Exp Numbe r of Rxn Cost per Rxn Phylobucks Total Amount Required Phylobucks Remaining Balance Phylobucks Rxn Type Soil Collection Char 1 1 1 3 Soil Water content 1 4 Soil pH determ 2 1 Cultivation 1st 2 2 Cultivation 2nd 2 3 Cultivation 3rd Cryogenic Isolate Freeze Genomic DNA Isolation 3 1 3 2 PCR of Isolates 3 4 DNA Purification DNA Quantification 3 5 3 6 DNA Sequencing 4 1 Microscopic Exam 4 2 Wet Mount 4 3 Gram Stains 4 4 4 5 Antibiotic Production Test Antibiotic Resistance Screen Meta DNA Isolation 5 1 5 2 PCR Gel 5 3 Electrophoresis 5 4 Purf 16S rDNA Quantification of PCR Method A Method B 5 5 5 6 TOPO Cloning Streak Purification 5 7 Plasmid DNA prep Restriction Analysis 5 8 5 9 DNA Sequencing 6 1 Assembly of Cons Seq NCBI BLAST search 6 2 6 3 RDP II search 7 1 RDP II Search 7 2 MEGA4 tree 1 1 1 91 34 40 1 26 17 16 16 16 3 3 3 4 3 8 10 3 12 4 6 3 8 70 0 16 16 16 16 16 16 5 5 5 1 1 1 1 5 3 20 2 5 10 3 5 32 21 10 5 10 5 10 2 20 3 3 3 5 0 0 0 0 0 5 5 5 91 34 40 20 26 85 48 32 80 30 9 15 63 80 50 30 60 128 0 40 12 60 24 210 0 80 0 0 0 0 0 1495 1490 1485 1394 1360 1320 1300 1274 1189 1141 1109 1029 999 990 975 847 784 704 654 624 564 564 524 512 452 428 218 218 138 138 138 138 138 138 Culture Dependent Outline Our strategy is a three tiered approach to culturing potential novel antibiotic producers an antibiotic resistance screen an antibiotic production screen and a competition assay The resistance tier will include assessment of the 16 candidates resistance to naturally occurring antibiotics as well as those that target sexually transmitted bacteria In the second tier a antibiotic production screen will be conducted on all sixteen candidates to assess their efficacy against E coli serving as a UPEC analog M luteus and two E coli mutants Based on the radial size of inhibition found in the production screen the top five candidates will be chosen for the third tier the comeptition assay This will comprise of testing the efficacy of each bacteria in inhibiting the growth of other candidates The purpose for the third tier is to assess the relationships between the top candidates and how they potentially inhibit growth of pathogens Are they resistant to each others antibiotics and fend off pathogens communally Are one or two responsible for killing off a pathogen while also inhibiting the growth of the other candidates Or do we have a Wild West scenario where all can inhibit pathogen growth while at the same time inhibiting each other In experiment 2 1 an extra 42 plates were used to obtain more colonies for future purification 2 3 was repeated 8 times because of insufficient diluting 12 times to overcome filamentous plates and to ensure purity The budget for 4 4 has been significantly expanded because of the foreseen requirement for the accurate characterization of antibiotic producers and for the use of four indicator strains Additionally five plates will be needed for competition assays 4 5 budget allows for three plates of antibiotic resistance screens and 20 phylobucks are allocated for a cryogenic isolate freeze There are no adjustments to the cost or expected costs of experiments 2 2 and the remaining protocol for the isolation of 16S rDNA and its subsequent sequencing CULTIVATION DEPENDENT JUSTIFICATION Culture Independent Alterations from the specified and expected amount occurred in experiments 4 1 4 2 4 3 5 2 5 3 5 4 5 5 5 6 and additional budgetary considerations were allocated for 5 7 and 5 8 However 5 9 6 1 6 2 6 3 7 1 and 7 2 are not expected to deviate from the expected costs 4 1 4 2 and 4 3 were only conducted by three members as one team member was absent from the laboratory 5 4 and 5 5 were conducted four times total because of the use of an improper reagent improper excision of a backup purified PCR product misplacement of the DNA sample all on independent trials with one successful procedure 5 2 had to be repeated after the loss of the backup purified PCR product and subsequently so did 5 3 5 6 was repeated twice because of an unsuccessful transformant result