Biology 423 L Sept 29 30 Conjugation Lab Hfr Mapping to Determine the Genetic Distance Between Genes in E coli Report due Oct 13 14 Readings Bacterial Genetics Hartwell Chapter 15 pp 543 562 Objectives The experiment you will be doing today involves the mating of two strains of Escherichia coli for the purpose of transferring genetic markers from one strain the donor or male to a second strain the recipient or female This process requires close cell cell contact and involves the physical transfer of DNA from the donor strain to the recipient strain Therefore the two strains must be incubated together for conjugative DNA transfer to occur With the discovery of bacterial conjugation methods for genetic mapping of the E coli genome developed quickly A surprising result that arose from these efforts was the discovery that the E coli chromosome was circular Many years passed before the bacterial chromosome was actually observed in the electron microscope confirming its circular topology Introduction The donor strain for today s experiment contains the F plasmid integrated into the chromosome to form a high frequency recombination or Hfr strain The Hfr being used is called HfrH and the F plasmid is integrated at about 98 minutes on the E coli genetic map The integration of the F plasmid into the bacterial chromosome allows the transfer of chromosomal genes from the donor strain to the recipient strain Notice that the donor strain is sensitive to the antibiotic streptomycin This will be important in the design of today s experiment The recipient cell strain E coli AB1157 is resistant to streptomycin and contains many additional mutations some of which are relevant to today s experiment In particular notice that the recipient strain requires both leucine and proline in the media in order to grow since it contains mutations in the biosynthetic pathways for both of these amino acids The strain also requires several other amino acids that will be provided in the media Each of these genes could also be used to select for recombinants provided they are transferred before the gene encoding streptomycin sensitivity resistance The goal of today s experiment is to determine the time of entry of each of two genes on the E coli chromosome This is possible since an Hfr strain transfers genes linearly from a fixed point of origin Thus the experiment will provide information regarding the location of each gene relative to the origin of entry for the HfrH strain and relative to each other Experiments like the one you are doing today were used to map the entire E coli chromosome where as a result of this approach to mapping the genome the locations of genes are described in terms of minutes on the 100 minute E coli map Each lab group will do a single mating experiment and determine the frequency of recombinants for each of two genes as a function of the time the donor and recipient cells are in contact prior to interruption of the mating pair The donor strain will donate the leu gene and the pro gene to allow a recipient which acquires one or both of these genes by F mediated transfer to grow on an appropriate selective plate Be sure to carefully label your plates You should see different results depending on whether you are selecting for 1 leu recombinants or pro recombinants Materials Minimal A Medium 1X Mix 15 g agar in 885 ml H2O and autoclave Autoclave separately and add 100 ml of 10X minimal salts For 100 ml 10X salts K2HPO4 KH2PO4 NH4 2SO4 sodium citrate H20 Autoclave 10 5 g 4 5 g 1g 0 5 g After autoclaving agar and salts mix and add the following All stocks must be sterile Vitamins and amino acids must be filter sterilized 1 ml of 1 M MgSO4 7H20 10 ml of 20 glucose 0 5 ml of 1 Vitamin B1 thiamine 4 ml of 10 mg ml amino acid stock Histidine Arginine Threonine and Proline or Leucine as appropriate Streptomycin to 50 g ml from a 50 mg ml stock filter sterilized The agar and minimal salt solution must be autoclaved separately and then combined to pour plates It is generally convenient to make the salt solution in a more concentrated form e g 10X to add to an appropriate volume of a solution containing the agar L plates will select for leu recombinants and will lack leucine but will contain arginine proline histidine and threonine as well as streptomycin to counter select the donor strain P plates will select for pro recombinants and will lack proline but will contain arginine leucine histidine and threonine as well as streptomycin to counter select the donor strain 2X YT media per liter 16g tryptone 10g yeast extract 5 g NaCl mix and autoclave liquid media only Sterile saline Per liter NaCl 9g Mix and autoclave LB plates Per liter Tryptone 10 g Yeast extract 5 g 2 NaCl 5g Bactoagar 15 g Mix first three add agar and autoclave Cool to 60 degrees in a water bath before pouring plates Donor Strain relevant genotype phenotype HfrH lacI22 serA6 spoT1 thi 1 Strs E coli JC158 Recipient Strain E coli AB1157 F thr 1 leuB6 thi 1 lacY1 galK2 xyl5 mtl 1 proA2 his4 argE3 tsx 33 supE44 kdgK51 Strr Materials required per lab group Sterile 250 ml Erlenmeyer flask containing 45 ml 2X YT media 1 per group Empty Sterile 125 ml Erlenmeyer flask 1 per group 13 X 100 sterile test tubes 22 sterile 1 5 ml eppendorf tubes in beakers 1 per group LB plates 8 per group L lacking leucine selective plates 24 P lacking proline selective plates 24 Pipettemen P200 and P1000 Sterile Blue tips Sterile Yellow tips Plastic cuvettes 4 per group Spectrophotometer warmed up Sterile 10 ml pipets Pipette pumps for 10 ml pipettes Vortex mixer Sterile spreader Alcohol lamp Sterile Saline 25 ml per group in Falcon tubes Waste container 2 test tube racks Water bath at 37 degrees Timer Method Day 0 TA will set up a 5 ml overnight culture of AB1157 JC158 in 2X YT media and shake overnight at 37 degrees Day 1 Work in groups of 2 Note for TA In the morning the TA will subculture the overnight cultures of the donor strain and the recipient strain For each strain a 125 ml sterile flask containing 25 ml of 2XYT media will be inoculated with 0 5 ml of the overnight culture Set up a separate flask 3 of each strain for each group in the shaking water bath They will grow in a shaking water bath at 37oC to an OD600 0 5 1 0 approximately 2 4 x 108 cells ml This should take 1 5 hours TA Start 45 minutes before the lab period begins Students Before the lab period read the protocol carefully Make yourself a list of all the saline tubes you will need They