Selected Cell
Cell:
Value:
amino acid scheme
other media components
Pyogenes exp 1
pyogenes exp2
pyogenes exp3
combined exps final OD
ser_gly validation
filipe media validation
comparison others
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| Medium preparation (all separate media are prepared using the same stock solutions) | ||||||||||||||||||||||||||||||||
| CDM | CDM1 | CDM2 | CDM3 | CDM4 | CDM5 | CDM6 | CDM7 | CDM8 | CDM9 | CDM10 | CDM11 | CDM12 | CDM13 | CDM14 | CDM15 | CDM16 | CDM17 | CDM18 | CDM19 | CDM20 | CDM21 | CDM22 | CDM23 | CDM24 | CDM25 | CDM26 | CDM27 | total amount | ||||
| all amino acids | ala | arg | asn | asp | cys | cyn | glu | gln | gly | his | ile | leu | lys | met | phe | pro | ser | thr | try | tyr | val | cys/cyn | gln/glu | gly/ser | asn/asp | cys/cyn/ser | no AA | Sums | ||||
| Basal Solution 4* | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | Basal Solution 2* | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | Basal Solution 2* | 25 | 25 | 25 | 25 | 25 | 25 | 700 | |
| AGU mix 50* | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | AGU mix 50* | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | AGU mix 50* | 2 | 2 | 2 | 2 | 2 | 2 | 56 | |
| hydroxide (xanth HCO3) | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | hydroxide (xanth HCO3) | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | hydroxide (xanth HCO3) | 5 | 5 | 5 | 5 | 5 | 5 | 140 | |
| 100*vitamin solution | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 100*vitamin solution | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 100*vitamin solution | 1 | 1 | 1 | 1 | 1 | 1 | 28 | |
| 100*metal solution | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 100*metal solution | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 100*metal solution | 1 | 1 | 1 | 1 | 1 | 1 | 28 | |
| alanine*100 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | alanine*100 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | alanine*100 | 1 | 1 | 1 | 1 | 1 | 0 | 26 | |
| arginine*100 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | arginine*100 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | arginine*100 | 1 | 1 | 1 | 1 | 1 | 0 | 26 | |
| asparagine*100 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | asparagine*100 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | asparagine*100 | 1 | 1 | 1 | 0 | 1 | 0 | 25 | |
| aspartic acid*10 | 10 | 10 | 10 | 10 | 0 | 10 | 10 | 10 | 10 | 10 | 10 | aspartic acid*10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | aspartic acid*10 | 10 | 10 | 10 | 0 | 10 | 0 | 250 | |
| cysteïne-HCl*100 | 1 | 1 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | cysteïne-HCl*100 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | cysteïne-HCl*100 | 0 | 1 | 1 | 1 | 0 | 0 | 24 | |
| cystine*40 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 0 | 2.5 | 2.5 | 2.5 | 2.5 | cystine*40 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | cystine*40 | 0 | 2.5 | 2.5 | 2.5 | 0 | 0 | 60 | |
| glutamate*10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 0 | 10 | 10 | 10 | glutamate*10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | glutamate*10 | 10 | 0 | 10 | 10 | 10 | 0 | 250 | |
| glutamine*100 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 1 | 1 | glutamine*100 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | glutamine*100 | 1 | 0 | 1 | 1 | 1 | 0 | 25 | |
| glycine*100 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 1 | glycine*100 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | glycine*100 | 1 | 1 | 0 | 1 | 1 | 0 | 25 | |
| histidine*100 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | histidine*100 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | histidine*100 | 1 | 1 | 1 | 1 | 1 | 0 | 26 | |
| isoleucine*100 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | isoleucine*100 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | isoleucine*100 | 1 | 1 | 1 | 1 | 1 | 0 | 26 | |
| leucine*25 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | leucine*25 | 4 | 0 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | leucine*25 | 4 | 4 | 4 | 4 | 4 | 0 | 104 | |
| lysine*100 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | lysine*100 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | lysine*100 | 1 | 1 | 1 | 1 | 1 | 0 | 26 | |
| methionine*100 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | methionine*100 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | methionine*100 | 1 | 1 | 1 | 1 | 1 | 0 | 26 | |
| phenylalanine*50 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | phenylalanine*50 | 2 | 2 | 2 | 2 | 0 | 2 | 2 | 2 | 2 | 2 | 2 | phenylalanine*50 | 2 | 2 | 2 | 2 | 2 | 0 | 52 | |
| proline*100 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | proline*100 | 1 | 1 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | proline*100 | 1 | 1 | 1 | 1 | 1 | 0 | 26 | |
| serine*100 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | serine*100 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | serine*100 | 1 | 1 | 0 | 1 | 0 | 0 | 24 | |
| threonine*100 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | threonine*100 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | threonine*100 | 1 | 1 | 1 | 1 | 1 | 0 | 26 | |
| tryptophane*100 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | tryptophane*100 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 1 | 1 | tryptophane*100 | 1 | 1 | 1 | 1 | 1 | 0 | 26 | |
| tyrosine*100 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | tyrosine*100 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 1 | tyrosine*100 | 1 | 1 | 1 | 1 | 1 | 0 | 26 | |
| valine*100 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | valine*100 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | valine*100 | 1 | 1 | 1 | 1 | 1 | 0 | 26 | |
| 2.5M NaOH | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2.5M NaOH | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 2.5M NaOH | 0 | 0 | 0 | 0 | 0 | 0 | 1 | |
| 2M HCl | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 2M HCl | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2M HCl | 1 | 0 | 0 | 0 | 1 | 0 | 3 | |
| pH adjust | 21.5 | 22.5 | 22.5 | 22.5 | 31.5 | 22.5 | 2.5 | 31.5 | 22.5 | 22.5 | 22.5 | pH adjust | 22.5 | 25.5 | 22.5 | 22.5 | 23.5 | 22.5 | 22.5 | 22.5 | 22.5 | 21.5 | 22.5 | pH adjust | 24 | 32.5 | 23.5 | 32.5 | 25 | 66 | 698.5 | |
| Total | 100 | 100 | 100 | 100 | 100 | 100 | 79.5 | 100 | 100 | 100 | 100 | Total | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | Total | 100 | 100 | 100 | 100 | 100 | 100 | 2679.5 | |
| 3 separate growth experiments performed on 3 consecutive days | ||||||||||||||||||||||||||||||||
| Streptococcus pyogenes precultured in THY-medium | ||||||||||||||||||||||||||||||||
| washed in PBS (2x) | ||||||||||||||||||||||||||||||||
| resuspended in PBS | ||||||||||||||||||||||||||||||||
| S. pyogenes in PBS used to inoculate 3 ml of the different CDM media (inoculated with start-OD600 approx 0.075-0.1) | ||||||||||||||||||||||||||||||||
| grown at 37oC in closed 15 ml standing tubes | ||||||||||||||||||||||||||||||||
| final OD measured (final OD was reached in all cultures at <24 hours after inoculation) |
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| Basal Solution 4* | |||||||||||||
| stock | Solvent | Fold-concentration (x) | Compound | MW (mg/mmol) | CAS | Stock (g/L) | Final (mg/L) | Final (mM) | UR | formula / syn | note | Ams | UvA number ! |
| PCA buffer | water | 4 | K2HPO4 | 174.18 | 7758-11-4 | 4 | 1000 | 5.7 | Roth P749 | Potassium phosphate dibasic | Merck 1.05104 / Sigma P3786 | K70 | |
| PCA buffer | water | 4 | KH2PO4 | 136.09 | 7778-77-0 | 20 | 5000 | 36.7 | Merck 1.04873 | Potassium phosphate monobasic | Merck 1.04873 / Sigma P5655 | K74 | |
| PCA buffer | water | 4 | NH3 citrate | 243.22 | 3458-72-8 | 2.4 | 600 | 2.5 | Sigma A-1332 | NH3 citrate tribasic | Sigma A-1332 | A97 | |
| PCA buffer | water | 4 | Na-acetate | 82.03 | 127-09-3 | 4 | 1000 | 12.2 | Merck 1.06268 | Sodium acetate anhydrous | Merck 1.06268 / Sigma S2889 | N21 | |
| alternative sugar | water | 4 | glucose monohydrate | 198.17 | 14431-43-7 | 48.4 | 12078 | 61 | D-(+)-Glucose monohydrate | 49159 Fluka | |||
| vitamin | water | 4 | ascorbate | 176.12 | 50-81-7 | 2 | 500 | 2.8 | A5960 | L-ascorbic acid, Vitamin C | A5960 | A212 | |
| AGU mix 50* | |||||||||||||
| stock | Solvent | Fold-concentration (x) | Compound | MW (mg/mmol) | CAS | Stock (g/L) | Final (mg/L) | Final (mM) | UR | formula / syn | note | Ams | UvA number ! |
| agu | 2 M HCl | 50 | Adenine | 135.13 | 73-24-5 | 1.9 | 38.5 | 0.28491082661141126 | Adenine | Sigma A-2786 | A14 | ||
| agu | 2 M HCl | 50 | Guanine | 151.13 | 73-40-5 | 1.1 | 22 | 0.14557003903923774 | Guanine | Sigma G-11950 | G134 | ||
| agu | 2 M HCl | 50 | Uracil | 112.09 | 66-22-8 | 1.1 | 22 | 0.19627085377821393 | Uracil | Sigma U-1128 | U6 | ||
| hydroxide 20* | |||||||||||||
| hydroxide | 0.6 M NaOH | 20 | xanthine | 152.11 | 69-89-6 | 0.2 | 10 | 0.066 | Sigma X-7375 | xanthine | Sigma X-7375 | X7 | |
| PCA buffer | 0.6 M NaOH | 20 | Na bicarbonate | 84 | 144-55-8 | 50 | 2500 | 30 | Merck 1.06329 | Na H carbonate | dissolve in NaOH to make sure CO2 stays in solution | Sigma S5761 | N24 |
| 100*vitamin solution | according to protocols | ||||||||||||
| 100*metal solution | according to protocols | ||||||||||||
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| inoculated on May 15th 2012 | |||||||||||
| Streptococcus pyogenes precultured in THY-medium (2 x 10 ml) | |||||||||||
| washed in PBS (2x) | |||||||||||
| resuspended in 10 ml PBS (final OD 2.0) | |||||||||||
| 100 ul of S. pyogenes in PBS used to inoculate 3 ml of the different CDM media | |||||||||||
| grown at 37oC in 15 ml standing tubes | |||||||||||
| OD measured in 5x dilutions (200 ul culture, 800 ul PBS) | |||||||||||
| measured value (5x diluted !) | OD600 corrected for dilution | ||||||||||
| omission | OD600 @ 19 hours | OD 600 @ 24 hours | OD600 @ x hours | OD600 @ 19 hours | OD600 @ 24 hours | ||||||
| CDM complete | full medium | 0.263 | 0.253 | 0.258 | 1.29 | 1.29 | |||||
| CDM 1 | ala | 0.268 | 0.265 | 0.263 | 1.3325 | 1.315 | |||||
| CDM 2 | arg | 0.022 | 0.025 | 0.023 | 0.1175 | 0.11499999999999999 | |||||
| CDM 3 | asn | 0.276 | 0.277 | 0.262 | 1.3825 | 1.31 | |||||
| CDM 4 | asp | 0.279 | 0.275 | 0.267 | 1.3850000000000002 | 1.335 | |||||
| CDM 5 | cys | 0.241 | 0.239 | 0.234 | 1.2 | 1.1700000000000002 | |||||
| CDM 6 | cyn | 0.252 | 0.245 | 0.255 | 1.2425 | 1.275 | |||||
| CDM 7 | glu | 0.29 | 0.305 | 0.269 | 1.4874999999999998 | 1.3450000000000002 | |||||
| CDM 8 | gln | 0.251 | 0.241 | 0.24 | 1.23 | 1.2 | |||||
| CDM 9 | gly | 0.059 | 0.058 | 0.043 | 0.2925 | 0.21499999999999997 | |||||
| CDM 10 | his | 0.058 | 0.059 | 0.043 | 0.2925 | 0.21499999999999997 | |||||
| CDM 11 | ile | 0.023 | 0.022 | 0.018 | 0.11249999999999999 | 0.09 | |||||
| CDM 12 | leu | 0.023 | 0.022 | 0.016 | 0.11249999999999999 | 0.08 | |||||
| CDM 13 | lys | 0.021 | 0.026 | 0.019 | 0.1175 | 0.095 | |||||
| CDM 14 | met | 0.074 | 0.082 | 0.051 | 0.39 | 0.255 | |||||
| CDM 15 | phe | 0.028 | 0.026 | 0.026 | 0.135 | 0.13 | |||||
| CDM 16 | pro | 0.237 | 0.221 | 0.211 | 1.145 | 1.055 | |||||
| CDM 17 | ser | 0.124 | 0.123 | 0.118 | 0.6174999999999999 | 0.59 | |||||
| CDM 18 | thr | 0.028 | 0.029 | 0.017 | 0.14250000000000002 | 0.085 | |||||
| CDM 19 | try | 0.058 | 0.054 | 0.046 | 0.28 | 0.22999999999999998 | |||||
| CDM 20 | tyr | 0.021 | 0.022 | 0.094 | wrong measurement | 0.10749999999999998 | 0.47 | ||||
| CDM 21 | val | 0.022 | 0.022 | 0.033 | 0.10999999999999999 | 0.165 | |||||
| CDM 22 | cys/cyn | 0.037 | 0.037 | 0.059 | 0.185 | 0.295 | |||||
| CDM 23 | glu/gln | 0.022 | 0.024 | 0.02 | 0.11499999999999999 | 0.1 | |||||
| CDM 24 | gly/ser | 0.085 | 0.094 | 0.07 | 0.4475 | 0.35000000000000003 | |||||
| CDM 25 | asn/asp | 0.294 | 0.289 | 0.275 | 1.4575 | 1.375 | |||||
| CDM 26 | cys/cyn/ser | 0.035 | 0.031 | 0.03 | 0.165 | 0.15 | |||||
| CDM 27 | no amino acids | 0.018 | 0.029 | 0.012 | 0.1175 | 0.06 |
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| inoculated on May 16th 2012 | |||||||||||
| Streptococcus pyogenes precultured in THY-medium (2 x 10 ml) | |||||||||||
| washed in PBS (2x) | |||||||||||
| resuspended in 10 ml PBS (final OD 1,5) | |||||||||||
| 133 ul of S. pyogenes in PBS used to inoculate 3 ml of the different CDM media | |||||||||||
| grown at 37oC in 15 ml standing tubes | |||||||||||
| OD measured in 5x dilutions (200 ul culture, 800 ul PBS) | |||||||||||
| measured value (5x diluted !) | OD600 corrected for dilution | ||||||||||
| omission | OD600 @ 26 hours | OD 600 @ 24 hours | OD600 @ x hours | OD600 @ 26 hours | |||||||
| CDM complete | full medium | 0.234 | 0.228 | 1.155 | |||||||
| CDM 1 | ala | 0.27 | 0.246 | 1.29 | |||||||
| CDM 2 | arg | 0.052 | 0.026 | 0.195 | |||||||
| CDM 3 | asn | 0.252 | 0.259 | 1.2775 | |||||||
| CDM 4 | asp | 0.256 | 0.257 | 1.2825 | |||||||
| CDM 5 | cys | 0.242 | 0.233 | 1.1875 | |||||||
| CDM 6 | cyn | 0.239 | 0.219 | 1.145 | |||||||
| CDM 7 | glu | 0.278 | 0.269 | 1.3675000000000002 | |||||||
| CDM 8 | gln | 0.227 | 0.208 | 1.0875 | |||||||
| CDM 9 | gly | 0.093 | 0.068 | 0.4025 | |||||||
| CDM 10 | his | 0.087 | 0.089 | 0.43999999999999995 | |||||||
| CDM 11 | ile | 0.035 | 0.04 | 0.18750000000000003 | |||||||
| CDM 12 | leu | 0.051 | 0.046 | 0.2425 | |||||||
| CDM 13 | lys | 0.031 | 0.034 | 0.1625 | |||||||
| CDM 14 | met | 0.061 | 0.06 | 0.3025 | |||||||
| CDM 15 | phe | 0.032 | 0.031 | 0.1575 | |||||||
| CDM 16 | pro | 0.252 | 0.227 | 1.1975 | |||||||
| CDM 17 | ser | 0.126 | 0.131 | 0.6425000000000001 | |||||||
| CDM 18 | thr | 0.039 | 0.041 | 0.2 | |||||||
| CDM 19 | try | 0.053 | 0.052 | 0.2625 | |||||||
| CDM 20 | tyr | 0.026 | 0.033 | 0.1475 | |||||||
| CDM 21 | val | 0.025 | 0.026 | 0.1275 | |||||||
| CDM 22 | cys/cyn | 0.044 | 0.043 | 0.21749999999999997 | |||||||
| CDM 23 | glu/gln | 0.029 | 0.027 | 0.14 | |||||||
| CDM 24 | gly/ser | 0.077 | 0.076 | 0.3825 | |||||||
| CDM 25 | asn/asp | 0.267 | 0.245 | 1.28 | |||||||
| CDM 26 | cys/cyn/ser | 0.035 | 0.034 | 0.17250000000000001 | |||||||
| CDM 27 | no amino acids | 0.029 | 0.02 | 0.1225 |
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| inoculated on May 17th 2012 | |||||||||
| Streptococcus pyogenes precultured in THY-medium (2 x 10 ml) | |||||||||
| washed in PBS (2x) | |||||||||
| resuspended in 7 ml PBS (final OD 5,0) | |||||||||
| 40 ul of S. pyogenes in PBS used to inoculate 3 ml of the different CDM media | |||||||||
| grown at 37oC in 15 ml standing tubes | |||||||||
| OD measured in 5x dilutions (200 ul culture, 800 ul PBS) | |||||||||
| measured value (5x diluted !) | OD corrected for dilution | ||||||||
| omission | OD 600 @ 24 hours | pH @ 24 hours | OD @ 24 hours | ||||||
| CDM complete | full medium | 0.254 | 0.254 | 4.6 | 1.27 | ||||
| CDM 1 | ala | 0.282 | 0.287 | 1.4224999999999999 | |||||
| CDM 2 | arg | 0.016 | 0.023 | 0.0975 | |||||
| CDM 3 | asn | 0.31 | 0.304 | 1.535 | |||||
| CDM 4 | asp | 0.289 | 0.288 | 1.4425 | |||||
| CDM 5 | cys | 0.266 | 0.268 | 1.335 | |||||
| CDM 6 | cyn | 0.223 | 0.223 | 1.115 | |||||
| CDM 7 | glu | 0.273 | 0.279 | 1.3800000000000001 | |||||
| CDM 8 | gln | 0.219 | 0.224 | 1.1075 | |||||
| CDM 9 | gly | 0.043 | 0.046 | 0.22249999999999998 | |||||
| CDM 10 | his | 0.038 | 0.04 | 0.195 | |||||
| CDM 11 | ile | 0.02 | 0.026 | 0.11499999999999999 | |||||
| CDM 12 | leu | 0.023 | 0.025 | 0.12 | |||||
| CDM 13 | lys | 0.022 | 0.027 | 0.1225 | |||||
| CDM 14 | met | 0.052 | 0.05 | 0.255 | |||||
| CDM 15 | phe | 0.019 | 0.024 | 0.10749999999999998 | |||||
| CDM 16 | pro | 0.225 | 0.223 | 1.12 | |||||
| CDM 17 | ser | 0.129 | 0.131 | 5.96 | 0.65 | ||||
| CDM 18 | thr | 0.019 | 0.031 | 0.125 | |||||
| CDM 19 | try | 0.039 | 0.047 | 0.21499999999999997 | |||||
| CDM 20 | tyr | 0.013 | 0.029 | 7.11 | 0.10500000000000001 | ||||
| CDM 21 | val | 0.015 | 0.025 | 0.1 | |||||
| CDM 22 | cys/cyn | 0.021 | 0.034 | 0.1375 | |||||
| CDM 23 | glu/gln | 0.018 | 0.028 | 0.11499999999999999 | |||||
| CDM 24 | gly/ser | 0.064 | 0.073 | 0.3425 | |||||
| CDM 25 | asn/asp | 0.297 | 0.312 | 1.5225 | |||||
| CDM 26 | cys/cyn/ser | 0.022 | 0.032 | 0.135 | |||||
| CDM 27 | no amino acids | 0.011 | 0.021 | 0.08 |
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| OD600 @ 19 hours | OD600 @ 26 hours | average OD600 | st. dev. | ||||||
| omission | 1st exp | 2nd exp | 3th exp | ||||||
| CDM complete | full medium | 1.29 | 1.155 | 1.27 | 1.2383333333333335 | 0.07285830998131465 | |||
| CDM 1 | ala | 1.3325 | 1.29 | 1.4224999999999999 | 1.3483333333333334 | 0.06765414498264923 | |||
| CDM 2 | arg | 0.1175 | 0.195 | 0.0975 | 0.1366666666666667 | 0.051498381851601216 | |||
| CDM 3 | asn | 1.3825 | 1.2775 | 1.535 | 1.3983333333333334 | 0.12947811912957846 | |||
| CDM 4 | asp | 1.3850000000000002 | 1.2825 | 1.4425 | 1.37 | 0.08104782538723664 | |||
| CDM 5 | cys | 1.2 | 1.1875 | 1.335 | 1.2408333333333335 | 0.08178987304876645 | |||
| CDM 6 | cyn | 1.2425 | 1.145 | 1.115 | 1.1675000000000002 | 0.06666145812986689 | |||
| CDM 7 | glu | 1.4874999999999998 | 1.3675000000000002 | 1.3800000000000001 | 1.4116666666666668 | 0.06597032161004908 | |||
| CDM 8 | gln | 1.23 | 1.0875 | 1.1075 | 1.1416666666666666 | 0.07714974616506096 | |||
| CDM 9 | gly | 0.2925 | 0.4025 | 0.22249999999999998 | 0.30583333333333335 | 0.09073771725877472 | |||
| CDM 10 | his | 0.2925 | 0.43999999999999995 | 0.195 | 0.30916666666666665 | 0.12334740910669076 | |||
| CDM 11 | ile | 0.11249999999999999 | 0.18750000000000003 | 0.11499999999999999 | 0.13833333333333334 | 0.042597926397106876 | |||
| CDM 12 | leu | 0.11249999999999999 | 0.2425 | 0.12 | 0.15833333333333333 | 0.07298687096549165 | |||
| CDM 13 | lys | 0.1175 | 0.1625 | 0.1225 | 0.13416666666666668 | 0.024664414311581145 | |||
| CDM 14 | met | 0.39 | 0.3025 | 0.255 | 0.31583333333333335 | 0.06848053251350585 | |||
| CDM 15 | phe | 0.135 | 0.1575 | 0.10749999999999998 | 0.13333333333333333 | 0.025041632002194676 | |||
| CDM 16 | pro | 1.145 | 1.1975 | 1.12 | 1.1541666666666668 | 0.039554814287686015 | |||
| CDM 17 | ser | 0.6174999999999999 | 0.6425000000000001 | 0.65 | 0.6366666666666667 | 0.01701714821388517 | |||
| CDM 18 | thr | 0.14250000000000002 | 0.2 | 0.125 | 0.15583333333333335 | 0.039237524556645136 | |||
| CDM 19 | try | 0.28 | 0.2625 | 0.21499999999999997 | 0.2525 | 0.03363406011768449 | |||
| CDM 20 | tyr | 0.10749999999999998 | 0.1475 | 0.10500000000000001 | 0.12 | 0.023848480035423638 | |||
| CDM 21 | val | 0.10999999999999999 | 0.1275 | 0.1 | 0.1125 | 0.013919410907075037 | |||
| CDM 22 | cys/cyn | 0.185 | 0.21749999999999997 | 0.1375 | 0.18000000000000002 | 0.04023369234857756 | |||
| CDM 23 | glu/gln | 0.11499999999999999 | 0.14 | 0.11499999999999999 | 0.12333333333333334 | 0.014433756729740658 | |||
| CDM 24 | gly/ser | 0.4475 | 0.3825 | 0.3425 | 0.39083333333333337 | 0.05299371031861518 | |||
| CDM 25 | asn/asp | 1.4575 | 1.28 | 1.5225 | 1.42 | 0.12552390210633188 | |||
| CDM 26 | cys/cyn/ser | 0.165 | 0.17250000000000001 | 0.135 | 0.1575 | 0.01984313483298456 | |||
| CDM 27 | no amino acids | 0.1175 | 0.1225 | 0.08 | 0.10666666666666667 | 0.023228933107943914 | |||
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| Streptococcus pyogenes is very sensitive to low pH ! | |||||||||||||||||||||
| Final pH in CDM complete after pyogenes growth (OD 1.25-1.5) is approx. 4.5, resulting in non viable pyogenes cells (thus not suitable for re-inoculation) | |||||||||||||||||||||
| Since final pH in CDM - serine after pyogenes growth (OD 0.5-0.6) is approx. 6.0 (due to less growth/lower OD), these pyogenes cells can be re-inoculated | |||||||||||||||||||||
| To confirm that serine/glycine is indeed not essential for pyogenes we will culture pyogenes for 3 subsequent batch cultures in CDM - serine/glycine, by reinoculating the pyogenes cells grow in CDM - serine and CDM - glycine | |||||||||||||||||||||
| reinoculation from CDM-serine to complete CDM is just a positive control to confirm viability of the cells. | |||||||||||||||||||||
| 1st inoculation with S. pyogenes precultured in THY medium, washed in PBS | |||||||||||||||||||||
| 2nd inoculation with pyogenes cells grown in CDM-serine or CDM - glycine, or CDM -GLY/SER (during 1st inoculation) | |||||||||||||||||||||
| 3th inoculation with pyogenes cells grown in CDM-serine or CDM -GLY/SER (2nd inoculation) | |||||||||||||||||||||
| final OD600 | |||||||||||||||||||||
| time (h) | 0 | 24 | 24.0009 | 24.001 | 48 | 48.0009 | 48.001 | 72 | |||||||||||||
| CDM complete | 0.07 | 1.595 | 0.081 | 1.338 | 0.106 | 1.28 | |||||||||||||||
| CDM - serine | 0.07 | 0.57 | 0.081 | 0.74 | 0.106 | 0.93 | |||||||||||||||
| reinoculation from CDM - ser | 0.57 | 0.081 | 0.74 | 0.106 | |||||||||||||||||
| CDM - glycine | 0.07 | 0.413 | 0.03754545454545454 | 1.16 | 0.027 | 1.498 | after 48 hours reinoculated from CDM -gly -ser ! | ||||||||||||||
| reinoculation from CDM - gly | 0.413 | 0.0375 | |||||||||||||||||||
| CDM - gly -ser | 0.07 | 0.458 | 0.042 | 0.795 | 0.027 | 0.996 | |||||||||||||||
| reinoculation from CDM - gly -ser | 0.458 | 0.04163636363636364 | 1.16 | 0.026500000000000003 | |||||||||||||||||
| CDM - threonine | 0.07 | 0.14 | 0.028 | 0.982 | 0.0066 | 1.696 | |||||||||||||||
| reinoculation from CDM -thr | 0.14 | 0.028000000000000004 | 0.033 | 0.0066 | |||||||||||||||||
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| Validation of results using independently prepared media (prepared by Filipe dec 2011) | ||||||||||||||||||||||||
| inoculated on 5th june 2012 | inoculated on 6th june 2012 | inoculated on 7th june 2012 | ||||||||||||||||||||||
| Streptococcus pyogenes M49 (isolate from UvA) precultured in THY-medium (2 x 10 ml) | Streptococcus pyogenes M49 (isolate from Antje Sieg, Rostock) precultured in THY-medium (10 ml) | |||||||||||||||||||||||
| washed in PBS (2x) | washed in PBS (2x) | since Filipe's media are old and do not contain bicarbonate following recipe used | ||||||||||||||||||||||
| resuspended in 10 ml PBS (final OD 2.0) | resuspended in 5 ml PBS (final OD 4.5) | 3 ml of medium from Filipe (complete, -GLU, -GLN, -SER, -TRY, -TYR) | ||||||||||||||||||||||
| S. pyogenes in PBS used to inoculate 3 ml of the different CDM media | S. pyogenes in PBS used to inoculate 3 ml of the different CDM media (start OD 0.05) | 1 ml of CDM 27 from Koen (CDM 27 is CDM lacking all amino acids) | ||||||||||||||||||||||
| grown at 37oC in 15 ml standing tubes | grown at 37oC in 15 ml standing tubes | 200 ul of 20x bicarbonate solution | ||||||||||||||||||||||
| OD measured in 5x dilutions (200 ul culture, 800 ul PBS) | OD measured in 5x dilutions (200 ul culture, 800 ul PBS) | medium incubated in 37oC used as blanc (to take color change effect medium into account!) | ||||||||||||||||||||||
| since Filipe's media are old and do not contain bicarbonate following recipe used | since Filipe's media are old and do not contain bicarbonate following recipe used | testing whether -GLN results are resulting from a difference between the M49 strain from Rostock and Amsterdam | ||||||||||||||||||||||
| 3 ml of medium from Filipe (complete, -GLU, -GLN, -SER, -TRY, -TYR) | 3 ml of medium from Filipe (complete, -GLU, -GLN, -SER, -TRY, -TYR) | |||||||||||||||||||||||
| 1 ml of CDM 27 from Koen (CDM 27 is CDM lacking all amino acids) | 1 ml of CDM 27 from Koen (CDM 27 is CDM lacking all amino acids) | This time experiments performed with pyogenes cells pre-adapted to CDM (by growing in CDM - serine) | ||||||||||||||||||||||
| 200 ul of 20x bicarbonate solution | 200 ul of 20x bicarbonate solution | |||||||||||||||||||||||
| medium incubated in 37oC used as blanc (to take color change effect medium into account!) | ||||||||||||||||||||||||
| omission | final OD (5x diluted) | OD600 | omission | final OD undiluted | OD600 | |||||||||||||||||||
| CDM complete | full medium | 0.417 | 0.403 | 2.0500000000000003 | CDM complete | full medium | 1.157 | 1.192 | 1.1745 | |||||||||||||||
| CDM 7 | glu | 0.363 | 0.364 | 1.8175 | CDM 7 | glu | 1.113 | 1.103 | 1.108 | Rostock strain | Amsterdam strain | blanc | ||||||||||||
| CDM 8 | gln | 0.46 | 0.456 | 2.29 | CDM 8 | gln | 0.247 | 0.264 | 0.2555 | CDM complete | 1.76 | 1.64 | 0.005 | |||||||||||
| CDM 17 | ser | 0.304 | 0.299 | 1.5074999999999998 | CDM 17 | ser | 0.676 | 0.68 | 0.678 | CDM - GLN | 1,72* | 1.67 | 0.0063 | |||||||||||
| CDM 19 | try | 0.039 | 0.039 | 0.195 | CDM 19 | try | 0.074 | 0.072 | 0.073 | CDM - GLN | 1,67** | |||||||||||||
| CDM 20 | tyr | 0.421 | 0.415 | 2.09 | CDM 20 | tyr | 0.463 | 0.462 | 0.4625 | |||||||||||||||
| * inoculated with pyogenes cells pre-adapted to CDM (by growing in CDM-serine at 6/6/12) | ||||||||||||||||||||||||
| media prepared by Koen, inoculated by M49 from Rostock | ** inoculated with pyogenes cells pre-adapted to CDM-GLN (from culture at 6/6/12) | |||||||||||||||||||||||
| CDM 7 | glu | 1.047 | 1.029 | |||||||||||||||||||||
| CDM 8 | gln | 1.045 | 0.942 | |||||||||||||||||||||
| media Filipe confirm GLU/GLN/SER and TRY data with pyogenes Amsterdam stock | media Filipe confirm GLU/SER and TRY data with pyogenes Rostock stock | |||||||||||||||||||||||
| strangely TYR does not seem essential in Filipe's medium preparation | media Filipe shows GLN to be essential for pyogenes Rostock stock | |||||||||||||||||||||||
| strangely TYR does not seem essential in Filipe's medium preparation | ||||||||||||||||||||||||
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| Essential amino acids acc. to | Slade etal. 1951 | Genomic model | CDM growth Rostock | CDM growth Amsterdam (Koen van Grinsven) | major disagreements | ||||
| Ala | n | y | n | n | |||||
| Arg | y | y | y | y | |||||
| Asn | n | n | n | n | |||||
| Asp | n | n | n | n | |||||
| Cys | y | n | n | n | |||||
| Gln | n | n | y | n | |||||
| Glu | n | n | n | n | |||||
| Gly | y | n | y | n | |||||
| His | y | y | y | y | |||||
| Ile | y | y | y | y | |||||
| Leu | y | y | y | y | |||||
| Lys | y | y | y | y | |||||
| Met | y | y | y | y | |||||
| Phe | y | y | y | y | |||||
| Pro | y | n | n | n | |||||
| Ser | y | n | y | n | |||||
| Thr | y | n | y | y | |||||
| Trp | y | y | y | y | |||||
| Tyr | y | y | y | y | |||||
| Val | y | y | y | y | |||||
