Microbiology Exam 2
During cellular growth what happens to the histone core and DNA?
1. How do RNAP's get acess to DNA 2. There is a transition in the DNA from solenoid to the beads on a string model. 3. Histone acetylation activates transcription. 4. Histone deacetylation deactivates transcription.
What happens in the absence of insulin?
1. In the absence of insulin glucose can not enter the cell. 2. Glucose signals the cell to insert glut4 transporters into the cell membrane that allows glucose to enter.
Eukaryotic promoter structure
1. Initiator box: where txn begins ( Initiator region/INR) 2. TATA box at the -25 position, AT Rich , recognize by TATA box factor 3. Upstream elements: are at least 10 bp long; recognize specific proteins ( also called proximal control elements.) Involve regulation of transcription. Can be more than one upstream element More= more complex control of the element.
The ability to use a vector for molecular biology?
1. Insert genes into the vector 2. Detect the presence of the vector and select for cells containing the vector. 3. Detect the presence of inserted gene into the vector.
What happens when insulin binds to a receptor molecule on the outside of the cell?
1. Insulin binds to a receptors on the outside of the cell. 2. This creates a signal transduction pathway. 3. Which singnals exocytosis of the Glut4 transporter to be embbeded into the cell membrane. 4. Then glucose enters the cell via the Glut4 transporters.
Specialized transduction in the Lambda Phage.
1. Lambda phages infects E.Coli and injects its viral particles at a specific site in the bacterial chromosome = prophage 2. Normally when the E. Coli chromsome is damaged the lambda phage will excise out its own DNA to produce more phages to infect other cells. 3. Sometimes the Lambda phage excises itself incorrectly and takes some bacterial chromosome with it. 4. Replicated new lambda particles burst out of the host cell and include ( Viral particles plus the bacterial DNA) and infect more cells.
What does leuicinne- or (Leu-) mean?
1. Leucine must be provided in the media. 2. Add the gene for leucine synthesis to the shuttle vector = select for yeast transformants
Whats color screening with the lac Z gene?
1. MCS is inserted into the middle of the lacZ gene. 2. MCS specifically inserted not to interrupt cloning sequence or production of the lac Z gene. 3. We can detect the presence of LacZ with B- galactodase. ONPG X-gal
How does the cell handel so many genes at once?
1. Multiple RNAP's
Improving Natural Insulin
1. Natural insulin tends to form hexamer clumps when injected at high concentrations. 2. Hexamer clumps will not bind to insulin receptors in the body.
Detecting and selecting for vectors?
1. Need a selectable marker: bla gene - encodes for beta-lactamase confers resistance to ampicilin. 2. (- Ampicillin) - All the colonies grown on the plate. 3. (+ Ampicillin) - Only a select amount of colonies grow (They have the amp resistance gene) AMP s - Do Not contain the vector AMP r- Do contain the vector
Can you cut a vector at any site and insert foreign DNA?
1. Need to make sure not to interrupt ABr or Rep genes. Multiple cloning site (MCS)- Stretch of DNA in commercial vectors that contain a number of unique restriction enzymes. ( Do not cut anywhere else in the plasmid.) Also known as a polylinker.
What does it mean when the Histone has a + Charge ?
1. No Aceytlation= + charge on histdine tail will interact with the (-) negative charge on DNA wrapping it tightly around in the solenoid form. 2. Acetylation = Mask the + charge on Lysine loosing the interaction between DNA and the hi-stones. HATS cause acetylation HD's cause deacytlation Acetylation is reversible and dynamic. Acetylation is reversible and dynamic.
Non-template strand
1. Non-template strand = coding strand of DNA equivalent in sequence to mRNA.
On a argrose gel what will you see different on in the bands?
1. On the argarose gel with the recombinant you should see an additional band. 2. This is how you know this is your recontaminant. 3. An extra piece of genetic material with a different weight.
Transfer of the F plasmid to the Recipient Cell
1. Once the conjugation bridge from one strand of the dsDNA is nicked at the oriT ( Origin of transfer.) 2. Linear ssDNA from the donor cell moves into the recipient via the conjugation bridge. 3. Unbroken single stranded DNA in the donor cell is used as a template to make dsDNA again. 4. ssDNA in the recipient is used as a template to make dsDNA through rolling circle replication. one strand is replicated at a time.
What 4 things does the F plasmid have incorporated into it?
1. OriV: Origin of vegetative replication. 2. Tra genes: transferable genes 3. OriT Origin of transferable genes 4. rep Replication genes
What is the difference between prokaryotic and eukaryotic RNAP's
1. Prokaryotic cells only have 1 RNAP 2. Eukaryotic cells have 3 RNAP's
RNA pol l, ll ,and lll
1. RNA pol l Larger rRNA 2. RNA pol ll- Protien-coding genes 3. RNA pol lll- tRNA genes, 5S RNA , and small RNA species
Once you find a cell with your vector how do you know it has your GOI?
1. Recombinant- a vector with foreign DNA inserted into it. 2. Restriction Digest- EcoRI site 1. Grow the cells overnight to make sure they make lots of copies of the plasmid. 2. Extract the plasmids from the cell. 3. Use RE to digest the plasmid. 4. Run on an argarose gel to look for a backbone that corresponds to the plasmid backbone and your insert.
Hormones
1. Regulatory molecules that travel around the body of plants and animals. 2. Produced by glands. 3. Hormones travel via the bloodstream. 4. coordinate physiological behavior.
Enhancers
1. Regulatory sequences outside of the promoter that bind " transcription factors and enchance transcription. They can work from a distance and they can be upstream or downstream.
Where does replication start in the bacterial chromosome?
1. Replication starts at the oriV and it is bi-directional. 2. F plasmid replicates in synchrony with the host chromosome. 3. When there is cell division the plasmid also replicates.
What are the players in prokaryotic transcription
1. Sigma factors 2. RNAP's 3. Activators and Respressors
Porkaryotes Charateristics
1. Simple 2. Fast growth 3. Single celled 4. Small cells 5. One circular chromosome 6. Haploid- One copy of each gene
Chromosome structure in Eukaryotes
1. Sister Chromatids 2. Attached to a centromere
Traits of a good cloning vector
1. Small and easy to handle 3 to 7 kb 2. Easily moved from cell to cell. 3. Easy to grow the host strain and easy to purify in large amount ( High copy number) -ColE1 plasmid example. -Small (64 kbs) -Moved by conjugation multiple copies. - Multicopi ( 40 per cell)
Traits of good cloning vectors
1. Small and easy to handle 3 to 7 kb 2. Easily moved from cell to cell 3. Easy to grow the host strains and easy to purify in large amounts ( high copy) ColE1 plasmid is a good example Small (6466Kb) Moved by conjugation Multiple copies (40 per cell) The original ColE1 is not used in Mol. Bio It has been changed to keep the good stuff (ex. replication) , get rid of the unnecessary stuff (colicin) And has been given things that are useful for cloning ( antibiotic resistance)
What is the shape of solenoid fibers.
1. Solenoid fibers are looped and loops are attached to non hi-stone proteins scaffold. 2. 18 loops are wound into a miniband 3. Minibands are stacked to form chromosome
How does the F plasmid get integrated into the host cells genome.
1. Sometimes the F plasmid gets inseerted into the host cell genome. 2. The insertion happens at specific sites called insertion sequences. 3. Spots where the bacterial chromosome and the F plasmid are identical.
Phototroph
1. Strain that can make use or grow a specific substance (wild type)
What type of activity does Telomerase have?
1. Telomerase has reverse transcription activity making DNA from an RNA template elongates the single strand. 2. Shifts down DNA and repeats, adding back a few bp repeats after every round of replication. 3. DNAP fills in the complementary sequence.
Structure of Chromosome ARM
1. Telomeres on the ends. 2. Each arm has origins of replication 3. A centromere more or less centralized joined the two sister chromatids. 4. Multiple Ori Also the site of micro-tubule attachment.
What does the bla gene contin?
1. The bla gene contains antibiotic resistance.
Insulin
1. The cell needs energy 2. Brain uses 120g of glucose per day (60% to 70% of glucose) 3. Glucose is delivered through the blood stream carried by insulin. 4. Insulin binds to receptors on the cell which allows glucose to enter. 5. Glucose is then metabolized or -Stored as fats as triglycerides -Glycogen in the liver and muscles.
Why use chloroplast instead of the nuclear DNA?
1. The chloroplast divides independently of the cell. 2. Genes in the chloroplasts are frequentley transcribed and translated because they are used for photosynthesis. GOAL- Clone into robust plant crops
Eukaryotic RNAP's
1. The core enzyme is grey and conserved (structure and function) and contains multiple subunits. 2. Different in accessory protiens 3. Different account for different gene expression. Across 3 domains of life 4 RNAP's are conserved.
Pol 1 and lll
1. They transcribe housekeeping genes that are consecutively expressed.
Pol ll
1. Transcribe genes that are regulated.
Other regions of Chromosome
1. Unique DNA- sequence only occur once in a genome ( coding sequence) Bacterial- Almost all DNA is unique. Humans 65% Frogs 22% The rest is repeated sequences.
Inserting genes into vectors
1. Use restriction enzymes that yield sticky ends. 2. Cut both the plasmid and the target DNA. 3. Ligate the two pieces of DNA together with Ligase. Restriction enzyme does not need to cut in multiple places on the vector.
Agi and clouds and
1. Used in 1980 to mitigate hail damage Agi in clouds. 2. Psudeomonas have been found in the upper atmosphere. 3. Have been found at the center of hail from all across the globe.
How do we get the production of insulin from pre proinsulin to insulin?
1. We start off with pre proinsulin (We moved through the Endoplasmic reticulum.) 2. Proinsulin (We moved through the Golgi Apparatus) 3. Insulin
Challenges to producing insulin in Bacteria?
1. While insulin is encoded by one gene, it needs several enzymes for its processing. 2. If cloned into bacterial gene we would get preproinsulin as the final product.
Why do yeast need a centromere sequence?
1. Yeast are eukaryotes and they go through mitosis. so they need a centromere sequence. 2. Site where microtublues bind and pull apart the plasmid so after DNA replication each cell will have a copy after division. - If we are trying to get EUKARYOTIC genes into yeast plasmids may be too small. Eukaryotic genes are very large. 50kb is the upper limit to successful plasmids.
How do you clone large pieces of DNA?
1. You clone large pieces of DNA into yeast using YAC. 1. Yeast Artificial chromosome (up to 1 mbp) Linear DNA which mimics a yeast chromosome. Posses 5 Things. 1. Yeast Ori 2. Centromere 3. MCS 4. Selectable marker 5. Telomeres on each end Sequences on the end of chromosomes.
Why do you need a restriction enzyme that cuts once?
1. You need a RE that cuts once so that you can insert your GOI. 2. If a restriction enzyme cuts in multiple places the plasmid will be open at various locations.
Once you have ligated your vector?
1. You transform your cell by incubating the vector with with cells that are chemically or electrically competent. 2. Or through conjugation a cell that has (tra) genes.
Current research to increase industrial production of PHA?
1. clone genes into plant chloroplasts. 2. Aribidopis- Simple fast growing plant used for molecular biology. ( Has 10x more genes than bacteria)
Recombinant DNA
1. rDNA that uses enzymes to cut and paste together DNA sequences of interest. 2. Then recombined DNA can be placed into vehichles called vectors that ferry the DNA into suitable host cells where it can be copied and Expressed.
Differences in yeast
1. yeast are very resistant to antibiotics. 2. Strains exist that cannot make their own leucine (Leu-) (Auxotroph vs Phototroph)
Epigenetics
1.Functionally relevant change to the genome that does not involve a change in the nucleotide sequence. 2. How can the same genetic material produce so many different cell types. 3. Mechanisms of different gene expression.
How can InaZ p. syringae used on crops?
1.It is sprayed on crops that have the bacteria that produce the INF. 2. Eventually they will replace the wild type bacteria and decrease the ice crop damage at low temperatures.
What does diploid mean for a cell?
1.It means the cell has a copy of two chromosomes and its labeled 2N 2. And these two copies are Homologous chromosomes.
What is snowmax?
1.Snow max is an active protien, which enhances the conversion of water droplets from a snowgun into snow. 2. The protien is derived from the tiny bacterium P. syringae
Electroporation
2) Electroporation- High Voltage shock Charging the membrane allows for the formation of pores as they act as capacitors. Membrane pores will seal as the shock is turned off.
Transffer of the F plasmid to the recipient
2) Linear ssDNA from the donor cell enters the recipient cell via conjugation bridge. 3) Unbroken ssDNA strand remaining in the donor cell is used as a template to make DsDNA. 4) ssDNA in the recipient is used as a template to make DsDNA Rolling circular replication: one strand is replicated at a time.
What does a Bacteriophage do during transduction?
A Bacteriophage infects a host cell and destroys it. Most resulting viral particles will carry virus DNA. In general only 1 in 10,000 carry virus particles only carry bacterial DNA by mistake (0.01) Generalized transduction ( example P1: Phage) P1 only attacks E.Coli P1 carries (80-90 genes) Any particular bacterial gene 1 in 500,000 viruses.(.0002) Get 1,000,000,000 phage per ml bacteria so transduction happens quite often. (2000)
How do we set up a Gel.
A) Melt Argarose B) Make a mold that we pour argarose into C) Add to your mold a comb to make wells
Alcohol production
Alcohol production is one of the earliest uses of biotechnology. China 7000BC : fermented made of grapes, hawthore berries, honey. Iran: First evidence of wine 5400 BC Alcoholic beverages: Ancient Egypt, Babylon< Mexico
Restriction Enzymes (RE)
An enzyme that binds to DNA at a specific sequence and cuts the DNA. Endonuclease Isolated from bacteria. Recognition Site- Specific base sequence where RE binds binds -inverted repeats. Naming restriction enzymes Bam HI Eco RI
If i place bacteria on an antibiotic resistant plate what grows?
Anything that grows must contain a vector in it. Blue colonies will be non-recombinant ( without any vectors) White colonies will be recombinant ( they will have vectors.)
Separate DNA based on size need an Argarose gel
Argarose gel- gelatin like polysaccharide found in seaweed. Contains a matrix of cross linked fibers that hinder the movements of large particles. The smaller pieces of DNA move through the matrix at greater speeds. Smaller DNA fragments move further down than the larger particles.
Bacteria and Biotech
Bacteria are critical to production of foods yogurts and (other foods) 1) Alcohol 2) GMO plants/animals/organisms 3)Plastics 4) Hormones 5) Dyes 6) Antibiotics
Genetic transfer in Bacteria
Bacteria divide by Binary Fission (Asexal) Donor cell can be dead or alive Recipient cell is always alive.
Bacteria that produces the RE
Bacteria that produces the recognition site will not degrade its own DNA due to modification enzymes. Add chemical tags to specific DNA bases after replication.
What are Bacteriophages examples of?
Bacteriophages are examples of Transduction.
So what do you use Sticky or blunt ends?
Blunt ends are "not directional" GOI= Gene of interest No overhangs Litigation into the plasmid that has been cut with a blunt cutter. Litigation is independent of the cut sequence.
Why are BLUNT ends not useful?
Blunt ends are more difficult to ligate together since no base pairing occurs between the DNA ends. T4 Ligase- Is a common type of ligase used to litigate pieces of DNA together. Ligase- Catalyze the formation of a phosphodiester bond between the 5' hydroxyl termini in duplex DNA. Isolated from the T4 bacteriophage. Circularied linear T4 DNA after injection into the host cells.
DNA Transformation in the Lab
Cells must be made competent in the lab by weakening the cell wall (artificial) or "turning up" uptake mechanisms. 1) Chemical treatment- Cold shock and calcium treatment followed by heat shock and cold shock. Cold and chemicals weaken the cell wall and help DNA stick to the surface. _ Calcium= + Charge, DNA is negative and the cell surface is negative, lessens electrostatic repulsions. Helps open up pores cold shock stops the process
Function of DNA packaging
Chromatin is the functionally active form of DNA and there are two types. Euchromatin: Lightly packed (less condensed) Enriched in genes Actively transcribed. Heterochromatin: Always condensed Not transcribed.
If X- Gal is added to Media?
Colonies will contain a non-recombinant plasmid will be blue because the Lac-Z gene will not be interrupted. Colonies containing a recombinant of the Lac-Z gene will be white because the Lac-Z gene will be interrupted.
Screening Recombinant vectors
Color screening via LacZ gene. MCS is inserted into the middle of the lacZ sequence. MCS purposefully inserted not to interrupt the reading frame or production of the LacZ enzyme. Without insertion of the MCS the lacZ protein is created. Analogs of B Galactose- ONPG X-Gal: yield indigo color if LacZ is present
Penicilin
Could make 1 g/mL $300 per kilogram Penicillin Chrysogenum Modification in: Increased expression of penicilin gene cluster. Increased peroxisome Elevated levels of transporters to secrete peniclin.
What color is DNA originally in the solution?
Cut DNA fragments are usually colorless in the solution. How can we see if its moving? How do we know if its floating? We use a dense loading dye. We load the DNA into the wells then we add the Dye.
Type II Enzymes
Cut DNA in the middle of the recognition site . Exact cut position is always know. Two different cut patterns are possible. Blunt ends: Fully base paired. Sticky Ends: Ends have single stranded overhangs.
How do you set up a Argarose Gel?
D) Place your gel in a chamber with buffered electrodes) hooked up to a power supply E) Make sure your gel is submerged ( you want current to run through the gel) F) Add a loading buffer to our DNA solution to help, makes it more dense than the buffer so it sinks in, see how far the DNA solution has run down the gel.
Griffith Concluded
Dead pathogenic S type gave off a transforming principle that change the live R type to S type. Transforming principle was the polysaccharide capsule. Others demonstrated the same transformation in cultures using cell free S types
V Cholera
Different cholera strains attach to chitinous surfaces and they make mixed bio-films. At a high cell density it competence system turns on. Cells turn on their T6SS and start puncturing other cells. Non Immune cells will lyse. The DNA from the lysed cells will be internalized by the immune cells.
Naming restriction enzymes
Echeria Ecoli strain RY13 I 1st enzyme iso. EcoR1 in dimer complex with DNA cutting two strands of DNA.
Separating DNA after restriction digestion
Electrophoresis movement of charged molecules toward an electrode of the opposite charge. Used for either nucleic acids or proteins charge and shape DNA- negatively charged phosphate backbone.
What are the insertion sequences
F plasmid can insert at either orientation. Hfr- High frequency recombination- transfer chromosomal genes at a high frequency.
Insertion sequences and the F plasmid
F plasmid can insert either orientation. Hfr- High frequency recombination transfer transfer CHROMOSOMAL genes in a high frequency.
Recombination
Original DNA- Destroy cell and purify the DNA And the fragments to a new recipient cell and it becomes transformed that has the genetic information. When it incorporates the DNA into its genome this is a recombined cell.
Transformation and Circular DNA
If incoming DNA is circular it will be retained and replicated if it has is own origin of replication (Ori) Plasmid- Circular molecule of DNA that replicates itself. Found naturally in many bacteria. Source of antibiotic resistant genes. Often used for genetic engineering.
DNA Buffer
If you put DNA into a buffer with an anode and cathode. Both fragments ( Negative charge) will move toward the (+) electrode at the same speed. Red fragment of DNA is larger it requires more force to move. Red fragment has a greater negative charge due to the phosphate groups. We havent actually separated them just made them move.
Plasmid properties Incompatibility
Incompatibility- The ability of two different plasmids to exist at the same time. Many bacteria stably contain more than one type plasmid. These plasmids are called compatible. Some plasmids can not be stably maintained in the same bacterial cell and one is generally lost. If they share the same replication of control then its is likely then only one of the plasmids at a time will be chosen for replication, Same case with partioning.
Targeting a specific gene in a DNA sample
Lets say we want to cut a specific gene out of our DNA. Nucleases enzymes that cleave nucleic acids by targeting a specific phospodiester bond. Deoxyribonuclease- Cuts DNA- This is what is usually implied when we use the word nuclease. Ribonuclease cuts RNA. Exonuclease cuts at the ends. Endonuclease: cuts in the middle of a nucleic acid chain.
How do we move our isolated DNA
Molecule of DNA which can replicate and is used to carry cloned DNA into a host cell. Plasmids are used widely for cloning applications.
Alcohol production
Mostly produced by fermentation of grains and fruits by yeast.
Detecting and Selecting for Vectors
Need a s electable marker Bla gene- encodes beta-lactomase Confers resistance to ampicillin. Medium minus Ampicillin. Colonies do not have the Amp (s) cells do NOT contain the vector. Medium plus ampicillin Amp (r) cells contain the vector.
Can you cut a vector at any site?
Need to be careful not to interrupt AB(r) or rep genes. Multiple cloning site (MCS)- Stretch of DNA in commercial vectors that contains a number of unique RE sites ( Do not cut anywhere in the plasmid.) Also known as a poly-linker.
Plasmid copy Number
Number of copies of a specific plasmid that a bacteria cell has. Cells with F plasmid have one or two copies per chromosome. Cells w/ CoIE plasmids can have up to 50 copies. CoIE- Plasmid- High copy number that encodes for the colicin E toxin
Specialized transduction
Occurs with the Lamda Phage 1) Lamda infects E. Coli and inserts its viral DNA at a specific site in the bacterial chromosome = Prophage 2) Normally when the E. Coli is damaged, the lamda phage excises its own DNA and replicates more phages to infect other cells. 3) Sometimes the Lamda DNA is excised incorrectly and some of the bacterial DNA is taken along with the host. 4) Newly replicated Lamda particles infect the host cell and infect a new cell, the DNA viral plus a portion of the hosts chromosomes. Lamda phage is possible to have viral and bacterial DNA.
Explain transformation and vectors
Once you have your recombination vector, you transform cells by incubating the vector with cells that are chemically and electrically competent.
Once you find a cell with you vector how do you know your GOI has it?
Recombinant: Is a vector with foreign DNA inserted into it. Grow the cells overnight to make lots of copies of the plasmid. Extract the plasmid from the cells. Use RE to digest the plasmid. Run on a argarose gel to find the plasmids that match your backbone and your insert.
Phenol and Extraction
Removal of proteins by phenol extraction. 1) DNA , RNA , and protiens are in water. 2) Add phenol DNA, RNA, and proteins separate into a water layer and a phenol layer. 3) Shaken them then everything is mixed together. 4) Allow the DNA and RNA to seperate back into thier own layer BUT PROTIENS will stay in the phenol layer.
Purification of DNA
Remove Protiens by phenol extraction DNA is negative charged due to the polar group on the phosphate backbone. DNA is polar in water since like dissolves like. Phenol is less polar than water Phenol is less sou-able than water. DNA is less sou-able in water. In phenol protein molecules will change shape, so the less polar amino acids interacit with the less polar amino acid. Phenol can cause chemical burns.
Purification of RNA from DNA
Remove RNA with ribonuclease. Enzyme that degrades RNA but not DNA. Ribonuclease treatment. DNA remains the same and RNA is degraded down to nucleotides.
Replication of the F plasmid
Replication starts at the OriV and is bi directional. F plamsid replicates in synchrony with the chromosome.
Restriction modification system (RE and Methylase)
Restriction enzymes evolved to recognize foreign DNA. They restrict the phages ability to infect the cell. How come restriction enzymes do not restrict bacterial DNA? Modification enzymes such as methylase add chemical tags to specific DNA bases recognition sites after replication. Restriction enzymes cant cut the site if its methylated. Methylase adds methyl groups to bases after replication. Together restriction enzymes and methylases are the restriction modification system.
EcoRI MODIFICATION Enzyme
Restriction modification system are ( RE and methylase) EcoRI is enzyme that adds on the methyl groups. ( This protects against the cell destroying its own DNA.) EcoRI will no longer be able to recognize this sequence.
Trying to express your cloned DNA into another organism?
Shuttle vector- Must be able to survive in more than one type of host organism. ( Often used to move genes in yeast). - Have to ADD 3 things 1. Yeast Ori - controls DNA replication in yeast on yeast origin of replication. 2. Centromere Sequence- Required for partitioning during cell division after replication. 3. Yeast selectable marker- Antibiotics target bacterial molecules.
Recognition sites continued
Sites range from 4 to 8 bases. Probability of and RE cutting. Each recognition site has a 1/4 chance of being the correct nucleotide. Number of base pair recognition sites 4= (1/4) * (1/4)* (1/4) * (1/4) = 1/256 6= 1 in 4,096 8 = 1 in 65, 536
Streptococcus pneumonia
Smooth form that produces a polysaccaride capsule and is pathogenic. Live Type S. R: Form that is non pathogenic, non capsulated. Live Type R.
How does the F plasmid transfer bacterial genes?
Sometimes the F plasmid get integrated into the host cells genome. The insertion happens at specific sites in the bacterial chromosome. Inserstion sequence: Spots that are nearly identical DNA sequence between the F plasmid and the bacterial chromosome. E coli has 19 sites where it can integrate into the host cells genome. 13 IS2 and 6 IS3
How does the F plasmid transfer bacterial Genes?
Sometimes the F plasmid gets integrated into the host cells genome. This insertion occurs at specific sites in the bacterial chromosome. Insertion sequences (IS)- spots of nearly identical DNA sequence between the F plasmid and the bacterial chromosome. E. coli has 19 sequences where the F plasmid can integrate itself. 13 IS2 6 IS3
DNA techniques Isolation and Cloning
Steps to isolating DNA from bacteria 1) Cell Lysis- Mechanical sheering breaks cells down by physical force. Chemical treatment Lysozyme: enzyme that breaks down the structural layer of the cell wall. Detergent- denature proteins, disrupt cell membrane permeability. After cell Lysis- cell debris, protiens and nucleic acids.
Sticky ENDS
Sticky ends can be " Non directional as well" GOI= Gene of Intrest Cut with one restriction that makes the sticky end Ligation into plasmid that been cut into ECORI Ligation is aided by base pairing. What if you don't want this to Happen?
High frequency Strain
Strain that has the F plasmid incorporated into its genome. (Hfr chromosome) Allows for a high rate of transfer of the host bacterial chromosomes genes to the recipient. Considered a donor cells because it contains the tra genes. ( Which are the genes for transfer.) Conjugation between the HFr and the F- cell Hfr strain attaches to the recipient using a sex pilus and transfers DNA via the conjugation bridge.
What does the arrow head mean on the Transfer of the genes?
The arrow is the direction of transfer: The arrowhead side of the F plasmid is what transfers first, followed by chromosome., following by the remaining plasmid. Bacterial genes transferred by the Hfr are dependent on the direction of transfer and the length of time the conjugation bridge is stable.
Arrow head
The arrowhead is the direction the F plasmid transfers first, followed by chromosome, followed by remaining F plasmid. Bacterial genes transferred by the Hfr strain are dependent on the direction of transfer and the length of time the conjugation bridge is stable.
How did they figure out the transforming substance was DNA?
The chemical nature of the transforming substance was DNA.
What is good and bad about zymonas?
The good: 1. Produce alcohols faster that yeast 2. More resistant to alcohols. The Bad: 1. Only use glucose as a substrate. to break down paper waste need enzymes to break down cellulose to glucose.
After we run the Gel and Dye ?
To visualize DNA we soak it in ethidium bromide. (EtBr) EtBr binds to DNA EtBr florescence when exposed to UV light.
Movement of the plasmid
Transferability- The ability of plasmids to transfer themselves from one cell to another. Mobility Plasmids can differ in their mobility in terms of conjugation. Some of them are too small to have all the genes necessary for conjugation. Some only have a few tra genes.
Incompatibility
Two plasmids that cannot stably coexist in the same time cell are considered to be incompatiable (Inc) group. RP4 is in the IncP group RSF1010 is in the IncQ group RSF1010 can be stably maintained with RP4, but not with another IncQ group plasmid.
Do Restriction Enzymes always cut at the Recognition Site?
Type 1 Enzymes Cut DNA far away from the recognition site. Cut at random sites Not terribly useful. DNA loops around and is cut by restriction enzyme bound to the recognition site.
Type 2 Diabetes
Type 2 - Multi-factor syndrome combined with genetic susceptibility and environmental factors. Factors such as Obesity , age, and physical inactivity result in insulin resistance in cells requiring insulin for glucose for absorption. (27 M)
Why do you ant to use a restriction enzyme that cuts Once?
Use a restriction enzyme that cuts in one spot so you make sure that you get your gene of interest. If the RE cuts in multiple spots you could have pieces of your plasmid floating away.
F- Plasmid Contains
oriV: Origin of vegetative replication Tra: Transfer genes oriT- Origin of DNA transfer rep: Replication genes
Sticky end vs blunt end during cloning?
What id you want to put a bacterial genome into a plasmid. 1) First you cut the gene out using a restriction enzyme. 2) Second you cut the plasmid with the same restriction enzyme. 3) Third you put the two pieces together 4) Fourth you add Ligase.
Purification of DNA
a) Centrifugation to separate the cell debris. Particles will seperate by their S values S-Value- Semidementional value- determined by size, density and shape. "Heavier" stuff settles Faster. DNA, RNA , and protiens are in the solution and the cell debris is at the bottom.
Conjugation
. Donor ability is due to the F-Plasmid Plasmid- Circular molecule of dsDNA that replicates independently of the host chromosome. Donor= F+ the fertility factor Recipient= F- 1) The donor makes contact with the recipient through the F pilius which retracts. 2) Next the conjugation bridge forms and the plasmid DNA moves through the conjugation bridge from the donor to the recipient cell.
Size of 1 Eukaryotic Chromosome
1 Eukaryotic chromosome = 1 mm of DNA DNA exist in an even smaller place the nucleus= 5 uM of DNA. DNA is packed into chromatin by coiling around histones.
Steps to isolating DNA from Bacteria
1) Cell Lysis - Mechanical or chemical 2) Purification of DNA- A. Centrifuge DNA B. Remove the Protien C Remove the RNA D. Precipitate DNA 3) Cutting the Nucleic Acids A. Deoxyribonuclease/ Ribonuclease B. Endonuclease C. Exonuclease D. Restriction Enzymes (IR) a. Type I (Far) Type II (close) i. Blunt ii Sticky methylation 4) Electrophoresis- DNA is negative Argarose allows separation by size Standards Etidium Bromide Restriction Mapping
Purification of DNA
1) Centifuge- To seperate the cell debris. 2) Remove protiens by phenol extraction. Phenol chemical that dissolves protiens. 3) Remove RNA with ribonuclease. Ribonuclease enzyme that degrades RNA but not DNA. 4) Precipitation of DNA using alcohol and salt combination displaces large DNA molecules from the water.
What do you need to know to use vectors for molecular biology ?
1) Detect for the presence for a vector. 2) Directly select for cells contain the vector 3) Insert genes into the vector. 4) Detect the presence of inserted gene into the vector.
Plasmid Properties
1) Host Range- Including all types of bacteria in which the plasmid can replicate varies widely. Some have a narrow host range (few closely related bacteria.) F-plasmid - Rnage in E.Coli and related enteric bacteria ( Salmonella and Shingela) Some have a wide host range P-type plasmids- Spread multiple resistance to antibiotics. 2) Copy number- Average number of copies a cell has.
Types of Movements of Plasmids
1) Nontransmissible- Cannot contact another cell or transfer DNA to another cell. 2) Conjugative - Encodes genes for effective contact but can NOT transfer DNA. 3) Mobilizable_ Encodes genes to transfer DNA but can NOT make contact. 4) Self-transmissiable- Encodes for genes for both contact and the transfer of DNA.
Transfer of the F plasmid
1) Once the conjugation bridge forms, one strand of dsDNA plasmid is nicked at the oriT (origin of transfer)
Generalized transduction continues
1) Phage infects a donor cell 2) An occasional phage packages bacterial cells. 3) Phage with bacterial DNA infects other cells. 4) Donor DNA enter recipeints cells.
3 Categories of Hormones?
1) Protiens- Easiest to genetically engineer (growth hormone, insulin.) 2. Steroids- Derived from lipids sex hormones, corticosteroids. 3) Amines- Tyro sine derivatives Adrenaline Epinephrine , Noephinephrine, and Dopamine
Fate of DNA once it enters the cell
1) Restriction- DNA is considered foreign and degraded nucleases or by restriction enzymes. 2) Recombination- If DNA is linear it must be incorporated into the host cells genome to be retained. Incoming DNA must be similar in sequence to the hosts DNA. Incoming DNA replaces the host DNA. 3) Replication- If incoming DNA is a replicon, it will be retanined.
Transfromation
1) Transformation- Uptake of pure or naked DNA no cell contact is necessary. Original donor cell is dead 2) Transduction- Transfer of DNA by a virus, NO cell to cell contact is necessary. Original donor cell is dead. 3) Conjugation- DNA transfer by donor cell to recipient by cell to cell contact Donor must be alive.
What is natural transformation, and competance
1) When a cell can uptake DNA from the environment without special treatment. 2) Competance_ Is the stage at which bacteria can uptake DNA from the environment. 3) Natural competance- Bacterial that can naturally reach a stage of competence.
When does the F plasmid replicate?
1)The F plasmid replicates during conjugation 2)Original plasmid has double stranded circular DNA. 3) The origin on transfer is Nicked 4) Single strand of DNA enters the female cell. 5) Synthesis of DNA complementary to the unbroken strand.
Auxotroph
1. A mutant cannot make or use a growth substance compared to the wild type. 2. Anabolic constructive metabolism auxotrophs: cannot multiply without a particular growth supplement. (ex) - Arg - It cannot grow without arginine supplement (ex) Mal- it cannot grow without the maltose supplement.
What does a nucleosome resemble?
1. A nucleosome resembles a bead on a string model. 2. A chain of nuclesomes is wound heclically into a solenoid. 3. 30nm of structure with 6 nucleosomes
What is a telomere?
1. A region of repetitive nucleotide sequences at the end of each chromosome. 2. The telomere sequences is GGGATT 3. 6 base pair sequence occurs 2000 times Protects chromosome from deteriorating Helped by telomerase
What is Telomerase?
1. A ribonucleoprotien ( a protien containing RNA) 2. RNA is complementary to the 6 base pair repeat 3. RNA binds to the empty end of the template. 4. DNAP fills in the complementary sequence. 5. Shifts down DNA and repeats adding back a few 6 bp repeats after every round of replication.
A yeast without a shuttle vector?
1. A yeast without a shuttle vector cannot grow on a minimal media without AA supplementation. ( Cannot produce leuicine.) 2. A yeast with a shuttle vector can grow on a minimal media without AA supplementation because now it can produce is own leucine.
What are the products from biotechnology?
1. Alcohol- pathway engineering gene designation- pro, ter, RBS, ORF protien destination folding and cofactors 2. Plastics- transgenetic plants 3. Ice- Gene knockout 4. Hormones- Trans genetic bacteria
Chromatin modification
1. Alteration of chromatin can change whether a gene is transcribed or not. Nucleosome sliding 2. Can cuase partial histone Histone dis placement then replacement
Histone Modification
1. Alteration of histones can change whether the gene is transcribed or not. 2. Histone tails can be moddifed.
Plurpotent stem cell
1. Are often termed stem cells because they have the ability to differentiate into any type of cells in the body. 1. Open to closed and Diffrentiation 2. Closed to open is Reporgramming.
Insulin production
1. Hormone that controls blood sugar levels. 2. Composed of two polypeptide chains an Alpha and Beta chain. 3. Two polypeptide chains held together by sulfide bonds.
Histones and nucleosomes
1. Histones- Are proteins that organize DNA into nucleosomes. 2. Nucleosomes- Basic unit of DNA packing in eukaryotes- repeating sub-unit of chromatin 3. Chromatin- Complex of DNA and protien.
Bioplastics
"Biopol"- Bioplastics industrially produced by company zeneca from a mutant strain of Alcaigenes eutrophus. 100% biodegradable Used to make slow release capsules of drugs. Very expensive to be made in bulk.
Diseases Diabetes Type 1
1. Autoimmune related the immune system destroys B cells in the pancreases resulting in immune deficiency.
Nuclesomes
1. Basic Unit of DNA packing in Eukaryotes 2. contains 200 bp of DNA 3. 140bp + 8 histone proteins 60bp + 9th histone= linker region between nucleosomes
Gel Electrophoresis
1. Cathode has the negative charge 2. Anode- has the positive charge 3. The DNA molecules moves towards the anode.
Prokaryotes
1. Cell Wall 2. Cell Membrane 3. Cytoplasm 4. Ribosomes 5. Chromosomal DNA
Eukaryotes
1. Cell membrane 2. Chromosomes 3. Nucleous 4. Cytoplasm 5. Other organelles
Engineering insulin to prevent Clumping.
1. Change the amino acid sequence of the peptide so that clumping no longer occurs. 2. Proline is located at the surface where to amino acids bind. 3. If change the proline to aspartic acid the two molecules will repel each other.
Strategies for cloning insulin
1. Chemically synthesize DNA for the alpha chain. 2. Insert into plasmid near a bacterial promoter. 3. transform into E. Coli 4. Bacteria grow and make lost of insulin (alpha) chain 5. Break open bacteria and purify the alpha chain. 6. Do steps 1-5 for the Beta chain. 7. Mix alpha and beta chains to form disulfide bonds.
How does RNAP interact with the DNA?
1. Chromatin remodeling allows RNAP's to interact with the DNA.
If X-Gal is inserted what will the colors be?
1. Colonies containing non recombinant plasmids will be blue becuase the lac Z gene will not be interrupted. 2. Colonies containg the recombinant plasmid will be white because the Lac Z gene will be intereupted. (Inserted gene in between the MCS) Non- recombinant= cloning vector without any foreign pieces of DNA.
Eukaryotic Characteristics
1. Complex 2. Slow Growth 3. Large Cells 4. Multi- Cellular 5. Multi Linear chromosomes REPLICATION DELIMA 6. Diploid two copies of each cell. BACKUPS
What is pathway engineering?
1. Construction by engineering of a complete biochemical pathway. 2. Try and clone and express genes for the biochemical breakdown for cellulose degradation from other organisms to zymonas.
What Chromosome structure can you find in Prokaryotes?
1. Convalently closed circular. Bacterial chromosome. 2. Arranged around a protein scaffold super-coiled.
Overview of cloning DNA into a plasmid and checking for recombination?
1. Cut vector in (MCS) and target DNA with the same RE and ligate two molecules together. 2. Transform ligation into the host cells. 3. Place reaction on a media containing Amp and X-gal. 4. Select AMP r and the white colonies.
Structures found in a Eukaryotic Cell
1. Cytoplasm 2. Vacuole 3. Nuclear Membrane 4. Centrioles 5. Smooth Endoplasmic reticulum 6. Cell Membrane 7. Mitochandria 8. Microfilament 9. Nucleous 10.Nucleolous 11. Cell Membrane 12. Rough Endoplasmic reticulum 14. Golgi Apparatus 15. Vacule
Explain how packing increases starting with DNA and moving to the chromosome.
1. DNA , 10 bp , PR: 1 2. Beads on a string- 60 bp, PR:6-7 Nucleosome- Chromatin 3. Solenoid (Mixture of nucleosomes) 1,200 bp PR: 40 4. Loops 60,000 bp PR: 680 5. Miniband 6. Chromosome- 1 million minibands form a chromatid.
Pseudogenes
1. Defective unless duplicate copies of genomes. 2. Exist in one or two copies 3. Copies can exist anywhere on the chromosome.
Gene structure and transcription
1. Defining a gene 2. Accessing a gene 3. Transcriptional machinery Multiple RNA polymerases Regulation of RNAP's Promoters/enhancers/ transcription factor
What is the problem with Linear chromosomes and replication?
1. During each successful round, the ends of the chromosomes would get progressivley shorter.
Insulin production
1. ER- Leader sequence is removed 2. Golgi- cleaves proinsulin 3. Stored and granuels for secretion.
What happens to the intron and exons in Eukaryotic transcrition?
1. Elimination of intron transcription segments 2. And the splicing of Exon transcription segments. Exons- coding region Introns- Non coding and may make up 90% of a gene.
Explain the blue and white dots on the plate?
1. Everything growing on the plate contains a vector because of antibiotic resistance. 2. White dot is the vector + insert. 3. Blue dot is just the vector
Genes for two chains?
1. Genes for two chains are fused to lacZ because its easy to purify with B-galactosidase and the small chains of insulin would get lost on their own.
What are the labels for the histone proteins?
1. H2A, H2B, H3, H4 and they are all associated with the nuclesome core. 2. DNA wraps around it 1-4 times.
Repetitive sequences?
1. Hallmarker of "higher" organisms 2. Moderately Repetitive- a) hundreds to thousands of copies b) rRNA and tRNA c) 25% of the human genome 3. Highly Repetitive a) Hundred thousand to millions of copies b) 10% of human DNA useless c) Exp, Alu element 300bp sequence 500,000 human copies 6-8% of our DNA Does NOT code any protiens
Example of the ColE1
1. Has genes for colicin production 2. Remove this gene and 3. Replace it with antibiotic resistance. Like ampicillin resistance.
Fred Griffiths experiments
Griffith injeced cells into mice and then recorded if the mice died and then recovered the bacteria and then recovered it either dead or alive. R- Nonvirulent (Alive Mouse) S- Virulent ( Dead Mouse) Heated Killed S ( Alive Mouse) Heated R and S (Dead Mouse)
What did Fred Griffiths experiments discover in 1928?
He discovered transformation by studying streptoccocous aureus.
What is the Hfr
Hfr ( High frequency strain) Strain that has the F plasmid incorporated into its genome. (Hfr chromosome) Allows for high frequency transfer of bacterial chromosomes genes to the recipients cell. Considered a donor cell because it has the genes for transfer. (tra) Conjugation between Hfr and cell and F- cell Hfr strain attached to recipient using a sex pillus and transfers DNA via a conjugation bridge.
Plasmid Properties
Host range- Includes all types of bacteria in which the plasmid can replicate
Fate of transferred DNA from Hfr
If entire chromosome is not transferred: 1) Recombination 2) Restriction Rarely is the entire host chromosome transferred to the recipient. Chromosome is considered a replicon so, neither recombination nor restriction are needed for retention. recipient is considered F+ However bacteria do not like to be diploid.
Fate of DNA from the Hfr Strand
If entire chromosome is not transferred: Recombination Restriction Rarely, the entire chromosome is transferred to the recipient. Chromosome is considered a replicon so neither recombination or restriction are needed for retention. Recipient cell is considered an F+ How ever bacteria do not like to be diploid.
Precipitate DNA
Precipitate DNA using alcohol and Salt combination displaces large DNA from the water. NA+ ions interact with the phosphate ions making them less polar. Alcohols are less Polar they increase the interaction between NA+ and PO3-
Transcription Factors
Proteins that bind to enhancer and promoter regions. There are two types of TF's 1) General- Needed by a RNA polymerase for all genes that transcribe it. (TATA binding protein and TBP) General transcription factors are not needed for all RNA pol.
Is the entire bacterial chromosome transferred from an Hfr to the recipient?
RARELY : It would take 90 minutes for transfer the full bacterial chromosome and the conjugation bridge is normally broken within 15 minutes.
Restriction enzymes that yield blunt ENDS?
RE enzymes that cut blunt ends Can stick any two pieces of DNA together and ligate them. Can be slower and lower yield since its not only relying on DNA sequence to bring pieces together. Ligase is what seals the DNA backbone.
Which Ends sticky or blunt are more useful for microbiology?
RE that cut sticky ends are more useful to molecular biology because two pieces of DNA cut with the same restriction enzyme can be ligated together. Exp) GGATTC
Is the entire bacterial chromosome transferred from the Hfr to the recipient.
Rarely it would take 90 minutes to transfer the bacterial host chromosome and the conjugation bridge is usually broken within 15 to 30 minutes.
Plasmid properties Copy Number
Usually highly regulated: If not regulated the number of plasmid cells would build and burden the cell. Replication of the plasmid may not be able to keep up with the cell division. We can engineer plasmids to be a high copy.
Transducing particle
Viral particle that contains bacterial DNA. Random bacterial host DNA packaged by a virus can be transferred to another recipient.
How can we use Argorose to check for a vector?
We can run an argarose Gel to make sure that the recombinant vector has the GOI. If there recombinant has the GOI then an additional band should show up on the argarose Gel that has the recombinant with the GOI.
How do we know what size our DNA fragments where?
We have a standard or a ladder Mixture of pieces of DNA with a known size. Compare sample to ladder of known sizes.
Inserting genes
We insert genes into a vector. RE that yield sticky ends Cut both the plasmid and target DNA Ligate two pieces of DNA together with ligase Make sure restriction enzyme does not cut into multiple places.
Running the Gel
We switch the electrodes ON Loading buffer does not show DNA itself but how far it has migrated. Remember to turn it off before the dye runs off the bottom of the page.
