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Team:Heidelberg/Tyrocidine week19 ms
From 2013.igem.org
Detection of short synthetic NRPs
03.09.2013 Pre-test
Pre-test: solubilize the synthetic dipeptide in Methanol; result: very bad solubility => not used for MS comparisons
03.09. - 04.09.2013 Preparation of samples 1-10 for mass spectrometry at neonate screening
Inocculation of BAP1 (Dipeptide I) and BAP1 (Tripeptide I) [Both tested on NRPS content with SDS-PAGE] in LB media. Washing and transfer of cells to M9 minimal media (evening 01.09.2013) and IPTG induction (noon 02.09.2013) of 50 ml (each) culture.
Taking first 15 ml samples after 2.5 hours. Centrifugation (3750 rpm, 4°C, 45 min) and separation of pellet and supernatant (2x ~7,5ml). Freezing with nitrogen and Lyophilization for 12 hours at ~-45°C and ~0,1 mbar.
Second sample (taken 12 hours after induction) and third one (21 hours after induction) have been treated as the first one.
Inocculation of native BAP1 cells in LB medium, washing in M9 and growing of cultures in M9 as negative reference for mass spec. Same treatment as positive samples.
| Sample number | Content | MS- Ornithine content [µmol/L] |
|---|---|---|
| 1 | Di A in BAP1 in M9+Cm Pellet 21 hrs after induction | 6,05 |
| 2 | Tri I in BAP1 in M9+Cm Pellet 12 hrs after induction | 29,81 |
| 3 | Tri I in BAP1 in M9+Cm Pellet 21 hrs after induction | 40,24 |
| 4 | Tri I in BAP1 in M9+Cm Pellet 2.5 hrs hrs after induction | 27,72 |
| 5 | Di A in BAP1 in M9+Cm Pellet 2.5 hrs after induction | 6,49 |
| 6 | Di A in BAP1 in M9+Cm Pellet 12 hrs after induction | 4,77 |
| 7 | Di A in BAP1 M9+cm Medium 1 2.5 hrs after induction | 4,78 |
| 8 | Tri I in BAP1 M9+Cm Medium 1 2.5 hrs after induction | 166,07 |
| 9 | Di A in BAP1 M9+Cm Medium 2 2.5 hrs after induction | 7,09 |
| 10 | Tri 1 in BAP1 M9+Cm Medium 2 2.5 hrs after induction | 173,36 |
Samples 1-6 contained different pellets, which have been resuspended in 500 µL PBS Buffer (1X) and treated with 3x20[s] ultra-sonification. Finally the samples have been centrifuged and supernatant decanted to 1.5 ml tubes for mass spectrometry at neonate screening.
Samples 7-10 contained lyophilized Medium and have been resuspended in 500 µL methanol for MS screening. As samples 1-6, supernatant was separated from solid contents and decanted to 1.5ml tubes. 100 µL were handed for MS at neonate screening. The rest has been frozen for hydrolysis.
05.09.-06.09.2013 Preparation of samples 11-21 for mass spectrometry at neonate screening
Samples treated as samples 1-10.
| Sample number | Content | MS result (Ornithine) |
|---|---|---|
| 11 | Di A in BAP1 M9+Cm after 12 hrs after induction Medium 1 | 15,5 |
| 12 | Di A in BAP1 M9+Cm after 21 hrs after induction Medium 1 | 10,2 |
| 13 | Di A in BAP1 M9+Cm after 21 hrs after induction Medium 2 | 10,7 |
| 14 | Di A in BAP1 M9+Cm after 21 hrs after induction Medium 3 | 7,8 |
| 15 | TriI in BAP1 M9+Cm after 12 hrs after induction Medium 1 | 1138,0 |
| 16 | TriI in BAP1 M9+Cm after 21 hrs after induction Medium 1 | 879,1 |
| 17 | TriI in BAP1 M9+Cm after 21 hrs after induction Medium 2 | 1343,4 |
| 18 | TriI in BAP1 M9+Cm after 21 hrs after induction Medium 3 | 1324,4 |
| 19 | BAP1 native M9 after 12 hrs after induction Medium 1 | 73,8 |
| 20 | BAP1 native M9 after 12 hrs after induction Medium 2 | 18,4 |
| 21 | BAP1 native M9 after 12 hrs after induction Pellet | 8,2 |
Samples with "H" are hydrolysates (6 M HCL at 105° over night). Those were hydrolysed over night, evaporated and neutralized for MS. Resolved supernatant for "media" samples has been analysed separately for the methanol supernatant and solid phase (marked with "sol").
| Sample number | Content | MS result |
|---|---|---|
| H 1 | Di A in BAP1 in M9+Cm Pellet 21 hrs after induction | not needed |
| H 2 | Tri I in BAP1 in M9+Cm Pellet 12 hrs after induction | not needed |
| H 3 | Tri I in BAP1 in M9+Cm Pellet 21 hrs after induction | not needed |
| H 4 | Tri I in BAP1 in M9+Cm Pellet 2.5 hrs after induction | not needed |
| H 5 | Di A in BAP1 in M9+Cm Pellet 2.5 hrs after induction | not needed |
| H 6 | Di A in BAP1 in M9+Cm Pellet 12 hrs after induction | not needed |
| H 7 | Di A in BAP1 M9+cm Medium 1 2.5 hrs after induction | not needed |
| H 8 | Tri I in BAP1 M9+Cm Medium 1 2.5 hrs after induction | not needed |
| H 9 | Di A in BAP1 M9+Cm Medium 2 2.5 hrs after induction | not needed |
| H 10 | Tri 1 in BAP1 M9+Cm Medium 2 2.5 hrs after induction | not needed |
| H 11 | Di A in BAP1 M9+Cm 12 hrs after induction Medium 1 | not needed |
| H 11 sol | Di A in BAP1 M9+Cm 12 hrs after induction Medium 1 | not needed |
| H 12 | Di A in BAP1 M9+Cm 21 hrs after induction Medium 1 | not needed |
| H 12 sol | Di A in BAP1 M9+Cm 21 hrs after induction Medium 1 | not needed |
| H 13 | Di A in BAP1 M9+Cm 21 hrs after induction Medium 2 | not needed |
| H 13 sol | Di A in BAP1 M9+Cm 21 hrs after induction Medium 2 | not needed |
| H 14 | Di A in BAP1 M9+Cm 21 hrs after induction Medium 3 | not needed |
| H 14 sol | Di A in BAP1 M9+Cm 21 hrs after induction Medium 3 | not needed |
| H 15 | TriI in BAP1 M9+Cm 12 hrs after induction Medium 1 | not needed |
| H 15 sol | TriI in BAP1 M9+Cm 12 hrs after induction Medium 1 | not needed |
| H 16 | TriI in BAP1 M9+Cm 21 hrs after induction Medium 1 | not needed |
| H 16 sol | TriI in BAP1 M9+Cm 21 hrs after induction Medium 1 | not needed |
| H 17 | TriI in BAP1 M9+Cm 21 hrs after induction Medium 2 | not needed |
| H 17 sol | TriI in BAP1 M9+Cm 21 hrs after induction Medium 2 | not needed |
| H 18 | TriI in BAP1 M9+Cm 21 hrs after induction Medium 3 | 273,3 |
| H 18 sol | TriI in BAP1 M9+Cm 21 hrs after induction Medium 3 | not needed |
| H 19 | BAP1 native M9 12 hrs after induction Medium 1 | not needed |
| H 19 sol | BAP1 native M9 12 hrs after induction Medium 1 | not needed |
| H 20 | BAP1 native M9 12 hrs after induction Medium 2 | not needed |
| H 21 | BAP1 native M9 12 hrs after induction Pellet | not needed |
09.09.-11.09.2013 further preparation for MS at chemistry
Preparation of cultures containing di- and tripeptide for MS (inocculation of LB, washing in M9, transfer to M9 and IPTG induction). IPTG induction at 12 p.m. 10.09., samples taken after 8.5 and 24 and 48 hours. Procedure as mentioned above (lysis, lophylisation etc.), but took pellet from 10 ml and lyophilized 7.5 ml supernatant again.
Results of hydrolysis (05.09.-06.09): A Maillard reaction occurred! Everything brown and many different products (as bonds between amino acids and sugars) arised during cooking in HCL (this protocol seems to be only used for pure peptide solutions). Therefore we have to search for other enzymatic reactions or other strategies to assess ornithine concentrations. Tried to rescue samples by an alkaline reaction with 5 M NaOH, but samples got only slightly lighter.
| Sample number | Content | MS result |
|---|---|---|
| 22 | DiA in BAPI Pellet in M9+Cm+IPTG 8.5 hrs after induction 10.09.2013 | 13,0 |
| 23 | TriI in BAPI Pellet in M9+Cm+IPTG 8.5 hrs after induction 10.09.2013 | 33,5 |
| 24 | Di A in BAPI Pellet1 in M9+Cm+IPTG 24 hrs after induction 11.09.2013 | 19,4 |
| 25 | TriI in BAPI Pellet1 in M9+Cm+IPTG 24 hrs after induction 11.09.2013 | 88,0 |
| 26 | Di A in BAPI Pellet2 in M9+Cm+IPTG 24 hrs after induction 11.09.2013 | 11,2 |
| 27 | TriI in BAPI Pellet2 in M9+Cm+IPTG 24 hrs after induction 11.09.2013 | 119,4 |
| 28 | DiA in BAPI Medium in M9+Cm+IPTG 8.5 hrs after induction 10.09.2013 | 7,8 |
| 29 | TriI in BAPI Medium in M9+Cm+IPTG 8.5 hrs after induction 10.09.2013 | 582,1 |
| 30 | Di A in BAPI Medium1 in M9+Cm+IPTG 24 hrs after induction 11.09.2013 | 13,9 |
| 31 | Di A in BAPI Medium2 in M9+Cm+IPTG 24 hrs after induction 11.09.2013 | 6,3 |
| 32 | TriI in BAPI Medium1 in M9+Cm+IPTG 24 hrs after induction 11.09.2013 | 520,2 |
| 33 | TriI in BAPI Medium2 in M9+Cm+IPTG 24 hrs after induction 11.09.2013 | 504,5 |
| 34 | Di A in BAPI Pellet in M9+Cm+IPTG 48 hrs after induction 12.09.2013 | 263,3 |
| 35 | Di A in BAPI Medium in M9+Cm+IPTG 48 hrs after induction 12.09.2013 | 498,2 |
| 36 | TriI in BAPI Pellet in M9+Cm+IPTG 48 hrs after induction 12.09.2013 | 373,7 |
| 37 | TriI in BAPI Medium in M9+Cm+IPTG 48 hrs after induction 12.09.2013 | 356,6 |
12.09.further preparation and delievery to MS at chemistry
Resuspension of lyophilized samples in 500 microliters methanol. Delievery of samples 25 and 26 to MS. Other samples to neonate screening.
