Selected Cell
Cell:
Value:
medium setup
other media components
faecalis inoculation 1
faecalis inoculation 2
faecalis inoculation 3
faecalis inoculation 4
faecalis inoculation 5
validation with media filipe
summary
continuous passaging
validation GLY_SER
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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 | ||||||||||||||||||||||||||||||||
| Enterococcus faecalis precultured in THY-medium | ||||||||||||||||||||||||||||||||
| washed in PBS (2x) | ||||||||||||||||||||||||||||||||
| resuspended in PBS | ||||||||||||||||||||||||||||||||
| 1st inoculation: E. faecalis in PBS used to inoculate 3 ml of the different CDM media (inoculated with start-OD600 approx 0.03) | ||||||||||||||||||||||||||||||||
| grown at 37oC in closed 15 ml standing tubes | ||||||||||||||||||||||||||||||||
| final OD measured (final OD was reached in all cultures at <24 hours after inoculation) | ||||||||||||||||||||||||||||||||
| 2nd inoculation: cells from the first inoculation in CDM 0-27 were used to inoculate the different CDM media (start OD 0.03), in case final OD600 from the first inoculation was <0,5, cells grown in complete CDM (=CDM0) were used for inoculation | ||||||||||||||||||||||||||||||||
| 3rd inoculation: cells from the second inoculation in CDM 0-27 were used to inoculate the different CDM media (start OD 0.03), in case final OD600 from the second inoculation was <0.5, cells grown in complete CDM (=CDM0) were used for 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 30th 2012 | |||||||||||
| Enterococcus faecalis precultured in THY-medium (2 x 10 ml) | |||||||||||
| washed in PBS (2x) | |||||||||||
| resuspended in 10 ml PBS (final OD 4.0) | |||||||||||
| 30 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 fysiological salt) | |||||||||||
| E. faecalis | |||||||||||
| measured value (5x diluted !) | OD600 corrected for dilution | ||||||||||
| omission | OD600 @ 23 hours | OD600 @ 23 hours | |||||||||
| CDM complete | full medium | 0.506 | 0.487 | 2.4825 | |||||||
| CDM 1 | ala | 0.457 | 0.447 | 2.2600000000000002 | |||||||
| CDM 2 | arg | 0.018 | 0.019 | 0.0925 | |||||||
| CDM 3 | asn | 0.483 | 0.477 | 2.4 | |||||||
| CDM 4 | asp | 0.526 | 0.518 | 2.6100000000000003 | |||||||
| CDM 5 | cys | 0.478 | 0.468 | 2.3649999999999998 | |||||||
| CDM 6 | cyn | 0.486 | 0.48 | 2.415 | |||||||
| CDM 7 | glu | 0.46 | 0.548 | 2.52 | |||||||
| CDM 8 | gln | 0.483 | 0.484 | 2.4175 | |||||||
| CDM 9 | gly | 0.056 | 0.056 | 0.28 | |||||||
| CDM 10 | his | 0.048 | 0.047 | 0.2375 | |||||||
| CDM 11 | ile | 0.023 | 0.022 | 0.11249999999999999 | |||||||
| CDM 12 | leu | 0.038 | 0.037 | 0.1875 | |||||||
| CDM 13 | lys | 0.294 | 0.299 | 1.4825 | |||||||
| CDM 14 | met | 0.042 | 0.07 | 0.28 | |||||||
| CDM 15 | phe | 0.46 | 0.457 | 2.2925 | |||||||
| CDM 16 | pro | 0.482 | 0.477 | 2.3975 | |||||||
| CDM 17 | ser | 0.369 | 0.362 | 1.8275 | |||||||
| CDM 18 | thr | 0.497 | 0.491 | 2.4699999999999998 | |||||||
| CDM 19 | try | 0.035 | 0.037 | 0.18000000000000002 | |||||||
| CDM 20 | tyr | 0.464 | 0.464 | 2.3200000000000003 | |||||||
| CDM 21 | val | 0.023 | 0.026 | 0.1225 | |||||||
| CDM 22 | cys/cyn | 0.487 | 0.476 | 2.4074999999999998 | |||||||
| CDM 23 | glu/gln | 0.025 | 0.033 | 0.14500000000000002 | |||||||
| CDM 24 | gly/ser | 0.092 | 0.092 | 0.45999999999999996 | |||||||
| CDM 25 | asn/asp | 0.472 | 0.465 | 2.3425000000000002 | |||||||
| CDM 26 | cys/cyn/ser | 0.247 | 0.245 | 1.23 | |||||||
| CDM 27 | no amino acids | 0 | 0 | 0 |
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| inoculated on May 31th 2012 | |||||||||
| Enterococcus faecalis taken from respective CDM medium first inoculation | |||||||||
| in case in first inoculation final OD<0.5, new inoculations were performed from complete CDM (=CDM0) | |||||||||
| 60 or 100 ul of E. faecalis 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 fysiological salt) | |||||||||
| all inoculations grew, because of residuals in the complete CDM with which they were inoculated !! (60 ul in 3 ml) | |||||||||
| therefore these OD's are not reliable for the "essential" amino acids, but the 3th inoculation and the validation with filipe's medium could be done with these partly grown cultures…. | |||||||||
| omission | OD600 5x diluted | OD600 | |||||||
| CDM complete | full medium | 0.484 | 0.472 | 2.3899999999999997 | |||||
| CDM 1 | ala | 0.452 | 0.45 | 2.255 | |||||
| CDM 2 | arg | 0.068 | 0.063 | 0.3275 | |||||
| CDM 3 | asn | 0.495 | 0.477 | 2.4299999999999997 | |||||
| CDM 4 | asp | 0.522 | 0.502 | 2.56 | |||||
| CDM 5 | cys | 0.487 | 0.471 | 2.395 | |||||
| CDM 6 | cyn | 0.474 | 0.469 | 2.3575 | |||||
| CDM 7 | glu | 0.47 | 0.465 | 2.3375000000000004 | |||||
| CDM 8 | gln | 0.497 | 0.49 | 2.4675 | |||||
| CDM 9 | gly | 0.181 | 0.205 | 0.9650000000000001 | |||||
| CDM 10 | his | 0.238 | 0.235 | 1.1824999999999999 | |||||
| CDM 11 | ile | 0.099 | 0.1 | 0.49750000000000005 | |||||
| CDM 12 | leu | 0.217 | 0.218 | 1.0875 | |||||
| CDM 13 | lys | 0.389 | 0.389 | 1.945 | |||||
| CDM 14 | met | 0.17 | 0.168 | 0.8450000000000001 | |||||
| CDM 15 | phe | 0.456 | 0.45 | 2.265 | |||||
| CDM 16 | pro | 0.466 | 0.479 | 2.3625000000000003 | |||||
| CDM 17 | ser | 0.371 | 0.376 | 1.8675 | |||||
| CDM 18 | thr | 0.503 | 0.5 | 2.5075000000000003 | |||||
| CDM 19 | try | 0.269 | 0.269 | 1.3450000000000002 | |||||
| CDM 20 | tyr | 0.475 | 0.465 | 2.3499999999999996 | |||||
| CDM 21 | val | 0.147 | 0.146 | 0.7324999999999999 | |||||
| CDM 22 | cys/cyn | 0.507 | 0.486 | 2.4825 | |||||
| CDM 23 | glu/gln | 0.065 | 0.066 | 0.3275 | |||||
| CDM 24 | gly/ser | 0.197 | 0.192 | 0.9725 | |||||
| CDM 25 | asn/asp | 0.472 | 0.467 | 2.3475 | |||||
| CDM 26 | cys/cyn/ser | 0.289 | 0.288 | 1.4425 | |||||
| CDM 27 | no amino acids | 0.028 | 0.035 | 0.1575 |
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| inoculated on June 1st 2012 | |||||||||
| Enterococcus faecalis taken from respective CDM medium second inoculation | |||||||||
| which all grew (1/50th of complete CDM present) | |||||||||
| grown at 37oC in 15 ml standing tubes | |||||||||
| OD measured in 5x dilutions (200 ul culture, 800 ul fysiological salt) | |||||||||
| omission | OD600 5x diluted | OD600 | |||||||
| CDM complete | full medium | 0.452 | 0.446 | 2.245 | |||||
| CDM 1 | ala | 0.397 | 0.389 | 1.965 | |||||
| CDM 2 | arg | 0.043 | 0.042 | 0.21249999999999997 | |||||
| CDM 3 | asn | 0.467 | 0.461 | 2.3200000000000003 | |||||
| CDM 4 | asp | 0.474 | 0.461 | 2.3375000000000004 | |||||
| CDM 5 | cys | 0.507 | 0.511 | 2.545 | |||||
| CDM 6 | cyn | 0.403 | 0.409 | 0.397 | 2.015 | ||||
| CDM 7 | glu | 0.446 | 0.426 | 2.18 | |||||
| CDM 8 | gln | 0.447 | 0.435 | 2.205 | |||||
| CDM 9 | gly | 0.137 | 0.134 | 0.6775 | |||||
| CDM 10 | his | 0.07 | 0.068 | 0.34500000000000003 | |||||
| CDM 11 | ile | 0.05 | 0.051 | 0.2525 | |||||
| CDM 12 | leu | 0.083 | 0.082 | 0.41250000000000003 | |||||
| CDM 13 | lys | 0.347 | 0.339 | 1.7149999999999999 | |||||
| CDM 14 | met | 0.052 | 0.05 | 0.255 | |||||
| CDM 15 | phe | 0.445 | 0.441 | 2.215 | |||||
| CDM 16 | pro | 0.472 | 0.462 | 2.335 | |||||
| CDM 17 | ser | 0.355 | 0.352 | 1.7674999999999998 | |||||
| CDM 18 | thr | 0.506 | 0.499 | 2.5124999999999997 | |||||
| CDM 19 | try | 0.056 | 0.053 | 0.2725 | |||||
| CDM 20 | tyr | 0.395 | 0.392 | 1.9675 | |||||
| CDM 21 | val | 0.056 | 0.053 | 0.2725 | |||||
| CDM 22 | cys/cyn | 0.476 | 0.455 | 2.3275 | |||||
| CDM 23 | glu/gln | 0.032 | 0.028 | 0.15 | |||||
| CDM 24 | gly/ser | 0.174 | 0.17 | 0.8599999999999999 | |||||
| CDM 25 | asn/asp | 0.4 | 0.396 | 1.9900000000000002 | |||||
| CDM 26 | cys/cyn/ser | 0.322 | 0.315 | 1.5925 | |||||
| CDM 27 | no amino acids | 0.051 | 0.05 | 0.2525 |
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| inoculated on June 4th 2012 | |||||||||
| Enterococcus faecalis taken from respective CDM medium third inoculation | |||||||||
| grown at 37oC in 15 ml standing tubes | |||||||||
| OD measured in undiluted | |||||||||
| omission | OD600 PBS blanc | OD600 CDM27 blanc | OD600 | ||||||
| CDM 8 | gln | 1.816 | 1.823 | 1.789 | 1.786 | 1.8195000000000001 | |||
| CDM 9 | gly | 0.345 | 0.346 | 0.321 | 0.323 | 0.3455 | |||
| CDM 10 | his | 0.215 | 0.208 | 0.183 | 0.183 | 0.2115 | |||
| CDM 12 | leu | 0.23 | 0.232 | 0.203 | 0.207 | 0.231 | |||
| CDM 24 | gly/ser | 0.487 | 0.487 | 0.469 | 0.454 | 0.487 | |||
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| Confirmation using media Filipe Santos (media made dec 2011) | |||||||||
| inoculated on June 2nd 2012 | |||||||||
| Enterococcus faecalis taken from respective CDM medium second inoculation | |||||||||
| which all grew (1/50th of complete CDM present) | |||||||||
| grown at 37oC in 15 ml standing tubes | |||||||||
| OD measured in 5x dilutions (200 ul culture, 800 ul fysiological salt) | |||||||||
| OD600 2,5x diluted | OD600 | ||||||||
| CDM complete | full medium | 0.4196 | 0.423 | 1.05325 | |||||
| CDM 1 | ala | 0.415 | 0.407 | 1.0274999999999999 | |||||
| CDM 2 | arg | 0.037 | 0.041 | 0.0975 | |||||
| CDM 3 | asn | 0.418 | 0.411 | 1.03625 | |||||
| CDM 4 | asp | 0.317 | 0.365 | 0.8524999999999999 | |||||
| CDM 5 | cys | 0.316 | 0.315 | 0.7887500000000001 | |||||
| CDM 6 | cyn | ||||||||
| CDM 7 | glu | 0.409 | 0.415 | 1.03 | |||||
| CDM 8 | gln | 0.352 | 0.35 | 0.8775 | |||||
| CDM 9 | gly | 0.084 | 0.102 | 0.083 | 0.22416666666666668 | ||||
| CDM 10 | his | 0.011 | 0.09 | 0.12625 | |||||
| CDM 11 | ile | 0.021 | 0.021 | 0.052500000000000005 | |||||
| CDM 12 | leu | 0.026 | 0.025 | 0.06375 | |||||
| CDM 13 | lys | 0.255 | 0.251 | 0.6325000000000001 | |||||
| CDM 14 | met | 0.012 | 0.005 | 0.02125 | |||||
| CDM 15 | phe | 0.347 | 0.346 | 0.86625 | |||||
| CDM 16 | pro | 0.181 | 0.179 | 0.72 | proline sample by mistake 4 times diluted for OD measurement | ||||
| CDM 17 | ser | 0.291 | 0.284 | 0.71875 | |||||
| CDM 18 | thr | 0.295 | 0.303 | 0.7474999999999999 | |||||
| CDM 19 | try | 0.01 | 0.011 | 0.026249999999999996 | |||||
| CDM 20 | tyr | 0.291 | 0.285 | 0.72 | |||||
| CDM 21 | val | 0.031 | 0.011 | 0.05249999999999999 | |||||
| CDM 22 | cys/cyn | ||||||||
| CDM 23 | glu/gln | ||||||||
| CDM 24 | gly/ser | ||||||||
| CDM 25 | asn/asp | ||||||||
| CDM 26 | cys/cyn/ser | ||||||||
| CDM 27 | no amino acids |
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| OD600 @ 23 hours | OD600 @ hours | average OD600 | st. dev. | |||||||
| omission | 1st exp | 2nd exp | 3th exp | 4th exp | 5th exp | media Filipe | OD 600 | |||
| CDM complete | full medium | 2.4825 | 2.3899999999999997 | 2.245 | 1.05325 | 2.3725 | 0.11971319893812868 | |||
| CDM 1 | ala | 2.2600000000000002 | 2.255 | 1.965 | 1.0274999999999999 | 2.16 | 0.16889345754054536 | |||
| CDM 2 | arg | 0.0925 | 0.3275 | 0.21249999999999997 | 0.0975 | 0.13416666666666666 | 0.06788470618138769 | |||
| CDM 3 | asn | 2.4 | 2.4299999999999997 | 2.3200000000000003 | 1.03625 | 2.3833333333333333 | 0.056862407030772985 | |||
| CDM 4 | asp | 2.6100000000000003 | 2.56 | 2.3375000000000004 | 0.8524999999999999 | 2.5025 | 0.14506464076404002 | |||
| CDM 5 | cys | 2.3649999999999998 | 2.395 | 2.545 | 0.7887500000000001 | 2.435 | 0.09643650760992958 | |||
| CDM 6 | cyn | 2.415 | 2.3575 | 2.015 | 2.2624999999999997 | 0.21626083787870604 | ||||
| CDM 7 | glu | 2.52 | 2.3375000000000004 | 2.18 | 1.03 | 2.345833333333333 | 0.17015311731888225 | |||
| CDM 8 | gln | 2.4175 | 2.4675 | 2.205 | 0.8775 | 2.3633333333333333 | 0.13938107236398098 | |||
| CDM 9 | gly | 0.28 | 0.9650000000000001 | 0.6775 | 0.22416666666666668 | 0.3938888888888889 | 0.24719584753194587 | |||
| CDM 10 | his | 0.2375 | 1.1824999999999999 | 0.34500000000000003 | 0.12625 | 0.23625 | 0.10938035701166829 | |||
| CDM 11 | ile | 0.11249999999999999 | 0.49750000000000005 | 0.2525 | 0.052500000000000005 | 0.13916666666666666 | 0.10263202878893768 | |||
| CDM 12 | leu | 0.1875 | 1.0875 | 0.41250000000000003 | 0.06375 | 0.22125000000000003 | 0.17680762851189424 | |||
| CDM 13 | lys | 1.4825 | 1.945 | 1.7149999999999999 | 0.6325000000000001 | 1.7141666666666666 | 0.23125112612338178 | |||
| CDM 14 | met | 0.28 | 0.8450000000000001 | 0.255 | 0.02125 | 0.18541666666666667 | 0.14272095442973096 | |||
| CDM 15 | phe | 2.2925 | 2.265 | 2.215 | 0.86625 | 2.2575 | 0.03929058411375436 | |||
| CDM 16 | pro | 2.3975 | 2.3625000000000003 | 2.335 | 0.72 | 2.3649999999999998 | 0.031324910215354156 | |||
| CDM 17 | ser | 1.8275 | 1.8675 | 1.7674999999999998 | 0.71875 | 1.820833333333333 | 0.05033222956847171 | |||
| CDM 18 | thr | 2.4699999999999998 | 2.5075000000000003 | 2.5124999999999997 | 0.7474999999999999 | 2.4966666666666666 | 0.023228933107944043 | |||
| CDM 19 | try | 0.18000000000000002 | 1.3450000000000002 | 0.2725 | 0.026249999999999996 | 0.15958333333333333 | 0.12438808557628556 | |||
| CDM 20 | tyr | 2.3200000000000003 | 2.3499999999999996 | 1.9675 | 0.72 | 2.2125 | 0.21270578271405782 | |||
| CDM 21 | val | 0.1225 | 0.7324999999999999 | 0.2725 | 0.05249999999999999 | 0.14916666666666667 | 0.11239810200058245 | |||
| CDM 22 | cys/cyn | 2.4074999999999998 | 2.4825 | 2.3275 | 2.4058333333333333 | 0.0775134396948898 | ||||
| CDM 23 | glu/gln | 0.14500000000000002 | 0.3275 | 0.15 | 0.20750000000000002 | 0.10395311443145891 | ||||
| CDM 24 | gly/ser | 0.45999999999999996 | 0.9725 | 0.8599999999999999 | 0.6599999999999999 | 0.2828427124746191 | ||||
| CDM 25 | asn/asp | 2.3425000000000002 | 2.3475 | 1.9900000000000002 | 2.226666666666667 | 0.20497459192137285 | ||||
| CDM 26 | cys/cyn/ser | 1.23 | 1.4425 | 1.5925 | 1.4216666666666666 | 0.1821457749532879 | ||||
| CDM 27 | no amino acids | 0 | 0.1575 | 0.2525 | 0.1366666666666667 | 0.1275326755515359 | ||||
| inoculated from THY (washed cells) | inoculated from resp. CDM or complete CDM non-washed | inoculated from respecitve CDM non washed | inoculated from respecitve CDM non washed | inoculated from respecitve CDM non washed | ||||||
| OD's in red are not representative, since these cultures were inoculated with 60 ul of unwashed faecalis from CDM complete | ||||||||||
| ARG | ||||||||||
| HIS | ||||||||||
| ILE | ||||||||||
| LEU | ||||||||||
| MET | ||||||||||
| TRY | ||||||||||
| VAL | ||||||||||
| GLU/GLN |
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| continuous subculturing | subculturing to identical media (24-48 hours), | subculturing to media Filipe (24-48 hours) | |||||||||||||||||||||||
| inoculated at t=0 with washed cells precultured in THY medium | inoculated at t=0 with 60 ul unwashed cells in CDM complete (hence initial growth) | inoculated at t=0 with 60 ul depleted cells (inoculation 2 @ 24h) | |||||||||||||||||||||||
| omission | 0 | 24 | 24.01 | 48 | 48.01 | 72 | 0 | 24 | 24.01 | 48 | 48.01 | 72 | 72.01 | 96 | 0 | 24 | |||||||||
| CDM complete | full medium | 0.04 | 2.4825 | 0.04965 | 2.3899999999999997 | 0.039833333333333325 | 2.245 | CDM complete | full medium | CDM complete | full medium | ||||||||||||||
| CDM 1 | ala | 0.04 | 2.2600000000000002 | 0.045200000000000004 | 2.255 | 0.03758333333333333 | 1.965 | CDM 1 | ala | CDM 1 | ala | ||||||||||||||
| CDM 2 | arg | 0.04 | 0.0925 | CDM 2 | arg | 0.04965 | 0.3275 | 0.043666666666666666 | 0.21249999999999997 | CDM 2 | arg | 0.043666666666666666 | 0.0975 | ||||||||||||
| CDM 3 | asn | 0.04 | 2.4 | 0.048 | 2.4299999999999997 | 0.040499999999999994 | 2.3200000000000003 | CDM 3 | asn | CDM 3 | asn | ||||||||||||||
| CDM 4 | asp | 0.04 | 2.6100000000000003 | 0.0522 | 2.56 | 0.042666666666666665 | 2.3375000000000004 | CDM 4 | asp | CDM 4 | asp | ||||||||||||||
| CDM 5 | cys | 0.04 | 2.3649999999999998 | 0.047299999999999995 | 2.395 | 0.03991666666666667 | 2.545 | CDM 5 | cys | CDM 5 | cys | ||||||||||||||
| CDM 6 | cyn | 0.04 | 2.415 | 0.0483 | 2.3575 | 0.03929166666666666 | 2.015 | CDM 6 | cyn | CDM 6 | cyn | ||||||||||||||
| CDM 7 | glu | 0.04 | 2.52 | 0.0504 | 2.3375000000000004 | 0.03895833333333334 | 2.18 | CDM 7 | glu | CDM 7 | glu | ||||||||||||||
| CDM 8 | gln | 0.04 | 2.4175 | 0.04835 | 2.4675 | 0.041124999999999995 | 2.205 | CDM 8 | gln | CDM 8 | gln | ||||||||||||||
| CDM 9 | gly | 0.04 | 0.28 | CDM 9 | gly | 0.04965 | 0.9650000000000001 | 0.03216666666666667 | 0.6775 | 0.033875 | 0.3455 | 0.05758333333333333 | 1.625 | CDM 9 | gly | 0.03216666666666667 | 0.22416666666666668 | ||||||||
| CDM 10 | his | 0.04 | 0.2375 | CDM 10 | his | 0.04965 | 1.1824999999999999 | 0.03941666666666666 | 0.34500000000000003 | 0.02875 | 0.2115 | 0.03525 | 0.21 | CDM 10 | his | 0.03941666666666666 | 0.12625 | ||||||||
| CDM 11 | ile | 0.04 | 0.11249999999999999 | CDM 11 | ile | 0.04965 | 0.49750000000000005 | 0.03316666666666667 | 0.2525 | CDM 11 | ile | 0.03316666666666667 | 0.052500000000000005 | ||||||||||||
| CDM 12 | leu | 0.04 | 0.1875 | CDM 12 | leu | 0.04965 | 1.0875 | 0.03625 | 0.41250000000000003 | 0.034375 | 0.231 | 0.0385 | 0.256 | CDM 12 | leu | 0.03625 | 0.06375 | ||||||||
| CDM 13 | lys | 0.04 | 1.4825 | 0.049416666666666664 | 1.945 | 0.03241666666666667 | 1.7149999999999999 | CDM 13 | lys | CDM 13 | lys | ||||||||||||||
| CDM 14 | met | 0.04 | 0.28 | CDM 14 | met | 0.04965 | 0.8450000000000001 | 0.02816666666666667 | 0.255 | CDM 14 | met | 0.02816666666666667 | 0.02125 | ||||||||||||
| CDM 15 | phe | 0.04 | 2.2925 | 0.04585 | 2.265 | 0.03775 | 2.215 | CDM 15 | phe | CDM 15 | phe | ||||||||||||||
| CDM 16 | pro | 0.04 | 2.3975 | 0.04795 | 2.3625000000000003 | 0.03937500000000001 | 2.335 | CDM 16 | pro | CDM 16 | pro | ||||||||||||||
| CDM 17 | ser | 0.04 | 1.8275 | 0.06091666666666666 | 1.8675 | 0.031125 | 1.7674999999999998 | CDM 17 | ser | CDM 17 | ser | ||||||||||||||
| CDM 18 | thr | 0.04 | 2.4699999999999998 | 0.04939999999999999 | 2.5075000000000003 | 0.04179166666666667 | 2.5124999999999997 | CDM 18 | thr | CDM 18 | thr | ||||||||||||||
| CDM 19 | try | 0.04 | 0.18000000000000002 | CDM 19 | try | 0.04965 | 1.3450000000000002 | 0.029888888888888892 | 0.2725 | CDM 19 | try | 0.029888888888888892 | 0.026249999999999996 | ||||||||||||
| CDM 20 | tyr | 0.04 | 2.3200000000000003 | 0.046400000000000004 | 2.3499999999999996 | 0.03916666666666666 | 1.9675 | CDM 20 | tyr | CDM 20 | tyr | ||||||||||||||
| CDM 21 | val | 0.04 | 0.1225 | CDM 21 | val | 0.04965 | 0.7324999999999999 | 0.03052083333333333 | 0.2725 | CDM 21 | val | 0.03052083333333333 | 0.05249999999999999 | ||||||||||||
| CDM 22 | cys/cyn | 0.04 | 2.4074999999999998 | 0.04815 | 2.4825 | 0.041375 | 2.3275 | CDM 22 | cys/cyn | CDM 22 | cys/cyn | ||||||||||||||
| CDM 23 | glu/gln | 0.04 | 0.14500000000000002 | CDM 23 | glu/gln | 0.04965 | 0.3275 | 0.043666666666666666 | 0.15 | CDM 23 | glu/gln | ||||||||||||||
| CDM 24 | gly/ser | 0.04 | 0.45999999999999996 | CDM 24 | gly/ser | 0.04965 | 0.9725 | 0.021611111111111112 | 0.8599999999999999 | 0.028666666666666663 | 0.487 | 0.0487 | 0.722 | CDM 24 | gly/ser | ||||||||||
| CDM 25 | asn/asp | 0.04 | 2.3425000000000002 | 0.04685 | 2.3475 | 0.039125 | 1.9900000000000002 | CDM 25 | asn/asp | CDM 25 | asn/asp | ||||||||||||||
| CDM 26 | cys/cyn/ser | 0.04 | 1.23 | 0.041 | 1.4425 | 0.0360625 | 1.5925 | CDM 26 | cys/cyn/ser | CDM 26 | cys/cyn/ser | ||||||||||||||
| CDM 27 | no amino acids | 0.04 | 0 | CDM 27 | no amino acids | 0.04965 | 0.1575 | CDM 27 | no amino acids | 0.039833333333333325 | 0.2525 | ||||||||||||||
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| continuous subculturing (validation ARG / ASN / GLY ) | |||||||||
| Koen van Grinsven (UvA) | |||||||||
| inoculated at t=0 with washed cells precultured in THY medium | |||||||||
| time (h) | |||||||||
| omission | 0 | 24 | 24.01 | 48 | 48.01 | 72 | |||
| CDM 2 | arg | 0.05 | 0.0325 | 0.0029545454545454545 | 0.04 | 0.0036363636363636364 | 0.08750000000000001 | no visual growth (by eye) | |
| CDM 3 | asn | 0.05 | 2.3575 | 0.023575 | 2.415 | 0.02415 | 2.275 | ||
| CDM 8 | gln | 0.05 | 2.325 | 0.023250000000000003 | 2.3649999999999998 | 0.023649999999999997 | 2.3874999999999997 | ||
| CDM 9 | gly | 0.05 | 0.34500000000000003 | 0.031363636363636364 | 0.365 | 0.03318181818181818 | 0.5974999999999999 | ||
| CDM 10 | his | 0.05 | 0.155 | 0.014090909090909091 | 0.07 | 0.006363636363636364 | 0.0625 | no visual growth (by eye) | |
| CDM 12 | leu | 0.05 | 0.145 | 0.013181818181818182 | 0.085 | 0.007727272727272728 | 0.13 | no visual growth (by eye) | |
| CDM 24 | gly/ser | 0.05 | 0.4675 | 0.022261904761904764 | 0.46499999999999997 | 0.02214285714285714 | 0.8200000000000001 | ||
| CDM 25 | asn/asp | 0.05 | 2.3625 | 0.023624999999999997 | 2.305 | 0.02305 | 2.3200000000000003 | ||
