Grey-all bacteria Red- E. coli

Common Complex media TY (per liter): 6 gr casein (hydrolyzed milk protein) 3 gr yeast extract.4 gr CaCl 2 LB (per liter): 10 gr casein 5 gr yeast extract 10 gr NaCl These are cheap, easy to make and support high rates of growth. However, what is in the media, exactly, is unknown. For example, how much methionine is in 10 ml of LB?

M9 minimal medium For one liter Na 2 HPO g KH 2 PO 4 3g MgSO NaCl0.5g NH 4 Cl1g CaCl gr KCl.185 Carbon source 4 gr Amino acids, vitamins, bases, etc. can be added as needed.

Lab WT vs Real WT Sinorhizobium meliloti-”WT” Rm1021 after 30 years found to be a mutant in expR. Sinorhizobium meliloti strain Rm8530. Same as Rm1021, but expR is fixed. It no longer forms single colonies because the goop together due to lot of exopolysaccharide being made.

Phenotype/Genotype S. meliloti trpA region Phenotype: observable properties (Trp +, Trp – ) Genotype: genetic composition (trp -, met + ) Allele: different forms of a gene. Different alleles are given numbers: (trpA1, trpA3) Different genes that give the same general phenotype when mutated are given the same name with different alphabetical designations: (trpA, trpB). Different mutations combine letters and numbers. For e.g. a strain may be (trpA1, trpA2, trpB3, accD). Meaning it has 2 mutations in trpA and one in trpB and accD.

Deciphering a genotype E. coli strain JM107  (lac-proAB) thi gyrA96 endA1 hsdR17(rK – mK + ) relA1 supE44 – /F´ traD36 proAB + lacI q Z  M15  (lac-proAB): deleted for the genes from lac to proAB thi: can’t make thiamine gyrA96: a specific mutation in the gyrase A gene endA1: a specific mutation in the endA (endonuclease) gene relA1: a specific mutation in the relA gene supE44: a specific mutation that suppresses nonsense mutations – : no bacteriophage lambda F´ : has a plasmid called the F plasmid which carries other E. coli genes (that’s what the prime after the the F means). This F’ carries traD26, the proAB genes (fixes the  proAB) and a special lacI gene called lacI Q and a lacZ deletion: Z  M15

Deciphering genotypes continued E. coli strain HB101  (gpt-proA)62 leuB6 supE44 ara-14 galK2 lacY1  (mcrC-mrr) rpsL20(Str r ) xyl-5 mtl-1 recA13 What carbon source(s) can strain HB101not grow on? What amino acid(s) would you need to add to minimal medium to allow growth? Why are leuB62 and ara-14 written as they are? What does Str r mean? Is this the strain’s genotype or phenotype? The NEB catalog, or the GeneticStockCenter at Yale (or similar) is very helpful in deciphering genotypes

NEB strains

NEB E. coli mutations

Penicillin enrichment to help find auxotrophs

Killing by penicillin From Fitzgerald and Williams 1975

Results of penicillin enrichment From Fitzgerald and Williams 1975