Difference between revisions of "Team:Aachen/Notebook/Documentation/Methanol Single Expression"
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'''It seems like the mastermix has problems with longer product lengths''' | '''It seems like the mastermix has problems with longer product lengths''' | ||
− | * spread a little bit of the cryos on the corresponding plates, to test colony PCR with | + | * spread a little bit of the cryos on the corresponding plates, to test colony PCR with colonys |
==15-07-07== | ==15-07-07== | ||
====Expression test==== | ====Expression test==== | ||
− | * first expression test of | + | * first expression test of mdh |
− | * plating of | + | * plating of mdh in A30 in BL21 clones: |
** #IGEM# | ** #IGEM# | ||
** #MB3A# | ** #MB3A# | ||
** #8ADZ# | ** #8ADZ# | ||
** #HEXE# | ** #HEXE# | ||
− | * plating of | + | * plating of rfp control: |
** #6DA3# | ** #6DA3# | ||
Line 533: | Line 533: | ||
! construct ID !! construct description !! numbers of clones !! expected length [bp] !! elongation time !! result | ! construct ID !! construct description !! numbers of clones !! expected length [bp] !! elongation time !! result | ||
|- | |- | ||
− | | E || RBS. | + | | E || RBS.mdh.B0015 in pSB1A3 || 27 || 1631 || 1'38'' || clone 6, 7, 8, 10, 17, 25, 27 positive |
|- | |- | ||
− | | F || RBS. | + | | F || RBS.hps.B0015 in pSB1A3 || 23 || 1109 || 1'07'' || clone 6, 9, 21, 22, 23 positive |
|- | |- | ||
− | | G || RBS. | + | | G || RBS.phi.B0015 in pSB1A3 || 24 || 1028 || 1'07'' || clone 4, 12, 13, 15, 17, 18, 19 positive |
|- | |- | ||
− | | H || RBS. | + | | H || RBS.xpk.B0015 in pSB1A3 || 22 || 2951 || 2'57'' || clone 2, 6, 17, 22 |
|} | |} | ||
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! construct ID !! construct description !! clone number !! cryo ID !! plasmid ID !! sequencing results | ! construct ID !! construct description !! clone number !! cryo ID !! plasmid ID !! sequencing results | ||
|- | |- | ||
− | | E || RBS. | + | | E || RBS.mdh.B0015 in pSB1A3 || 6 || #CHWW# || #H39R# || VALID |
|- | |- | ||
| || || 25 || #84BB# || #RRN3# || VALID | | || || 25 || #84BB# || #RRN3# || VALID | ||
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| || || 27 || #WBCK# || #L4OY# || VALID, but discard to safe space | | || || 27 || #WBCK# || #L4OY# || VALID, but discard to safe space | ||
|- | |- | ||
− | | F || RBS. | + | | F || RBS.hps.B0015 in pSB1A3 || 6 || #HBDX# || #EZWV# || VALID |
|- | |- | ||
| || || 21 || #KW16# || #8181# || VALID | | || || 21 || #KW16# || #8181# || VALID | ||
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| || || 22 || #RSWY# || #HVXO# || VALID, but discard to safe space | | || || 22 || #RSWY# || #HVXO# || VALID, but discard to safe space | ||
|- | |- | ||
− | | G || RBS. | + | | G || RBS.phi.B0015 in pSB1A3 || 4 || #E89D# || #X9WT# || discard before sequencing |
|- | |- | ||
| || || 12 || #HWPO# || #H9BV# || discard before sequencing | | || || 12 || #HWPO# || #H9BV# || discard before sequencing | ||
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| || || 18 || #F6V1# || #DK8F# || discard before sequencing | | || || 18 || #F6V1# || #DK8F# || discard before sequencing | ||
|- | |- | ||
− | | H || RBS. | + | | H || RBS.xpk.B0015 in pSB1A3 || 2 || #3A1V# || #DDT3# || discard before sequencing |
|- | |- | ||
| || || 6 || #E63S# || #Q9B3# || discard before sequencing | | || || 6 || #E63S# || #Q9B3# || discard before sequencing | ||
Line 573: | Line 573: | ||
* check sequencing results of sequencing from 15-07-02 | * check sequencing results of sequencing from 15-07-02 | ||
* prepare overnights for expression test of platet cryos: | * prepare overnights for expression test of platet cryos: | ||
− | ** 2x #IGEM# ( | + | ** 2x #IGEM# (mdh in pSB1A30) |
** #6DA3# (RFP control) | ** #6DA3# (RFP control) | ||
* check out results table on top of this page! | * check out results table on top of this page! | ||
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'''Expression Tests''' | '''Expression Tests''' | ||
− | *we test Mdh (+) induced, Mdh (-) not induced, and | + | *we test Mdh (+) induced, Mdh (-) not induced, and Rfp control |
{|class="wikitable" | {|class="wikitable" | ||
! time (h)\ Sample !! (1) Mdh (-) !! (2) Mdh (+) !! (0) RFP | ! time (h)\ Sample !! (1) Mdh (-) !! (2) Mdh (+) !! (0) RFP | ||
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* 7 µl of samples were loaded to gel | * 7 µl of samples were loaded to gel | ||
* SDS gel showed no significant bands | * SDS gel showed no significant bands | ||
− | * repeat expression test of | + | * repeat expression test of Mdh with other three genes next week |
==15-07-13== | ==15-07-13== | ||
* plate cryos on LB+A plates for precultures: | * plate cryos on LB+A plates for precultures: | ||
− | ** | + | ** mdh: #IGEM# |
− | ** | + | ** hps: #4P9H# |
− | ** | + | ** phi: #TFE1# |
− | ** | + | ** xpk: #ONK4# |
** RFP: #6DA3# | ** RFP: #6DA3# | ||
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==15-07-15== | ==15-07-15== | ||
====Expression Tests==== | ====Expression Tests==== | ||
− | * | + | * main culture preparation |
{|class="wikitable" | {|class="wikitable" | ||
! ID !! OD Preculture !! Volume added to 50 ml LB+A [ml] | ! ID !! OD Preculture !! Volume added to 50 ml LB+A [ml] | ||
|- | |- | ||
− | | | + | | mdh 1 || 5.031 || 1.5 |
|- | |- | ||
− | | | + | | mdh 2 || 4.872 || 1.5 |
|- | |- | ||
− | | | + | | hps 1 || 5.382 || 1.4 |
|- | |- | ||
− | | | + | | hps 2 || 5.421 || 1.4 |
|- | |- | ||
− | | | + | | phi 1 || 5.826 || 1.2 |
|- | |- | ||
− | | | + | | phi 2 || 6.264 || 1.2 |
|- | |- | ||
− | | | + | | xpk 1 || 5.442 || 1.4 |
|- | |- | ||
− | | | + | | xpk 2 || 5.361 || 1.4 |
|- | |- | ||
| RFP Control || 4.542 || 1.7 | | RFP Control || 4.542 || 1.7 | ||
|} | |} | ||
− | * | + | * main culture OD values |
{|class="wikitable" | {|class="wikitable" | ||
! sample !! OD (1 h) !! OD (1,5 h) !! OD (2 h) !! induced | ! sample !! OD (1 h) !! OD (1,5 h) !! OD (2 h) !! induced | ||
|- | |- | ||
− | | | + | | mdh 1 || 0.234 || 0.408 || 0.568 || no |
|- | |- | ||
− | | | + | | mdh 2 || 0.254 || 0.469 || 0.655 || yes |
|- | |- | ||
− | | | + | | hps 1 || 0.344 || 0.624 || 0.814 || no |
|- | |- | ||
− | | | + | | hps 2 || 0.305 || 0.577 || 0.761 || yes |
|- | |- | ||
− | | | + | | phi 1 || 0.282 || 0.526 || 0.721 || yes |
|- | |- | ||
− | | | + | | phi 2 || 0.295 || 0.581 || 0.776 || no |
|- | |- | ||
− | | | + | | xpk 1 || 0.270 || 0.476 || 0.648 || no |
|- | |- | ||
− | | | + | | xpk 2 || 0.275 || 0.494 || 0.650 || yes |
|- | |- | ||
| Rfp || 0.348 || 0.640 || 0.816 || yes | | Rfp || 0.348 || 0.640 || 0.816 || yes | ||
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====Expression tests==== | ====Expression tests==== | ||
* samples taken at 6 h and 21 h after induction with 50 µl IPTG | * samples taken at 6 h and 21 h after induction with 50 µl IPTG | ||
− | * cell pellets were | + | * cell pellets were resuspended in 150 µl water |
* suspensions were diluted to OD 10 | * suspensions were diluted to OD 10 | ||
− | * 10 µl SDS added to | + | * 10 µl SDS added to 50 µl cell suspension (OD 10) |
* incubate at 95 °C for 15 min | * incubate at 95 °C for 15 min | ||
* finished SDS preparations stored in Falcons: | * finished SDS preparations stored in Falcons: | ||
− | ** | + | ** mdh: #MDQL# |
− | ** | + | ** hps: #PBEA# |
− | ** | + | ** phi: #BHBB# |
− | ** | + | ** xpk: #ZR8V# |
==15-07-17== | ==15-07-17== | ||
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====Expression tests==== | ====Expression tests==== | ||
− | ''' | + | '''Mdh - SDS gel''' |
* negative expression test | * negative expression test | ||
− | ''' | + | '''Hps - SDS gel''' |
* no positive expression test so far | * no positive expression test so far | ||
− | ''' | + | '''Phi - SDS gel''' |
* no positive expression test so far | * no positive expression test so far | ||
− | ''' | + | '''Xpk - SDS gel''' |
* positive expression test | * positive expression test | ||
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====Expression tests==== | ====Expression tests==== | ||
− | ''' | + | '''Hps - SDS gel''' |
* no reliable results | * no reliable results | ||
− | ''' | + | '''Phi - SDS gel''' |
* no reliable results | * no reliable results | ||
− | ====New expression tests of | + | ====New expression tests of Hps and Phi==== |
− | * precultures from cryos #4P9H# ( | + | * precultures from cryos #4P9H# (hps) and #TFE1# (phi) |
− | * preculture from cryo #IGEM# ( | + | * preculture from cryo #IGEM# (mdh) as control |
==15-07-22== | ==15-07-22== | ||
− | '''New expression tests of | + | '''New expression tests of Hps and Phi''' |
− | * make maincultures of | + | * make maincultures of Hps and Phi |
* 2 ml samples taken 6 h after induction with IPTG | * 2 ml samples taken 6 h after induction with IPTG | ||
==15-07-23== | ==15-07-23== | ||
− | ====New expression tests of | + | ====New expression tests of Hps and Phi==== |
* prepare samples and load them on the gel | * prepare samples and load them on the gel | ||
* run gel at 130 V for 110 min | * run gel at 130 V for 110 min | ||
Line 760: | Line 731: | ||
==15-07-27== | ==15-07-27== | ||
− | clone | + | clone mdh, hps and phi into J61002 with constitutive Anderson Promoter J23119 for <span style="color:red">expression tests with Anderson Promoter 19</span> |
'''restriction''' | '''restriction''' | ||
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Cut with XbaI/PstI | Cut with XbaI/PstI | ||
− | * RBS. | + | * RBS.mdh in A30 #BZBQ# |
− | * RBS. | + | * RBS.hps in A30 #4PAB# |
− | * RBS. | + | * RBS.phi in A30 #PEA6# |
Products are stored in Falcon #FPTS# | Products are stored in Falcon #FPTS# | ||
Line 791: | Line 762: | ||
==15-07-29== | ==15-07-29== | ||
− | Do colony PCR with | + | Do colony PCR with masterplates with #A9W9# & #XE3D# (chosen annealing temp. 58 °C). (negative with RFP 1143bp, positive Hps 939 bp, positive Phi 858 bp, positive MDH 1461 bp). |
− | * results of cPCR and red/white screening disagree. In both screenings positive: | + | * results of cPCR and red/white screening disagree. In both screenings positive: phi #3, hps #1,2,3,4,5 |
* do NEW masterplates and overnights. | * do NEW masterplates and overnights. | ||
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'''Colony PCR on new masterplates''' | '''Colony PCR on new masterplates''' | ||
− | * this time with one primer that binds on the backbone (#A9W9#) and an insert-specific reverse primer (for | + | * this time with one primer that binds on the backbone (#A9W9#) and an insert-specific reverse primer (for mdh: #MSP1#, hps:#HSP2#, phi:#PSP2#) |
− | *based on the colony PCR, overnights | + | *based on the colony PCR, overnights of mdh #4&5, phi #3&5 and hps #1,2,3 were used for cryos and plasmid purification. |
− | * | + | *cryo cultures: #4EMV# #3PVM#; #1MDN# #38L9#; #KL96# #B39O# #BEZO# |
* purified plasmids: #PS1M# #ONMP#; #LSHO# #KQ1W#; #MXYY# #1DPL# #KTZ8# | * purified plasmids: #PS1M# #ONMP#; #LSHO# #KQ1W#; #MXYY# #1DPL# #KTZ8# | ||
− | *test digest | + | *test digest of plasmids with EcoRI. Expected length of hps.J61002: 2731bp; phi.J61002: 2650; mdh.J61002: 3253bp; negative rfp.J61002: 2987 bp |
==15-08-03== | ==15-08-03== | ||
− | *run test digest from 30th July on gel: | + | *run test digest from 30th July on gel: mdh#4, Mdh#5, phi#3, phi#5, hps#1, hps#2, hps#3; all bands at expected lenght |
− | *repeat colony PCR for | + | *repeat colony PCR for hps with #A9W9# and #HSP2#; expected lenght is 621 bp, chosen annealing Temp 58 °C |
*overnight cultures of successful clones | *overnight cultures of successful clones | ||
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==15-08-05== | ==15-08-05== | ||
− | *Prepare three main cultures ( | + | *Prepare three main cultures (hps #KL96#, mdh #3PVM#, phi #1MDN#). RFP control #13YN# did not grow. We have to use #BYS8# |
*Prepare 2 SDS (12 % for Mdh; 15 % for Hps, Phi) gels for tomorrow. | *Prepare 2 SDS (12 % for Mdh; 15 % for Hps, Phi) gels for tomorrow. | ||
Line 825: | Line 796: | ||
*Preculture of #BYS8# | *Preculture of #BYS8# | ||
− | Tomorrow: Prepare cell pellets according to the protocol. Run one gel with Mdh and | + | Tomorrow: Prepare cell pellets according to the protocol. Run one gel with Mdh and Rfp as control and a second gel with Hps, Phi and Mdh as control. The SDS-PAGE with Mdh and RFP as control has to be done on Friday. |
==15-08-06== | ==15-08-06== | ||
− | *Main culture of the | + | *Main culture of the Rfp control (BYS8). OD at 10.45 is 0.3. OD at 11:30 is 0.788. Take 2 2 ml samples at 17:15 and freeze the pellet. |
*Load SDS gel (15 %) with the 6 h-samples of Mdh (#3PVM#), Phi (#1MDN#) and Hps (#KL96#). Gel runs at 120 V for 90 minutes. Expected Mdh 40 kDa; Phi 20 kDa & Hps 22.5 kDa. All samples are negative. | *Load SDS gel (15 %) with the 6 h-samples of Mdh (#3PVM#), Phi (#1MDN#) and Hps (#KL96#). Gel runs at 120 V for 90 minutes. Expected Mdh 40 kDa; Phi 20 kDa & Hps 22.5 kDa. All samples are negative. | ||
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*Take 22h samples for back-ups and freeze them. | *Take 22h samples for back-ups and freeze them. | ||
− | *Prepare two more SDS gels (12 % for Mdh and | + | *Prepare two more SDS gels (12 % for Mdh and Rfp tomorrow and one 15 % as backup for Hps,Phi & Mdh) |
==15-08-07== | ==15-08-07== | ||
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Prepare suspensions with OD 0.13 of the samples (6 and 22 hours) | Prepare suspensions with OD 0.13 of the samples (6 and 22 hours) | ||
− | *Do SDS-PAGE Mdh and | + | *Do SDS-PAGE Mdh and Rfp with 12 % acrylamid. |
*Hps, Phi & Mdh with 15 % acrylamid. Results can be found in SDS-Gels 15-08-07. | *Hps, Phi & Mdh with 15 % acrylamid. Results can be found in SDS-Gels 15-08-07. | ||
− | *Gel with Hps, Phi & Mdh is completely | + | *Gel with Hps, Phi & Mdh is completely negative. |
− | *Gel with Mdh & | + | *Gel with Mdh & Rfp has a very strong band in the Rfp 6 h Sample slightly abouve 26 kDa but Rfp has 25 kDa. |
Prepare for sequencing: 15-08-07 Sequencing Sheet | Prepare for sequencing: 15-08-07 Sequencing Sheet | ||
Line 852: | Line 823: | ||
Plated all four genes + RFP in pSB1A30 in BL21 Gold (DE3) cells on LB+A (#IGEM#, #4P9H#, #TFE1#, #ONK4#, #6DA3#) | Plated all four genes + RFP in pSB1A30 in BL21 Gold (DE3) cells on LB+A (#IGEM#, #4P9H#, #TFE1#, #ONK4#, #6DA3#) | ||
− | ===sequencing results of | + | ===sequencing results of mdh, phi & hps in J61002=== |
* #PS1M#: VALID | * #PS1M#: VALID | ||
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==15-08-08== | ==15-08-08== | ||
− | Do | + | Do overnight cultures from all genes in pSB1A30 (#IGEM#, #4P9H#, #TFE1#, #ONK4#, #6DA3#), cultivation startet 16:30. |
==15-08-09== | ==15-08-09== | ||
− | Do 50 ml mainculture of A30 constructs (injected with 2 ml overnight culture) in LB media with | + | Do 50 ml mainculture of A30 constructs (injected with 2 ml overnight culture) in LB media with ampicilin. Incubation started at 10:07. Induce with 60 µl 1000x stock IPTG at 12:15 (OD600 of samples: #IGEM# 0.79; #4P9H# 0.758; #TFE1# 0.871; #ONK4# 0.640; #6DA3# 0.310). |
After 6 h (18:15) take 2x2 ml samples of each cultivation and again next morning. | After 6 h (18:15) take 2x2 ml samples of each cultivation and again next morning. |
Latest revision as of 09:35, 18 September 2015