose in the pGLO experiment?
inducers
What are colony-forming-units? (CFUs)
is a measure of viable bacterial; measures only cells that are alive
LB (Luria and Bertani)
liquid and solid nutrient media, a nutrient broth and agar made from an extract of yeast and enzymatic digest of meat byproducts for bacterial growth
carcinogen
mutagen that causes cancer
repressors
negative regulatory proteins that bind to operator sites that block RNA polymerase binding and keep it from being able to perform transcription
Can the Ames test tell you if something is a carcinogen?
no; compounds that test positive would need to be tested further before being labeled as a carcinogen
How would you know if something is highly mutagenic?
o What does it tell you? o What are its advantages?
phenotype
observable characteristics or traits
araO1
operator site; araC binds to this site and represses transcription from the Pc promoter; in the presence of arabinose, araC bound @ this site activates expression of the PBAD promoter
araO2
operator site; araC bound @ this site can simultaneously bind to araI site to repress transcription from PBAD promoter
how to read a micropipette
p1000 = 220 µl p200 = 22 µl p20 = 2.2 µl
araB
phosphorylates ribulose
mutagen
physical or chemical agent that produces a mutation
What is the negative control in the Ames test experiment?
plates with water; expect no growth, purpose is to show no outside influence or contamination growth on plate equals spontaneous mutations
activators
positive regulatory sequence where proteins bind on DNA nearby promoter regions that facilitates RNA activity and transcription of nearby genes
positive vs negative gene expression
positive: the genes are expressed only when an activator is present. negative: the genes in the operon are expressed unless they are switched off by a repressor
What causes the bacteria to fluoresce?
presence of the arabinose induces araC gene to activate expression of the GFP; araC is bound at the PBAD promoter site, but without arabinose is in the incorrect conformation to recruit RNA polymerase and initiate transcription. in the presence of arabinose, the sugar binds to the araC protein and changes its conformation so that in combination with RNA polymerase, transcription is initiated and an mRNA transcript is produced causing the phenotype to be expressed
gene expression
process by which information from a gene is used in the synthesis of a functional gene product; control of these processes plays a critical role in determining what proteins are present in a cell and in what amounts
genetic recombination
process which allows bacteria cells to receive small amounts of DNA from other cells or from the environment 3 types: conjugation, transduction, transformation
araC
produces a protein needed for transcription of genes in the presence of arabinose repressor in the absence of arabinose, binds to araO2 and araI forming a loop, which prevents transcription of the ara operon; regulates 3 structural genes (A, B, D)
What is the organism's phenotype when GFP is expressed?
product glows green under UV light
transcription
protein production starts here (DNA to RNA)
transcription factors
proteins that help turn specific genes "on" or "off" by binding to nearby DNA; activators boost a gene's transcription. Repressors decrease transcription
Ori region
provides a recognition site for DNA polymerase; without it the plasmid will never be replicated
GFP gene
provides code for green fluorescent protein; under control of araC protein; production will only occur if RNA is allowed to bind to the gene; when bacteria is transformed w/ pGLO and grown in presence of arabinose, GFP gene turned on and bacteria glows green under UV light
araC gene
regulates expression of the PBAD promoter to either repress or induce transcription - in the absence of arabinose, obstructs the promoter and prevents RNA from binding to GFP gene -when arabinose present, allows araC to bind GFP gene -also prevents its own expression (autoregulator); if concentration falls to low, transcription of araC resumes until sufficient
operators
repressors that bind to pieces of DNA; When bound to its operator, a repressor reduces transcription (e.g., by blocking RNA polymerase from moving forward on the DNA).
What is the organism's phenotype when bla is expressed?
resistant to ampicillin, allowing the bacteria to survive
true reversion
reversed by another point mutation in which the nucleotide is change at the original mutated site; Reversion by removal of original insertion
What is the importance of adding minimal amounts of histidine? (with respect to detecting reversions)
salmonella is auxotrophic for histidine meaning it cannot synthesize histidine -salmonella is added to an agar that has a small amount of histidine in it so that there is enough for several rounds of replication so that mutations can be created
gene
sequence of DNA or RNA which codes for a molecule that has a function
plasmid
small independent circular DNA that replicates itself independent of chromosomal DNA; contain genes for drug resistance and can be transmitted between bacteria of the same and diff species
inducers
small molecules that binds to activator that act as on/off switches ex: an activator may not be able to activate the operon, but once the molecule binds, it can turn "on" the activation or vice versa
Ames test
test designed to determine how mutagenic a chemical is by taking an already mutated bacteria (auxotroph) and adding a chemical to see if it returns back to its original form (prototroph); Detects frame shifts and base substitutions; the more revertant colonies, the more mutagenic the substance is
How do we know the colonies on the +pGLO plates contain the pGLO plasmid?
the +pGLO plates also contain amp, without the presence of the plasmid containing the bla and the opi to replicate, the bacteria could not grow;
What is a wild type allele?
the allele that is the most common in the population
What would happen if you added arabinose to a tube containing pGLO plasmid? Why?
the bacteria would be transformed because the arabinose would induce the araC gene to turn "on" the GFP gene and promote transcription of the phenotype (glow green)
how does the phenotype (with respect to growth and fluorescence under UV light) of E. coli vary as a result of pGLO and environmental conditions?
the bla allowed it to be amp resistant to survive, the opi allowed the plasmid to replicate, the arabinose induced araC to promote transcription and activate the PBAD promoter to turn "on" the GFP gene which glowed green under a UV light, the transformation solution neutralized DNA and allowed for uptake of foreign DNA followed by the heat shock which increased the uptake significantly
+pGLO LB/amp
this control proves that the transcriptional control of the GFP gene is intact, and no GFP protein is produced in the absence of the arabinose sugar
role of heat shock in bacterial transformation
this procedure is necessary for bacterial uptake of foreign DNA; when E. coli is subjected to heat, a set of genes are expressed (heat shock genes) which aid the bacteria in surviving at such temp; absence of the heat shock will result in a 10 fold decrease in transformants; above 42C, bacteria's ability to uptake is reduced and at extreme temps it will die
RNA polymerase
transcribes the gene
How to you determine how much DNA (plasmid) you used?
use concentration of plasmid (.08µg/1 µl) to convert to total quantity (?µg/10µl) = .8µg
bacterial transformation
used to add new genes to bacteria, creating recombinant cells
transformation efficiency
used to describe how successfully DNA molecules enter bacterial cells
What was the purpose of conducting serial dilutions?
used to reduce a dense culture of cells to a more usable concentration
mutations
when a chemical interacts w/ DNA to produce permanent changes in the DNA sequence & structure 2. produce allelic variation 3. occur randomly
When are the genes in the ara operon expressed?
when arabinsoe is present, it allows araC to bind to araI, which activates expression of the PBAD promoter; this allows for transcription of the araBAD genes
promoters
where RNA polymerase binds; aka regulatory DNA sequence; in prokaryote, multiple genes grouped together share one promoter