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Grey-all bacteria
Red- E. coli
Common Complex media
TY (per liter): 6 gr casein (hydrolyzed milk protein)
3 gr yeast extract
.4 gr CaCl2
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
Na2HPO4
KH2PO4
MgSO4
NaCl
NH4Cl
CaCl2
KCl
Carbon source
12.8g
3g
0.5
0.5g
1g
0.015 gr
.185
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. Sam
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
•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).
S. meliloti trpA region
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+ lacIqZ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 lacIQ
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(Strr) 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 Strr 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