1. Introduction to Microbiology

2. Gene Expression: Review
Gene Expression is a process by which a gene’s DNA sequence is turned into functional protein
Marked by the production of mRNA in a cell
All cells contain the same genome, but not all genes are transcribed & translated
Specific cues to express genes include: temperature changes, nutrients in the external environment, hormones, or other complex signals
Gene Expression: Review

3. Gene regulation is a process by which genes are controlled (turned on and off) in response to signals
Cells regulate the genes that are expressed
Prokaryotic & eukaryotic cells regulate a bit differently
Transcriptional regulation (common to both cells) refers to controlling the amount of mRNA transcribed.
Gene Control

4. Transcriptional Regulation
Once the amount of mRNA is controlled, the amount of protein is indirectly controlled
RNA polymerase binds at the promoter (found “upstream” of gene sequences), which indicates the beginning site for transcription
Eukaryotic promoters commonly have a CAAT box (GGCCAATCT) about 80 nucleotides upstream of the start site of gene and a TATA box (TATAAAA) about 30 nucleotides upstream of start site of gene
Transcriptional Regulation

5. Transcription Factors
Transcription factors are DNA binding proteins that regulate the binding of RNA polymerase to stimulate transcription of a gene.
Bind to the TATA & CAAT boxes to stimulate transcription
Bind upstream to either enhance or repress transcription of a gene by assisting or blocking RNA polymerase binding
Transcription Factors

6. Enhancers Many regulated genes contain enhancers
They are regulatory DNA sequences, about 50 or more base pairs
They are found upstream (occasionally downstream), and can be bound by regulatory proteins
Can be located far, but a loop in DNA causes contact
The regulatory proteins are called activators
Enhancers

7. Activators are DNA-binding proteins that regulate one or more genes by increasing the rate of transcription
They interact with transcription factors and RNA polymerase to activate or enhance transcription of a gene
Bind to particular enhancers
Examples include certain hormones
Activators

8. A repressor is a DNA-binding protein that regulates the expression of one or more genes by decreasing the rate of transcription
Repressors bind within the promoter, preventing RNA polymerase from binding
Identifying promoters, enhancer sequences, and transcription factors is important for making biotech products!
Repressors

9. Learning Check What is the mark of gene expression?
What cues may turn genes on or off?
Define promoter and name 2 regions of eukaryotic promoters.
Define transcription factor.
Define enhancer.
Define activator.
Define repressor.
Learning Check

10. Prokaryotic Regulation
Studying bacterial regulation of genes is very important for many biotechnology applications.
Many bacterial genes are organized in arrangements called operons.
Prokaryotic Regulation

11. Prokaryotic Regulation
Operons are clusters of several related genes that are located together and controlled by a single promoter.
Many genes controlling nutrient metabolism by bacteria are organized as operons.
Prokaryotic Regulation

12. Bacteria, although only unicellular, have the ability to turn the expression of certain genes on or off in response to certain stimuli
Serratia marcescens is a bacterium that exhibits temperature dependant gene regulation of pigment production
Serratia marcescens

13. Culturing Prokaryotes
Bacteria can be easily grown on Petri dishes with nutrient agar (growth media)
Agar is a polysaccharide obtained from cell walls of some protist species (namely algae or seaweed)
Each bacterial cell divides to form circular-shaped colonies containing thousands or millions of cells
Culturing Prokaryotes

14. Culture medium –a solution that contains nutrients to support growth of microorganisms
Liquid – broth
Semisolid -- w/solidifying agent such as agar < 1%
Solid – w/solidifying agent such as agar 1.5% or greater
Agar – seaweed extract
-- Agar turns solid at 40°C and turns liquid at 100°C
Growing Bacteria

15. Types of Culture Media

16. Culturing Prokaryotes
Be sure to always label the bottom of the petri dish that you are working with
Also, make sure you store any agar plates UPSIDE DOWN to prevent any condensation from dripping back onto the plate thus inhibiting growth!
There are a few methods of plating we may use for culturing so follow lab protocol.
Culturing Prokaryotes

17. Labeling & Transfer Transfer Instruments – Needle Loop Pipette
Labeling – all plates and tubes must be labeled with your group name, date, name of media, organism being grown
Labeling & Transfer

18. Serratia marcescens Room Temperature (20-25°C) = prodigiosin
Prodigiosin: red surface pigment
Warmer temperatures (above 37°C and higher) = no pigment production
Bacteria appear white
Serratia marcescens

19. In addition to giving bacteria a red pigmentation, prodigiosin has been shown to exhibit some antibiotic activity against
Bacteria
Trypanosomes
Protozoa
fungi
Why would it be beneficial for a bacteraum to produce an antibiotic?
Prodigiosin

20. Is of interest to humans due to its pharmacological applications in treatment of infectious disease
Prodigiosin is insoluble in water
May play a role in dispersion of S. marcescens allowing it to reach potentially more favorable environments
Prodigiosin

21. Microbiology Laboratory Technique
Bacteria grow and divide rapidly
As bacteria multiply, the resulting mass of cells becomes visible
On a solid media, they look like a layer of slime
Each individual cell in the mass is a discrete organism capable of survival and reproduction without the others
Microbiology Laboratory Technique

22. Microbiology Laboratory Technique
The transfer of bacteria from stock culture to fresh medium is called innoculation
Cells are picked up from stock culture on a sterile innoculating instrument called a loop, and then deposited in either liquid broth or along the surface of an agar medium plate
The transferred cells are called the innoculum
Cells become visible within 16 hours
Microbiology Laboratory Technique

23. When performing microbiology laboratory procedures it is important to protect the media, stock cultures, and instruments you are using from contamination by microorganisms present in the environment
Asceptic Technique is employed to accomplish this
Asceptic Technique

24. Asceptic Technique 1st Always have long hair tied back
Wear closed toed shoes
Use the ethanol to wipe down all work areas before and after lab work
Wear gloves when working with bacteria
ALWAYS thoroughly wash hands when finished working with microorganisms!
Asceptic Technique

25. Asceptic Technique (sterile) uses sterilized equipment and specific procedures to prevent contamination of the equipment by contact with non-sterile objects
Since microorganisms naturally exist all around us, it is important to limit exposure of cultures and petrie plates to the air
Keep the lid on petri plates at all times except when you are ready to use them
Asceptic Technique

26. When innoculating, lift the lids off the plates just enough for you to work, and then replace them immediately when you are done
Use only sterile innoculating instruments to transfer bacteria
Once you have removed a sterile innoculating stick from its package, do not touch it or let it touch a nonsterile surface
Asceptic Technique

27. ALL inoculating sticks must be placed in appropriately labeled biohazard containers
DO NOT THROW ANYTHING IN THE NORMAL GARBAGE CANS!!!!! Items may only be placed in things marked biohazard
Disposal