Lab 20 Goals and Objectives: Exercise 58: Bacterial Counts on Foods Count colonies: calculate CFU/gram (meat, spinach) or CFU/ml (milk) *Remember, plates of 30-300 only! Share data, get class averages Exercise 59: Bacteriological Examination of Water Determine the MPN (Table 59.1) coliforms /100ml Streak one positive tube for isolation on EMB agar Exercise 60: The Membrane Filter Method Check filters for coliforms: (examples of Escherichia and Enterobacter on EMB provided to demonstrate appearance of coliforms) Count coliforms and compare to MPN from Ex 59 Biotechnology Practice Exercise Each pair get: Two Pipettors, Box of tips, Tube of colored water, Two squares of parafilm: Practice pipetting 5µl water: make a 3 X 3 5µl drop pattern on the parafilm and check that all drops look the same size EDVOKIT#300: Blue/White Cloning of a DNA Fragment Work in five groups total (double up with another pair?) Set up the ligation (tube 1) and ligation control (tube 2) reactions according to directions on pg 11. When done, put in rack to incubate.

Cloning A process of producing genetically modified organisms (Recombinant DNA technology) 1. Isolating and copying the genetic material of interest (DNA fragment ). *2. Building a construct (vector and desired gene) containing all the genetic elements for correct expression -Ligation reaction *3. Inserting the vector into the host organism, directly through injection or transformation. 4. Selecting the cells expressing that gene by growing the host cells in the presence of an antibiotic or chemical that is possible to separate the transgenic events from the non-transgenic 5. Growing successfully transformed organisms and screening for the presence of the new genetic material.

Cloning - Blue-white screening system This is a molecular technique that allows for the detection of successful ligations in vector-based gene cloning. DNA of interest is ligated into a plasmid vector. The MCR ( Multiple Cloning Region) - cloning site (restriction enzyme recognition site) is inserted into the β-galactosidase gene. Cloning the desired gene at that site will destroy β-galactosidase gene. Lac Z (β-galactosidase) gene. Βgal can hydrolyze Xgal substrate into blue product Inducible promoter IPTG to turn on LacZ expression pUC8 Multiple Cloning Region P O LacZ ampicillin resistance gene Cloning restriction site Origin of replication P O Desired gene ~ Lac Z

Digestion with EcoR1

Control Ligation Ligase Ligase Transformation Transformation EcoR1 DNA fragmentEcoR1 EcoR1 Digested with EcoR1 Digested with EcoR1 Ligase Ligase Transformation Transformation

Experiment Overview Ligation Transformation EDVO page 6

Biotechnology Tools Micropipettor Practice - touch tip to surface when dispensing - “blow out” sample and pull away before letting go of plunger Good consistent pipetting! Need more practice!

Set up ligation EDVO page 11