Difference between revisions of "Team:Aachen/Notebook/Documentation/Methanol Single Expression"
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! construct description !! sequencing confirmed plasmid !! confirmed cryo | ! construct description !! sequencing confirmed plasmid !! confirmed cryo | ||
|- | |- | ||
− | | RBS. | + | | RBS.mdh in pSB1A30 || #BZBQ# || #IGEM# |
|- | |- | ||
− | | RBS. | + | | RBS.hps in pSB1A30|| #4PAB# || #4P9H# |
|- | |- | ||
− | | RBS. | + | | RBS.phi in pSB1A30 || #PEA6# || #TFE1# |
|- | |- | ||
− | | RBS. | + | | RBS.xpk in pSB1A30|| #1NFT# || #ONK4# |
|} | |} | ||
Line 21: | Line 21: | ||
'''restrictions:''' | '''restrictions:''' | ||
# B0015 in pSB1C3 #TK6C# with XbaI and PstI | # B0015 in pSB1C3 #TK6C# with XbaI and PstI | ||
− | # | + | # hps in pSB1C3 #H4M9# with EcoRI and SpeI |
− | # | + | # phi in pSB1C3 #ANNA# with EcoRI and SpeI |
''calculation:'' | ''calculation:'' | ||
{|class="wikitable" | {|class="wikitable" | ||
− | ! components !! Volumes for (1.) B0015 [µl] !! Volumes for (2.) | + | ! components !! Volumes for (1.) B0015 [µl] !! Volumes for (2.) hps [µl] !! Volumes for (3.) phi [µl] |
|- | |- | ||
| ddH{{sub|2}}O || 14.7 || 15.2 || 15.5 | | ddH{{sub|2}}O || 14.7 || 15.2 || 15.5 | ||
Line 43: | Line 43: | ||
* restriction products stored in Falcon #TUSS# | * restriction products stored in Falcon #TUSS# | ||
'''ligation:''' | '''ligation:''' | ||
− | * '''(A)''' (1.) B0015 digest and (2.) | + | * '''(A)''' (1.) B0015 digest and (2.) hps digest into pSB1K30 digest (#SZKD#) |
− | * '''(B)''' (1.) B0015 digest and (3.) | + | * '''(B)''' (1.) B0015 digest and (3.) phi digest into pSB1K30 digest (#SZKD#) |
{|class="wikitable" | {|class="wikitable" | ||
! components !! Volumes for '''(A)''' [µl] !! Volumes for '''(B)''' [µl] | ! components !! Volumes for '''(A)''' [µl] !! Volumes for '''(B)''' [µl] | ||
Line 69: | Line 69: | ||
'''restriction''' | '''restriction''' | ||
− | * | + | *xpk in pSB1C3 (#KFEY#) with EcoRI and SpeI (X) |
− | * | + | *mdh in pSB1C3 (#9PBD#) with EcoRI and SpeI (M) |
{|class="wikitable" | {|class="wikitable" | ||
− | ! components !! Volumes for Xpk (X) [µl] !! Volumes for | + | ! components !! Volumes for Xpk (X) [µl] !! Volumes for mdh (M) [µl] |
|- | |- | ||
| ddH{{sub|2}}O || 16.5 || 15.4 | | ddH{{sub|2}}O || 16.5 || 15.4 | ||
Line 86: | Line 86: | ||
|} | |} | ||
− | * | + | *xpk restictions: tube X |
− | * | + | *mdh restrictions: tube M |
'''ligation''' | '''ligation''' | ||
− | * '''(A)''' B0015 digest from #OHOZ# and (2) | + | * '''(A)''' B0015 digest from #OHOZ# and (2) hps digest from #TUSS# into (K30) pSB1K30 digest from #SZKD# |
− | * '''(B)''' B0015 digest from #OHOZ# and (3) | + | * '''(B)''' B0015 digest from #OHOZ# and (3) phi digest from #TUSS# into (K30) pSB1K30 digest from #SZKD# |
− | * '''(C)''' B0015 digest from #OHOZ# and (X) | + | * '''(C)''' B0015 digest from #OHOZ# and (X) xpk digest from #OHOZ# into (K30) pSB1K30 digest from #SZKD# |
− | * '''(D)''' B0015 digest from #OHOZ# and (M) | + | * '''(D)''' B0015 digest from #OHOZ# and (M) mdh digest from #OHOZ# into (K30) pSB1K30 digest from #SZKD# |
{|class="wikitable" | {|class="wikitable" | ||
Line 104: | Line 104: | ||
| upstream Part Digest || 4 (B0015 digest B0015 from #OHOZ#) || 4 (B0015 digest B0015 from #OHOZ#) || 4 (B0015 digest B0015 from #OHOZ#) || 4 (B0015 digest B0015 from #OHOZ#) | | upstream Part Digest || 4 (B0015 digest B0015 from #OHOZ#) || 4 (B0015 digest B0015 from #OHOZ#) || 4 (B0015 digest B0015 from #OHOZ#) || 4 (B0015 digest B0015 from #OHOZ#) | ||
|- | |- | ||
− | | downstream Part Digest || 4 ( | + | | downstream Part Digest || 4 (hps digest 2 from #TUSS#) || 4 ((phi digest 3 from #TUSS#) || 4 (xpk digest X from #OHOZ#) || 4 (mdh digest M from #OHOZ#) |
|- | |- | ||
− | | pSB1K30 digest ( | + | | pSB1K30 digest (plasmid from #SZKD#) || 2 || 2 || 2 || 2 |
|- | |- | ||
| T4 DNA Ligase || 1 || 1 || 1 || 1 | | T4 DNA Ligase || 1 || 1 || 1 || 1 | ||
Line 115: | Line 115: | ||
==15-06-18== | ==15-06-18== | ||
− | * make | + | * make overnight cultures and masterplates |
** 12 clones of each construct were plated on master plates. 6 overnights were made. | ** 12 clones of each construct were plated on master plates. 6 overnights were made. | ||
Line 123: | Line 123: | ||
! gene / clones !! tube number !! expected length !! machine !! elongation time !! result | ! gene / clones !! tube number !! expected length !! machine !! elongation time !! result | ||
|- | |- | ||
− | | | + | | mdh.B0015 in pSB1K30 clones 1-12 || 37-48 || 1682 || 2 || 1'50" || Negative for all clones. Observed bands at 1227 bp (RFP) |
|- | |- | ||
− | | | + | | hps.B0015 in pSB1K30 clones 1-12 || 13-24 || 1160 || 1 || 1'13" || Negative for all clones. Observed bands at 1227 bp (RFP) |
|- | |- | ||
− | | | + | | phi.B0015 in pSB1K30 clones 1-12 || 1-12 || 1079 || 1 || 1'13" || Negative for all clones. Observed bands at 1227 bp (RFP) |
|- | |- | ||
− | | | + | | xpk.B0015 in pSB1K30 clones 1-12 || 73-84 || 3002 || 3 || 3'05" || Negative for all clones. Observed bands at 1227 bp (RFP) |
|} | |} | ||
* We picked only "red" clones, because we forgot to induce with IPTG. | * We picked only "red" clones, because we forgot to induce with IPTG. | ||
− | * | + | * with green light the red flourescence could be observed very well |
* we picked the few white clones and put them on a masterplate LB+K | * we picked the few white clones and put them on a masterplate LB+K | ||
* next step will be a new colony PCR on monday. | * next step will be a new colony PCR on monday. | ||
Line 144: | Line 144: | ||
! gene / clones !! tube number !! expected length !! machine !! elongation time !! result | ! gene / clones !! tube number !! expected length !! machine !! elongation time !! result | ||
|- | |- | ||
− | | | + | | mdh.B0015 in pSB1K30 clones 1-3 || 6-8 || 1682 || 2 || 1'50" || bands are too short |
|- | |- | ||
− | | | + | | hps.B0015 in pSB1K30 clones 1-3 || 1-3 || 1160 || 1 || 1'13" || bands are too short |
|- | |- | ||
− | | | + | | phi.B0015 in pSB1K30 clones 1-2 || 4-5 || 1079 || 1 || 1'13" || bands are too short |
|- | |- | ||
− | | | + | | xpk.B0015 in pSB1K30 clones 1-3 || 9-11 || 3002 || 3 || 4'00" || bands are too short |
|} | |} | ||
* restriction/ligation didn't work out as we expected. | * restriction/ligation didn't work out as we expected. | ||
− | * problem: pSB1K30 digest | + | * problem: pSB1K30 digest never works properly |
** next step: plate cryo #8T49# to get more plasmid of pSB1K30 | ** next step: plate cryo #8T49# to get more plasmid of pSB1K30 | ||
Line 194: | Line 194: | ||
|} | |} | ||
* ligate each RBS.CDS digest with the B0015 out of pSB1C3 into pSB1A30 digest | * ligate each RBS.CDS digest with the B0015 out of pSB1C3 into pSB1A30 digest | ||
+ | |||
{|class="wikitable" | {|class="wikitable" | ||
− | ! components !! (A) RBS. | + | ! components !! (A) RBS.mdh.B0015 in pSB1A30 volumes [µl] !! (B) RBS.hps.B0015 in pSB1A30 volumes [µl] !! (C) RBS.phi.B0015 in pSB1A30 volumes [µl] !! (D) RBS.xpk.B0015 in pSB1A30 volumes [µl] |
|- | |- | ||
| H{{sub|2}}O || 7 || 7 || 7 || 7 | | H{{sub|2}}O || 7 || 7 || 7 || 7 | ||
Line 210: | Line 211: | ||
|} | |} | ||
ligation products stored in falcon #CAJO# | ligation products stored in falcon #CAJO# | ||
− | * heatshock transformation into BL21. Don't forget | + | * heatshock transformation into BL21. Don't forget induction with IPTG for red/white screening! |
==15-06-24== | ==15-06-24== | ||
Line 245: | Line 246: | ||
! gene / clones !! tube number !! expected length !! machine !! elongation time !! result | ! gene / clones !! tube number !! expected length !! machine !! elongation time !! result | ||
|- | |- | ||
− | | RBS. | + | | RBS.mdh.B0015 in pSB1A30 clones 1 || 1 || 1682 || 2 || 0'25'' || no bands |
|- | |- | ||
− | | RBS. | + | | RBS.hps.B0015 in pSB1A30 clones 1-3 || 2, 3, 4 || 1160 || 2 || 0'25'' || no bonds |
|- | |- | ||
− | | RBS. | + | | RBS.phi.B0015 in pSB1A30 clones 1-6 || 5, 6, 7, 8, 9, 10 || 1079 || 2 || 0'25'' || very bad bands |
|- | |- | ||
− | | RBS. | + | | RBS.xpk.B0015 in pSB1A30 clones 1-3 || 11, 12, 13 || 3002 || 5 || 0'45'' || very bad bands |
|} | |} | ||
Line 267: | Line 268: | ||
! gene / clones !! tube number !! expected length !! machine !! elongation time !! result | ! gene / clones !! tube number !! expected length !! machine !! elongation time !! result | ||
|- | |- | ||
− | | RBS. | + | | RBS.mdh.B0015 in pSB1A30 clones 6, 8, 10, 11 || 1-4 || 1682 || 2 || 0'25'' || no bands |
|- | |- | ||
− | | RBS. | + | | RBS.hps.B0015 in pSB1A30 clones 3 || 5 || 1160 || 2 || 0'25'' || no bands |
|- | |- | ||
− | | RBS. | + | | RBS.phi.B0015 in pSB1A30 clones 2, 4, 6, 7, 8, 13, 17-21 || 6-16|| 1079 || 2 || 0'25'' || no bands |
|- | |- | ||
− | | RBS. | + | | RBS.xpk.B0015 in pSB1A30 clones 1, 7 || 17, 18 || 3002 || 5 || 0'45'' || no bands |
|} | |} | ||
− | *control with red clones: | + | *control with red clones: mdh (tube MR), hps (tube HR), phi (tube PR), xpk (XR) |
'''We have to think about a new strategy, as the Biobrick assembly seems not to work''' | '''We have to think about a new strategy, as the Biobrick assembly seems not to work''' | ||
Line 286: | Line 287: | ||
In a next step we will swap the RBS.CDS.B0015 constructs into pSB1K30 backbones. | In a next step we will swap the RBS.CDS.B0015 constructs into pSB1K30 backbones. | ||
− | Monday and Wednesday are the days with the most workload, while tuesday and thursday give space to plan further steps | + | Monday and Wednesday are the days with the most workload, while tuesday and thursday give space to plan further steps |
* cut #FW6L# with EcoRI and XbaI; reaction volume: 50 µl; water: 36.3 µl; DNA: 6.7 µl | * cut #FW6L# with EcoRI and XbaI; reaction volume: 50 µl; water: 36.3 µl; DNA: 6.7 µl | ||
Line 298: | Line 299: | ||
* digest RBS.CDS constructs with EcoRI and PstI | * digest RBS.CDS constructs with EcoRI and PstI | ||
− | ** RBS. | + | ** RBS.mdh from #YSVQ# |
− | ** RBS. | + | ** RBS.hps from #H4M9# |
− | ** RBS. | + | ** RBS.phi from #ANNA# |
− | ** RBS. | + | ** RBS.mdh from #KFEY# |
Line 311: | Line 312: | ||
* control digests on gel | * control digests on gel | ||
*results | *results | ||
− | *** #TUSS#: | + | *** #TUSS#:mdh (expected: 1220 bp / 2037 bp): bands at expected length |
− | *** #TUSS#: | + | *** #TUSS#:hps (expected: 698 bp / 2037 bp): bands at expected length |
− | *** #TUSS#: | + | *** #TUSS#:phi (expected: 617 bp / 2037 bp): bands at expected length |
− | *** #TUSS#: | + | *** #TUSS#:xpk (expected: 2540 bp / 2037 bp): bands at expected length |
*** #TUSS#:K30 (expected: 902 bp / 2222 bp): no bands at all | *** #TUSS#:K30 (expected: 902 bp / 2222 bp): no bands at all | ||
* ligation of fragments | * ligation of fragments | ||
** Ligation of #OHOZ#:1 with ... | ** Ligation of #OHOZ#:1 with ... | ||
− | *** #TUSS#: | + | *** ...#TUSS#:mdh (ML) |
− | *** #TUSS#: | + | *** ...#TUSS#:hps (HL) |
− | *** #TUSS#: | + | *** ...#TUSS#:phi (PL) |
− | *** #TUSS#: | + | *** ...#TUSS#:xpk (XL) |
− | Pipeting volumes for | + | Pipeting volumes for ligation |
{|class="wikitable" | {|class="wikitable" | ||
! component !! volume [µl] | ! component !! volume [µl] | ||
Line 336: | Line 337: | ||
| T4 Ligase || 1 | | T4 Ligase || 1 | ||
|} | |} | ||
− | * ligation products stored in | + | * ligation products stored in falcon #LIGJ# |
− | * | + | * transformation into BL21 Gold (DE3) |
==15-07-01== | ==15-07-01== | ||
Line 344: | Line 345: | ||
! gene / clones !! tube number / row in gel !! expected length !! machine !! elongation time !! result | ! gene / clones !! tube number / row in gel !! expected length !! machine !! elongation time !! result | ||
|- | |- | ||
− | | RBS. | + | | RBS.mdh.B0015 in pSB1A30 clones 6, 8, 10, 11 || 1-4 || 1682 || 2 || 0'25'' || no positive clones |
|- | |- | ||
− | | RBS. | + | | RBS.hps.B0015 in pSB1A30 clones 3 || 5 || 1160 || 2 || 0'25'' || no positive clones |
|- | |- | ||
− | | RBS. | + | | RBS.phi.B0015 in pSB1A30 clones 2, 4, 6, 7, 8, 13, 17-21 || 6-16|| 1079 || 2 || 0'25'' || clones 13, 19, 20 and 21 positive |
|- | |- | ||
− | | RBS. | + | | RBS.xpk.B0015 in pSB1A30 clones 1, 7 || 17, 18 || 3002 || 5 || 0'45'' || clone 7 positive |
|} | |} | ||
====RBS.CDS.B0015 in pSB1A3 (E, F, G, H)==== | ====RBS.CDS.B0015 in pSB1A3 (E, F, G, H)==== | ||
− | * make masterplates and | + | * make masterplates and overnight cultures |
====RBS.CDS in pSB1A30 (ML, HL, PL, XL)==== | ====RBS.CDS in pSB1A30 (ML, HL, PL, XL)==== | ||
Line 362: | Line 363: | ||
====RBS.CDS.B0015 in pSB1A30==== | ====RBS.CDS.B0015 in pSB1A30==== | ||
* purify plasmid of: | * purify plasmid of: | ||
− | ** | + | ** xpk clone 7 |
− | ** | + | ** phi clones 13, 19, 20, 21 |
====RBS.CDS.B0015 in pSB1A3 (E, F, G, H)==== | ====RBS.CDS.B0015 in pSB1A3 (E, F, G, H)==== | ||
Line 370: | Line 371: | ||
! construct ID !! construct description !! clone numbers !! expected length [bp] !! elongation time !! result | ! construct ID !! construct description !! clone numbers !! expected length [bp] !! elongation time !! result | ||
|- | |- | ||
− | | E || RBS. | + | | E || RBS.mdh.B0015 in pSB1A3 || 1-8 || 1631 || 0'25'' || nothing |
|- | |- | ||
− | | F || RBS. | + | | F || RBS.hps.B0015 in pSB1A3 || 9-16 || 1109 || 0'25'' || nothing |
|- | |- | ||
− | | G || RBS. | + | | G || RBS.phi.B0015 in pSB1A3 || 17-24 || 1028 || 0'25'' || nothing |
|- | |- | ||
− | | H || RBS. | + | | H || RBS.xpk.B0015 in pSB1A3 || 25-32 || 2951 || 0'45'' || clone 26 positive |
|} | |} | ||
* purify plasmid and make cryos of positive clones | * purify plasmid and make cryos of positive clones | ||
Line 382: | Line 383: | ||
− | * because | + | * because almost nothing worked: new transformation of ligation products #CAJO#:E/F/G/H |
** 50 µl competent DH5alpha | ** 50 µl competent DH5alpha | ||
** 2 µl ligation product | ** 2 µl ligation product | ||
Line 392: | Line 393: | ||
! construct ID !! construct description !! clone numbers !! expected length [bp] !! elongation time !! result | ! construct ID !! construct description !! clone numbers !! expected length [bp] !! elongation time !! result | ||
|- | |- | ||
− | | ML || RBS. | + | | ML || RBS.mdh in pSB1A30 || 33-40 || 1553 || 0'25'' || Clone: 34, 35, 37, 39 |
|- | |- | ||
− | | HL || RBS. | + | | HL || RBS.hps in pSB1A30 || 41-48 || 1031 || 0'25'' || Clone: 41 - 48 |
|- | |- | ||
− | | PL || RBS. | + | | PL || RBS.phi in pSB1A30 || 49-56 || 950 || 0'25'' || Clone: 49 - 56 |
|- | |- | ||
− | | XL || RBS. | + | | XL || RBS.xpk in pSB1A30 || 57-64 || 2873 || 0'45'' || clones 57 and 62 positive |
|} | |} | ||
* purify plasmid and make cryos of positive clones | * purify plasmid and make cryos of positive clones | ||
Line 406: | Line 407: | ||
! Construct !! Clone !! Plasmid ID !! Cryo ID !! Plasmid konzentration !! Sequencing result | ! Construct !! Clone !! Plasmid ID !! Cryo ID !! Plasmid konzentration !! Sequencing result | ||
|- | |- | ||
− | | H: RBS. | + | | H: RBS.xpk.B0015 in pSB1A3 || 26 || #8ZRY#|| #84BM# || 234.7 || VALID |
|- | |- | ||
− | | ML: RBS. | + | | ML: RBS.mdh in pSB1A30 || 34 || #1RZF# || #8AZD#|| 136.5 || discard |
|- | |- | ||
| || 35 || #PZRX# ||#MB3A# || 160.3 || VALID | | || 35 || #PZRX# ||#MB3A# || 160.3 || VALID | ||
Line 416: | Line 417: | ||
| || 39 || #6WTB# || #HEXE# || 133.8 || discard | | || 39 || #6WTB# || #HEXE# || 133.8 || discard | ||
|- | |- | ||
− | | HL: RBS. | + | | HL: RBS.hps in pSB1A30 || 41 || #YSAE# || #MKCP# || 165.4 || VALID |
|- | |- | ||
| || 42 || #T8KB# || #DR63# || 133.4 || discard | | || 42 || #T8KB# || #DR63# || 133.4 || discard | ||
|- | |- | ||
| || 43 || #H8KF# || #F4KN# || 158.1 || discard | | || 43 || #H8KF# || #F4KN# || 158.1 || discard | ||
− | | | + | |- |
| || 44 || #4PAB# || #4P9H# || 188.0 || VALID | | || 44 || #4PAB# || #4P9H# || 188.0 || VALID | ||
|- | |- | ||
− | | PL: RBS. | + | | PL: RBS.phi in pSB1A30 || 49 || #PEA6# || #TFE1# || 168.4 || VALID |
|- | |- | ||
| || 50 || #814A# || #QHB3# || 139.7 || discard | | || 50 || #814A# || #QHB3# || 139.7 || discard | ||
Line 432: | Line 433: | ||
| || 52 || #VBEZ# || #B9XO# || 135.2 || discard | | || 52 || #VBEZ# || #B9XO# || 135.2 || discard | ||
|- | |- | ||
− | | XL: RBS. | + | | XL: RBS.xpk in pSB1A30 || 57 || #4AE3# || #NF66# || 110.5 || VALID |
|- | |- | ||
| || 62 || #1NFT# || #ONK4# || 268.2 || VALID | | || 62 || #1NFT# || #ONK4# || 268.2 || VALID | ||
|- | |- | ||
− | | RBS. | + | | RBS.xpk.B0015 in pSB1A30 || 7 || #KANH# || #CNEK# || 110.1 || VALID |
|- | |- | ||
− | | RBS. | + | | RBS.phi.B0015 in pSB1A30 || 13 || #RX39# || #D8S8# || 85.0 || VALID |
|- | |- | ||
| || 19 || #3MNR# || #PZ6F# || 45.5 || discard | | || 19 || #3MNR# || #PZ6F# || 45.5 || discard | ||
Line 455: | Line 456: | ||
** list of items to take to iAMB: | ** list of items to take to iAMB: | ||
*** KAPA Mastermix | *** KAPA Mastermix | ||
− | *** | + | *** Primers |
*** Master plates | *** Master plates | ||
*** PCR strips | *** PCR strips | ||
Line 514: | Line 515: | ||
'''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 532: | 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 |
|} | |} | ||
Line 546: | Line 547: | ||
! 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 | ||
Line 552: | Line 553: | ||
| || || 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 | ||
Line 558: | Line 559: | ||
| || || 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 | ||
Line 564: | Line 565: | ||
| || || 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 572: | 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 | ||
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==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# | ||
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==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. | ||
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*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 | ||
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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