The Effect of Temperature on Growth Exercise 13 *TURN ON INCINERATORS*

The Effect of Temperature of Growth Many species of bacteria are evolved to live in every kind of environment Sensitive to all kinds of environmental factors Temperature, Chemical Levels, Oxygen Levels, pH, Nutrient Levels, ect. Temperature is most commonly linked to enzyme activity and structure

Bacterial species are categorized by their optimal growth temperatures into three broad categories: psychrophiles (“cold-lovers”) mesophiles (“middle-lovers”) thermophiles (“heat-lovers”)

Enzymes Special types of proteins that are responsible for a lot of work that goes on inside the cells Catalysts: speed up chemical reactions Very specific- only reacts with the specific type of substance that it was made for “Key and Lock” Substrate binds to Enzymes at the active site creating an enzyme/substrate complex

Enzymes Optimal temperature: temperature at which the enzyme functions at its highest rate If exposed to a temperature higher then optimal, the structure of the enzyme may be altered If the structure of the enzyme is altered, it can no longer attach to the substrate. Permanent alteration to the enzyme and loss of functionality is called denaturation. Thermoliable: unable to withstand slight increases in temperature above their optimal without denaturation Thermostable: can tolerate moderate temperature increases without permanent damage

Enzymes Temperatures below optimal slow down enzymatic activity Extremely low temps can preserve bacteria, slowing down their metabolism until temperatures reach optimum again Important application to food preparation and whether or not a food needs to be frozen or heated to reduce the health threat to consumers

Psychrophiles Bacterial species that will grow in a range from -5oC up to 20oC. These organisms include those living in cold water, soils, and in the refrigerator. (Most psychrophiles grow well at 0oC to 5oC, which is the setting for most refrigerators.)

Mesophiles Bacterial species which grow within the range from just below 20oC up to around 45oC. These grow well within the temperatures at which humans and other living things live, and at which many food are held for consumption. Due to their importance as plant and animal pathogens, they are further subdivided based on what part of the range constitutes their optimum: Room-temperature mesophiles: Growing from 20oC to 35oC, these are plant pathogens, as well as saprophytes which help with the decomposition of plant and animal bodies. Body temperature mesophiles: Found on warm-blood animals as mutualistic normal flora or pathogens, they prefer temperatures from 35oC to 45oC.

Thermophiles Bacterial species which have very thermostable enzymes flourish in hot environments, such as hot water heaters (e.g. Legionella pneumophilia) or warm springs and geysers such as “Old Faithful”. These organisms are found growing at 45oC or higher, even to above boiling temperatures at the high-pressure deep-sea geothermal cleft regions.

Thermal classifications of bacteria

Today’s Exercise Work in groups of 4 Cultures 1 TSA plate/student Cultures Geobacillus stearothermophilus – Thermophile Escherichia coli – BT mesophile Pseudomonas fragi: psychrophile Serratia marcescens: RT mesophile; develops a red pigment at room temp Each student picks a temperature 5°, 25°, 37°, 55°

Next Lab Period: Retrieve the plate and rate the amount of growth of each organism for all four plates in the data chart under “amt”, using the scale below: = no growth + scant growth ++ moderate growth +++ abundant growth ++++ extremely large amount of growth

Transformation in Bacteria Exercise 14

Background Transformation: Insertion of DNA into a bacterial cell Along with the bacterial singular, circular chromosome, bacteria can also carry other small DNA molecules called plasmids Contain genes that are useful to a bacteria’s survival Can include genes for certain proteins and antibiotic resistance

Plasmid DNA usually contains genes for more than one trait that are helpful to the bacteria’s 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.

In this laboratory, we will transform E In this laboratory, we will transform E. coli with a gene that codes for Green Fluorescent Protein (GFP). This gene is from the bioluminescent jellyfish Aequorea victoria. GFP causes the jellyfish to fluoresce and glow in the dark. The pGLO plasmid has been genetically engineered to carry the GFP gene which codes for the green fluorescent protein, GFP, and a gene (bla) that codes for a protein that gives the bacteria resistance to an antibiotic.

pGLO Plasmid

Today’s Exercise- Supplies 2 tubes of CaCl2 Label pGLO+ and pGLO= 2 LB agar plates 2 LB/AMP agar plates 2 LB/AMP/ARA 2 LB Broth Escherichia coli starter plate (stays at front desk)

Today’s Exercise Step 1 Using a sterile pipet, transfer 250 μL of CaCL2 into each tube. (already done for you) CaCL2 punches holes in the cell membrane, allowing the bacteria to take up plasmids 250μL CaCl2 250μL CaCl2 pGlo + pGlo =

Today’s Exercise Step 2 Using a sterile loop, pick a colony from the E. coli starter plate Immerse the loop in the solution in the pGLO+ tube and spin the loop to mix the cells into the solution. Repeat the same for the pGLO= tube pGlo + pGlo =

Today’s Exercise Step 3 Pipet 45 μL of your pGLO plasmid into your pGLO+ tube. (One student from each group will come up and I will pipet for you) Gently tap your tube to mix the plasmid into the solution. No plasmid needs to be added to pGLO= tube. pGLO plasmid pGlo = pGlo +

Today’s Exercise Step 4 Incubate the tubes on ice for 10 mins Make sure the tubes are in contact with the ice. Label your plates during this time LB pGLO+ LB/AMP pGLO+ LB/AMP/ARA pGLO+ LB pGLO= LB/AMP pGLO= LB/AMP/ARA pGLO= Today’s Exercise

Today’s Exercise Step 5 Step 6 Take both tubes and place them in the heat block (42°C) for EXACTLY 50 seconds. This will heat-shock your cells. Heat-shock will make the cell wall more permeable, allowing the cell to take up the plasmid Step 6 After the 50 sec heat shock, place the tubes IMMEDIATELY back into the ice. Incubate in the ice for 2 mins

Today’s Exercise Step 7 Step 8 Remove the tubes from the ice, add 250μL of LB broth to each tube. The LB heals the cell after the CaCl2 and heat shock by providing an influx of nutrients Incubate for 10 mins at room temperature Step 8 Tap the tubes gently to mix Using a sterile pipette, transfer 100 μL (0.1 mL) to the appropriate plates Swirl your plate gently to spread the solution on the plate. Place your plates in the class bucket for incubation Next week we will view our plates under the UV light

Lab Procedure: 12. Using a sterile loop for each plate, spread the suspensions evenly around the surface of the entire agar for confluent growth. 13. Place your plates in your classes bucket for incubation.

Lab Procedure:

Results:

Results: Antibiotics usually kill bacteria (are bacteriocidic) or inhibit their growth (bacteriostatic). Thus, there should be few, if any, bacterial colonies present on the ampicillin plate. The presence of any colonies on the ampicillin plate would suggest that those bacteria are resistant to the antibiotic ampicillin.

Results: There should be multiple colonies on both the LB/amp and LB/amp/ara plates that received the pGLO plasmid (approximately ~ 75 colonies). There should be no growth on the LB/amp (-) pGLO plate. There should be a lawn of bacteria on the LB (-) pGLO plate. The bacteria on the (+) pGLO LB/amp plate and the (-) pGLO LB plates should be whitish. The bacteria on the (+) pGLO LB/amp/ara plate should appear whitish when exposed to normal, room lighting, but fluoresce bright green upon exposureto the long-wave UV light.

Results: The sugar arabinose in the agarose plate is needed to turn on the expression of the GFP gene. The UV light is necessary to cause the GFP protein within the bacteria to fluoresce.

Results: The sugar arabinose turns on expression of the GFP gene by binding to a regulatory protein, araC, which sits on the PBAD promoter. When arabinose is present, it binds to araC, consequently changing the conformation of araC which facilitates transcription of the gene by RNA polymerase . When exposed to UV light, the electrons in GFP’s chromophere are excited to a higher energy state. When they drop down to a lower energy state they emit a longer wavelength of visible fluorescent green light at 509 nm.