Molecular Biology 2

bla gene

(beta-lactamase enzyme gives resistance to ampicillin) confers resistance to ampicillin, allowing transformed cell and its progeny to survive on media containing ampicillin

GFP gene

(green fluorescent protein) taken from jellyfish, it encodes for a green fluorescent protein, the gene of interest in this simulation

ori site

(origin) location for DNA polymerase to start replication of the plasmid

araC gene

(regulates GFP production) regulates transcription of the GFP gene, which is active in the presence of arabinose

Match the inoculated agar plates with their expected growth after incubation -pGLO on LB: -pGLO on LB/amp: +pGLO on LB/amp: +pGLO on LB/amp/ara:

-pGLO on LB: Very dense white bacterial colonies -pGLO on LB/amp: No growth +pGLO on LB/amp: White bacterial colonies +pGLO on LB/amp/ara: Green fluorescent bacterial colonies

The transformation process requires

-plasmid DNA to pass through the cell membrane. -this is achieved by making the cells competent

recombinant plasmid

-plasmid that has had a particular gene inserted into it using restriction enzymes -could then be transformed into a bacterial cell changing the cell's genotype to express the foreign gene product.

restriction enzymes (restriction endonucleases)

-produced by bacteria to cut the DNA of viruses that infect them. -We can use these natural enzymes in the lab to cut DNA samples to create DNA profiles. -only cut DNA at a specific site or sequence of bases. Because of this, any variations in the DNA sequence, say from mutations, will result in a different cutting pattern, resulting in different sizes of DNA fragments

Plasmid

-self-replicating extrachromosomal DNA molecule found in some bacterial cells (circular DNA naturally found in some bacteria) -They can be engineered to carry genes from one organism into a bacterial cell. -give bacteria a competitive advantage

DNA profiling (fingerprinting)

-uses restriction digestion and gel electrophoresis to identify individuals or samples based on differences in their DNA sequences. -DNA is cut with enzymes that recognize specific sequences and then the fragments are separated on a gel to reveal a specific pattern for an individual. -It works best with highly polymorphic sequences - Any source of DNA (i.e., bacteria, a hair, a speck of blood, or semen) can be used

How long does the PCR electrophoresis test run for?

1 hour at 70 V

What was added to make the master mix?

1 μL Taq polymerase 3 μL Primer 1 3 μL Primer 2 6 μL dNTP 30 μL taq buffer 57 μL water

What is needed for PCR

1) Template DNA that contains the target sequence 2) Primers: short oligonucleotides that define the ends of the target sequence 3) A heat stable polymerase: Taq polymerase 4) Buffer 5.) Nucleotides

The PCR cycle has 3 steps:

1. Denaturation 2. Anneling 3. Elongation

What is the importance of creating a master mix?

1. Increase efficiency 2. Ensures a high degree of consistency

What are the main purposes for adding a loading dye to the samples before they are loaded into the gel?

1. It contains a colored indicator, allowing migration of the samples to be monitored during electrophoresis 2. It contains glycerol , which allows the samples to sink into the wells.

DNA profiling has three key steps:

1. Prepare a restriction enzyme digest of the DNA samples. 2. Use an electrophoresis gel and chamber to separate the DNA fragments by size. 3. Use a staining process to visualize and then analyze the DNA fragments in the gel.

2 practical applications of gene cloning

1. Protein harvested (ex. human growth hormone, insulin** typically not produced by bacteria but bc it has that gene it will produce it ) 2. Copies of a gene (ex. genes for pest resistance inserted into plants, or gene used to alter bacteria for cleaning up toxic waste)

Which of the following explain the difference in gel results for the outbreak strain in patient D?

1. The bacterial DNA sequence is different from the DNA sequence of the outbreak strain. 2. The outbreak strain had more restriction digest sites than the bacterial DNA in patient D. NOT CORRECT: the rest

Gene cloning steps

1. gene inserted into plasmid 2. plasmid put into bacterial cell 3. host cell grown in culture to form a clone of cells containing the cloned gene of interest 4. the proteins are harvested and used for research

pGLO plasmid sites

1. ori site 2. bla gene 3. GFP gene 4. araC gene

How long was the gel electrophoresis run for in experiment 2

1.5-- 2 hours at 70 V

How long did we let the LB broth stand once added to the -pGLO and +pGLO tubes

10 min

How much E.coli and pGlO did we add

10 μL

As an epidemiologist, you are collecting statistics on the number of patients that are part of the current Salmonella outbreak. What percentage of these tested patients are positive for the outbreak strain?

20%

Using the DNA ladder provided, estimate the size of the largest DNA fragment in lane 4.

2322 base pairs I DON'T UNDERSTAND GO THROUGH THIS ONE (email her about this one)

How long did we incubate the bacteria plates in exp. 1

24 hours

How long were the tubes in exp. 2 incubated for?

90 minutes in 37 degree C water bath

Taq polymerase

A DNA polymerase enzyme adapted to working at high temperatures without denaturing.----builds the new DNA molecules from free deoxynucleotides added to the reaction (The process is carried out at high temperatures so a heat stable polymerase is used, generally taken from thermophilic (heat loving/heat thriving) bacteria.)

Electrophoresis chamber

A buffer-filled box where a separation gel is placed and a current is applied to separate DNA fragments by size.

10x Taq buffer

A concentrated solution used to maintain conditions suitable for the activity of the Taq DNA polymerase.

LB broth

A general growth medium used to grow a wide variety of bacteria.

Ethidium bromide

A molecule that binds to DNA bases and fluoresces under UV illumination to visually detect the location of DNA bands in the electrophoresis gel.

pGLO plasmid

A plasmid is a self-replicating extrachromosomal DNA molecule found in some bacterial cells. The pGLO plasmid contains the ori site, bla gene, GFP gene, and the araC gene.

Positive control in PCR experiment

A pure culture of Neisseria meningitidis

SYBR Green I

A safer alternative to Ethidium bromide used to visually detect the location of DNA bands in the electrophoresis gel.

Loading dye

A solution added to an electrophoresis sample to give it color and density.

Loading/Indicator dye

A solution added to an electrophoresis sample to give it color and density.

DNA ladder

A solution of DNA fragments of known size used to compare unknown DNA to estimate fragment size. (shown in number of base pairs)

LB/amp agar

Also contains the antibiotic ampicillin so only transformed, resistant bacteria will grow.

This segment of DNA is cut at restriction sites 1 and 2, which creates restriction fragments A, B, and C. Which of the following electrophoretic gels represents the separation of these fragments? See image

Answer: A, because the A strand is the smallest fragment and therefore will move the farthest, C is the second smallest fragment, and B is the longest fragment

What must happen before PCR?

Before amplification with PCR, DNA must first be isolated from the cells in a patient or environmental sample. This involves breaking down cell membrane structures to release the DNA from the cell. The extraction process uses enzymes to digest these structures. The sample is centrifuged to collect all the cell debris at the bottom of the tube, leaving the DNA suspended in the fluid above. In this simulation, the needed DNA from patient samples and bacterial cells from the positive control are provided.

What is the problem with plating the cells on a nonselective medium after a transformation procedure?

Both types of cells will grow, making it impossible to tell which colonies contain transformed cells (With a nonselective medium, differentiating transformed and non-transformed cells is not possible)

Transformed cell

Cell that has taken up and incorporated genetic material from outside the cell.

Competent cells

Cells treated to allow DNA to pass through the cellular membrane leading to transformation.

Master mix

Contains Taq polymerase, primers, deoxynucleotides, and polymerase buffer solution.

LB/amp/ara agar

Contains arabinose sugar as well as ampicillin so only transformed cells will grow and will express the GFP gene and fluoresce.

Annealing

Cool to allow primers to form hydrogen bonds with ends of target sequence.

dNTP mix

Deoxynucleotides needed for the PCR process.

Micropipette

Device used to precisely transfer small volumes of liquid.

What bacteria were we transforming in experiment 1 and what was the product of this transformation?

E.coli , the product was Green floruescnet protein

What was the restriction enzyme used in exp. 2?

EcoRI/Pstl enzyme mixture

DNA Polymerase

Enzyme that reproduces DNA strands.

ara operon

Genes normally regulated by the araC operon ("B" "A" and D) have been replaced with the gene for GFP.

What does the master mix solution contain?

Heat stable polymerase Free deoxynucleotides

Denaturation

Heating to 95°C is used to break the hydrogen bonds between nitrogenous bases and separate the strands of DNA.

What is the purpose of the LB/amp agar plate?

It is a selective medium containing an antibiotic to inhibit the growth of non-resistant cells.

What is the purpose of the LB/amp/ara agar plate?

It is a selective medium with an antibiotic which prevents growth of non-resistant cells and a sugar that allows for the expression of a plasmid gene.

Colony

Microbiology term used to describe a visible mass of bacteria growing together on an agar plate or slant. Colonies are derived from a single microorganism.

Horizontal transfer

Movement of genetic material between independent cells. Passing one or more genes through routes OTHER THAN parent---to--offspring

Thermocycler

PCR machine that heats and cools nucleic acid samples that facilitates replication to amplify the amount of nucleic acid sample.

Microcentrifuge

Small tabletop centrifuge that uses centripetal force to separate substances by density.

Microcentrifuge tube

Small tube with a lid holding less than 2 mL used in a microcentrifuge.

What would be the result of running the gel too long?

The DNA sample will move through the entire gel and exit at the bottom.

Following PCR, the products were loaded into a gel and electrophoresis was performed. Following visualization of the gel, no band of DNA was observed. What explains this result?

The desired sequence was not successfully amplified.

What does the EcoRi/PstI enzyme mixture do?

The enzyme mixture cuts DNA at specific locations, creating various-sized fragments.

What is the negative control for the experiment 1

The other tube does not have any plasmid (Negative - pGLO) and acts as a negative control.

DNA amplification

The production of multiple copies of a sequence of DNA

Extension

The sample is warmed back up to 72°C and DNA polymerase adds nucleotides to the 3 end of each primer.

What was the DNA samples from exp. 2 inside?

The samples from each patient were in a restriction enzyme buffer solution

How many times is PCR repeated?

These steps are repeated over and over till the desired amount of DNA is synthesized.

Once the restriction enzyme was placed into the DNA microcentrifuge tubes, then what was done?

They were placed into the centrifuge and centrifuged for 1 min

When can PCR be used?

This exponential copying of DNA samples is useful when there is little DNA to use for other means of detection or analysis. Examples include detection of infectious agents from patient samples, analyzing crime scene DNA for forensic purposes and for identifying remains of victims.

LB (Luria-Bertani) agar

This is a nonselective medium so all cells will grow.

What is the purpose of the LB agar plate?

This is a nonselective medium so all cells will grow.

What is the purpose of changing micropipette tips after each step?

To prevent cross contamination of the samples.

What steps were taken to increase the efficiency of the transformation process?

Treatment with calcium chloride Brief heat shock

How did we do heat shock treatment in the first experiment?

Tubes placed in ice bath for 10 min Move to 42 degree C for 50 seconds Move to ice bath for 2 min

What are cells plated on instead?

a selective medium (only allows transformed cells to grow) -Often, a plate containing an antibiotic is used, and the plasmid contains a gene that confers resistance to the antibiotic, allowing the transformed cell to grow and killing off any cells that are not transformed with the plasmid.

Bacteria reproduction

asexually, by simple division, so they sometimes rely on acquiring genetic material from other bacterial cells to increase genetic diversity using the process called horizontal gene transfer.

What loading dye was added in experiment 2

bromophenol blue (to help visualize where the samples are located in the gel) and glycerol

polymorphic sequences

differ a lot in their nucleotides

operon

group of genes operating together

Why was glycerol added to each sample in exp. 2

help samples sink to the bottom of the well and not float on top on the buffer

Heat shock treatment

increases the permeability of the cell membrane (Cells are kept on ice and then suddenly moved to a warm water bath for less than a minute and then back to ice)

What type of gene did we use in experiment 1

jellyfish gene whose product is green fluorescent protein

transformation

natural process, but it can be used in the laboratory to insert medically useful genes into bacterial cells that then produce a specific protein, like insulin or human growth hormone. The gene of interest is typically introduced into the cell as part of a plasmid.

Cold calcium chloride

neutralizes the negatively charged DNA (allow it to pass more easily through the nonpolar cell membrane)

Negative control in PCR experiment

plain water in place of a DNA sample

Is transformation common or rare?

relatively rare phenomenon. -After cells are subjected to a transformation procedure, not all cells will have the desired plasmid. -Some cells will be transformed, but most will not. It becomes necessary to identify the transformed cells.

Primer

short DNA fragments with a known sequence used to direct the polymerase to specific areas on the template DNA strand that flank the target DNA sequence.

https://www.youtube.com/watch?v=DbR9xMXuK7c

super helpful vid

To acquire enough DNA for analysis, what technique is used

the PCR technique is used. To start, a template DNA strand is needed

Why was the DNA and restriction enzyme centrifuged?

to pool these two small volumes

arabinose

turns the acaC operon on, turning on expression of the GFP gene