BIOB12H3 Lecture Notes - Lecture 3: Absorbance, Turbidity, Exponential Growth

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scarletwallaby454

10 Aug 2017

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Note: you will be using the lb and the lb/agar plates that you made last week for this laboratory. Other factors which are important for growth are oxygen and temperature. The culture can be oxygenated by rapid shaking in an incubator, and for e. coli, the optimal growth temperature is 37 c. Under these conditions, e. coli can reproduce in about 20 minutes. E. coli can grow in the absence of oxygen as it is a facultative anaerobe but it grows optimally in the presence of oxygen. Healthy wild type e. coli have a doubling time (or generation time: the time required to reproduce to double the number of cells) of about 20 minutes in rich medium at 37 c. Strains used for molecular biology have mutations in the genome and will grow slightly slower. You will be monitoring the growth of the molecular biology strain of e. coli called xl1blue at 37 c with aeration.

Related Questions

QUESTION 1: Escherichia coli is characterized as a facultative anaerobe. You set up an experiment to examine the growth of Escherichia coli in three environments: a GasPak jar (0% oxygen), a candle jar (10% oxygen), and the benchtop (22% oxygen). Aside from differing oxygen levels, all other culture conditions (such as temperature and media used) were kept constant. Which of the following represents the most likely outcome of the experiment?

A. E. coli will have heavy growth on the benchtop, moderate growth in the candle jar, and no growth in the GasPak jar.

B. E. coli will have heavy growth on the benchtop but moderate growth in the candle jar and the GasPak jar.

C. E. coli will have heavy growth in the GasPak jar and moderate growth in the candle jar and the benchtop.

D. E. coli should grow equally in all three growth environments.

QUESTION 2: Chloroflexus aurantiacus is a photoautotrophic bacterium that grows in hot springs. Which of the following serves as an energy source for C. aurantiacus?

A. The sun

B. Glucose (C₆H₁₂O₆)

C. C. aurantiacus does not need an energy source

D. Carbon dioxide (CO₂)

QUESTION 3: You are a manufacturer of polypropylene hypodermic syringes. Which of the following methods would be best for ensuring sterility of this heat-sensitive material?

A. Non-ionizing radiation

B. Ionizing radiation

C. Pasteurization

D. Autoclave

QUESTION 4: What is the primary difference between a disinfectant and an antiseptic?

A. Disinfectants are low-level germicides, whereas antiseptics are high-level germicides.

B. A disinfectant is microbiocidal, whereas an antiseptic is microbiostatic.

C. A disinfectant is used to decontaminate critical equipment, whereas an antiseptic is used to decontaminate semicritical equipment.

D. A disinfectant is used to decontaminate lifeless objects, whereas an antiseptic is applied to living tissue.

QUESTION 5: Your patient is receiving a spinal fusion to correct a skeletal defect. Which of the following germicides is recommended to sterilize the bone used for the transplantation?

The presence and growth of bacteria in food products causes a need for a âbest before dateâ of some products (such as milk) so that shoppers will buy the product and consume it before the number of bacteria grows too large and the product goes bad. When milk is stored in a fridge at 4oC, the average growth rate of the bacteria is 0.00019 cells/minute. Assuming an initial bacterial load of 500 cells and a critical threshold of 56570 cells, for how many days can the milk be stored and consumed before it goes bad? 9. At room temperature, the generation time of bacteria in milk is about 10 times shorter than that at 4oC. Given this information, for how many days can the milk be stored at room temperature before it goes bad? 10.Five E. coli cells are introduced in a medium containing 2 carbon sources; glucose and lactose. The generation times with glucose and lactose are 15 and 20 minutes, respectively. Initially, only glucose is used as a carbon source until it is exhausted after one hour of growth. How many cells would you have after 3 hours of growth?

goldbeetle120

HELP ASAP;

Please help me understand my lab results, so be detailed to clearly understand . INclude answers to these questions what role does ampicilin (amp) and arabinose (ara) play in the experiment. Because GFP was isolated from jelly fisht, what modification to the GFP DNA had to be made to result in expression in E. coli?

BACKGROUND INFORMATION

Transformation of GFP and the pGLO Plasmid

GFP is a commonly used reporter protein in research labs, as the fluorescence creates a marker protein that can be used in many types of cell biology and biochemical studies. In basic research, GFP is often fused to a specific target protein of interest, creating a chimeric reporter protein. GFP has been used as a reporter protein to study blood vessel and tumor progression in mice, brain activity in mice, and malaria eradication in mosquitoes for instance (see website mentioned in notes).

In the first week you will use a procedure to genetically transform bacteria with the gene that codes for Green Fluorescent Protein. Following the transformation procedure, the bacteria express their newly acquired jellyfish gene and produce the fluorescent protein, which causes them to glow a brilliant green color under ultraviolet light. During the second week you will purify the protein away from all of the other proteins produced by the bacteria using chromatography, and during the third week you will evaluate the success of the purification procedure and estimate the size (molecular weight) of GFP by using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Fundamental to the process of genetic transformation of bacteria are plasmids. In addition to one large chromosome, bacteria naturally contain one or more small circular pieces of DNA called plasmids. Plasmid DNA usually contains genes for one or more traits that may be beneficial to bacterial survival. In nature, bacteria can transfer plasmids back and forth allowing them to share these beneficial genes. This natural mechanism allows bacteria to adapt to new environments. The occurrence of bacterial resistance to antibiotics is due in large part to the transmission of plasmids which contain antibiotic resistance genes.

The pGLO plasmid (Fig. 5, not a naturally occurring plasmid) contains the gene for GFP, and also contains the gene for the enzyme Î²-lactamase (bla), which provides resistance to the antibiotic ampicillin. The Î²-lactamase protein is produced and secreted by bacteria that contain the plasmid. Î²-Lactamase inactivates the ampicillin present in the LB nutrient agar, allowing bacterial growth. Only transformed bacteria that contain the plasmid and express Î²-lactamase can survive on plates that contain ampicillin. Only a very small percentage of the cells take up the plasmid DNA and are transformed. Untransformed cells cannot grow on the ampicillin selection plates. pGLO also incorporates a special gene regulation system, which can be used to control expression of the fluorescent protein in transformed cells. The gene for GFP can be switched on in transformed cells by adding the sugar arabinose to the cellsâ nutrient medium. Selection for cells that have been transformed with pGLO DNA is accomplished by growth on antibiotic plates. Transformed cells will appear white (wild-type phenotype) on plates not containing arabinose, and fluorescent green when arabinose is included in the nutrient agar medium.

for the first week section of lab

To each of two microfuge tubes, add 250ul of transformation buffer. Keep these tubes on ice.

Using a sterile loop, pick one colony of bacteria from the LB plate.

Transfer this colony to one of the microfuge tubes. Gently vortex the tube until the colony is dispersed. Repeat for the second tube.

Add 0.08ug of DNA to one of these tubes (+DNA tube).

Incubate both tubes on ice for 10 minutes.

Incubate both tubes at 42Â°C for 50 seconds.

Incubate both tubes on ice for 2 minutes.

Add 250ul of LB broth to each tube and incubate at room temperature for 10 minutes.

Flick each tube gently. Add 100ul of this transformation mix to the appropriate plates.

Results:

+DNA: LB/amp--> 1 colony, Glow: uncertain

+DNA: LB/amp/ara--> 9 colonies, Glow: yes

-DNA: LB/amp--> 7 colonies, GLow: no

-DNA: LB--> many colonies, Glow: no

BIOB12H3 Lecture Notes - Lecture 3: Absorbance, Turbidity, Exponential Growth

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