Difference between revisions of "Team:Aachen/Notebook/Documentation/Methanol Monocistronic Diversity Library"

(→‎Constructing Precursor Plasmids)
(→‎Laboratory Notebook)
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digests stored in falcon #PSHF#
 
digests stored in falcon #PSHF#
 +
 +
 +
==15-08-09==
 +
 +
'''Gel control of MDH precursor plasmids''':
 +
*no good picture could be taken because the UV light destroyed the DNA; picture of it will be taken tomorrow in the Schwaneberg lab
 +
*master plates and overnights of the backup plasmids
 +
*plasmid purification and cryos of MDH precursor plasmids
 +
 +
==15-08-10==
 +
*make cryos and purify plasmids of MDH precursor plasmids
 +
*Measure concentrations of purified MDH precursor plasmids
 +
*Gel control of MDH precursor plasmids
 +
*Pipett sequencing of mdh precursor plasmids
 +
 +
* test digest of MDH precursor plasmids:
 +
 +
{|class="wikitable"
 +
! digest ID !! template ID !! expected lengths [bp] !! result
 +
|-
 +
| A || #CDPA# || 2226 / 1279 || 2226 / -
 +
|-
 +
| B || #ZYBC# || 2226 / 1279 || 2226 / 1700
 +
|-
 +
| C || #YNOB# || 2226 / 1279 || 2226 / 1700
 +
|-
 +
| D || #1KSW# || 2226 / 1279 || 2226 / 1700
 +
|-
 +
| E || #EBWO# || 2226 / 1279 || 2226 / -
 +
|-
 +
| F || #936C# || 2226 / 1279 || 2226 / -
 +
|-
 +
| G || #1MC6# || 2226 / 1279 || 2226 / 1279
 +
|-
 +
| H || #ESQD# || 2226 / 1279 || 2226 / 1279
 +
|-
 +
| I || #LWDR# || 2226 / 1187 || 2226 / 1187 + one long band
 +
|-
 +
| J || #4TSQ# || 2226 / 1187 || 2226 / 1187
 +
|-
 +
| K || #KTHK# || 2226 / 665 || 2226 / 665
 +
|-
 +
| L || #O9ZE# || 2226 / 584 || 2226 / 584
 +
|-
 +
| M || #6QE3# || 2226 / 2644 || 2226 / 2644 + one short band
 +
|-
 +
| N || #KVEY# || 2226 / 2644 || 2226 / 2644 + two long bands
 +
|}
 +
 +
digests stored in falcon #PSHF#
 +
 +
==15-08-11==
 +
* sequencing of following samples: #1MC6#, #ESQD#, #4TSQ#, #KTHK#, #O9ZE#
 +
* '''oligo assembly''': BBa_B1002-RDP (#HTSR#), BBa_B1006-RDP (#8AMS#), AP19-RDP (#MBWS#)
 +
**heated on 90 °C, then linear cool down in 45 minutes to room temperature
 +
 +
'''assembly of HPS Precursors'''
 +
{|class="wikitable"
 +
! gene !! RDP parts !! product in
 +
|-
 +
| Hps || (HPS.B1006.AP00) #RYPQ# + #VO4C# + #8AMS# + #M1ZY# + #OZD1# || #V9H9#
 +
|-
 +
| Hps || (HPS.B1006.AP04) #RYPQ# + #VO4C# + #8AMS# + #HB96# + #OZD1# || #XKX6#
 +
|-
 +
| Hps || (HPS.B1006.AP10) #RYPQ# + #VO4C# + #8AMS# + #4T9A# + #OZD1# || #SNDK# 
 +
|-
 +
| Hps || (HPS.B1006.AP19) #RYPQ# + #VO4C# + #8AMS# + #XYD9# + #OZD1# || #BC4V# 
 +
|}
 +
 +
*there is the very small probability that #SNDK# and #BC4V# were mixed up. Seqencing will show
 +
 +
*heat shocked in DH5alpha with 5 µl of DNA
 +
 +
==15-08-12==
 +
*there were colonies on the trafo plates of the HPS precursors
 +
*Oligos from 15-08-11: put them on 2% agarose:
 +
(good results: 0,8µl DNA and more than 3,5 µl 50bp Ladder)
 +
 +
{|class="wikitable"
 +
! part !! expected lenght !! result
 +
|-
 +
| BBa_B1002-RDP (#HTSR#) || 45 bp || fragments of ca. 50bp
 +
|-
 +
|  BBa_B1006-RDP (#8AMS#) || 50 bp || fragments of ca. 50bp
 +
|-
 +
|  AP19-RDP(#MBWS#) || 47 bp || fragments of ca. 50bp
 +
|}
 +
 +
===Assembly of PHI precursors===
 +
 +
{|class="wikitable"
 +
! gene !! RDP parts !! product in
 +
|-
 +
| Phi || (PHI.B1002.AP00)  || #VVSC#
 +
|-
 +
| Phi || (PHI.B1002.AP04) || #BNHW#
 +
|-
 +
| Phi || (PHI.B1002.AP10)  || #D1LL# 
 +
|-
 +
| Phi || (PHI.B1002.AP19)  || #EB8T# 
 +
|}
 +
*each 5µl transformed in DH5alpha
 +
*masterplates and colony PCR will follow
 +
 +
===colony PCR of MDH precursor clones===
 +
* primers: #MSP2# and #94LB#
 +
* elongation time: 1'00''
 +
* annealing temperature: 58°C
 +
* expected length: 920 bp
 +
{|class="wikitable"
 +
! ID !! description !! positive clones
 +
|-
 +
| A 1-12 || MDH.Ter.AP00  || 
 +
|-
 +
| B 1-15 || MDH.Ter.AP04 || 
 +
|-
 +
| C 1-12 || MDH.Ter.AP10 || 
 +
|-
 +
| D 1-11 || MDH.Ter.AP19 || 
 +
|}
 +
 +
===assembly of polycistronic versions===
 +
:T7 polycistronic version assembly product: #DSHM#
 +
:AP19 polycistronic version assembly product: #MCLC#
 +
*each 5 µl transformed in DH5 alpha and additionally, T7 version in BL21
 +
 +
==15-08-13==
 +
===tasks:===
 +
* colony PCR of HPS precursors
 +
* colony PCR of MDH precursors (again)
 +
* masterplates of T7 poly
 +
* masterplates of AP19 poly
 +
* masterplates of PHI precursors
 +
* overnight cultures of positive colony PCR clones
 +
 +
===Q5 amplification of Z-XPK.B0015-X'===
 +
*6 reactions á 100µl
 +
:::Mastermix:
 +
 +
{|class="wikitable"
 +
! component !! amount [µl]
 +
|-
 +
| ddH2O || 250,8
 +
|-
 +
| F-Primer #RKW3# || 24 
 +
|-
 +
| R-Primer #CNAT# || 24 
 +
|-
 +
| 2x Q5 Mastermix || 300
 +
|-
 +
| Template #8ZRY# || 1,2 
 +
|}
 +
:::Programme:
 +
{|class="wikitable"
 +
! Time !! Temp [°C]
 +
|-
 +
| 3:00 || 98
 +
|-
 +
| cyc. 0:25 || 98 
 +
|-
 +
| cyc. 0:20 || 58
 +
|-
 +
| cyc. 1:23 || 72 
 +
|-
 +
| 2:00 || 72
 +
|-
 +
| store || 8
 +
|}
 +
30 cycles
 +
 +
* all amplifications look good on the gel
 +
 +
===Colony PCRs===
 +
 +
'''MDH colony PCR:'''
 +
* Primers: #MSP2#, #94LB#
 +
{|class="wikitable"
 +
! step !! time !! Temp [°C]
 +
|-
 +
| denature || 5'00'' || 94
 +
|-
 +
| denature || 0'30'' || 94
 +
|-
 +
| anneal || 0'30'' || 55
 +
|-
 +
| elongate  || 1'00'' || 68 
 +
|-
 +
| elongate || 2'00'' || 68
 +
|-
 +
| store || forever || 16
 +
|}
 +
'''HPS colony PCR:'''
 +
* Primers: #HSP1#, #94LB#
 +
 +
{|class="wikitable"
 +
! step !! time !! Temp [°C]
 +
|-
 +
| denature || 5'00'' || 94
 +
|-
 +
| denature || 0'30'' || 94
 +
|-
 +
| anneal || 0'30'' || 55
 +
|-
 +
| elongate  || 0'40'' || 68 
 +
|-
 +
| elongate || 2'00'' || 68
 +
|-
 +
| store || forever || 16
 +
|}
 +
 +
'''Results:'''
 +
 +
{|class="wikitable"
 +
! construct ID !! construct description !! expected length [bp] !! positive clones
 +
|-
 +
| A 1-8 || MDH 00 precursor || 920 bp || (A16), A18
 +
|-
 +
| B 1-8 || MDH 04 precursor || 920 bp || B15, (B17), (B18) 
 +
|-
 +
| C 1-8 || MDH 10 precursor || 920 bp || -
 +
|-
 +
| D 1-8 || MDH 19 precursor || 920 bp || D12, D14
 +
|-
 +
| E 1-8 || HPS 00 precursor || 579 bp || - 
 +
|-
 +
| F 1-8 || HPS 04 precursor || 579 bp || - 
 +
|-
 +
| G 1-8 || HPS 10 precursor || 579 bp || - 
 +
|-
 +
| H 1-8 || HPS 19 precursor || 579 bp || - 
 +
|}
 +
 +
==15-08-14==
 +
===tasks:===
 +
* colony PCR of all PHI precursors
 +
* colony PCR of all HPS precursors
 +
* colony PCR of MDH AP10 precursor
 +
* colony PCR of AP19 polycistronic constructs (A)
 +
* colony PCR of T7 polycistronic constructs (G)
 +
* prep plasmid of:
 +
** MDH AP00 precursors (A16, A18)
 +
** MDH AP04 precursors (B15, B17, B18)
 +
** MDH AP19 precursors (D12, D14)
 +
* label gels of yesterday
 +
* BsaI clean-up
 +
 +
 +
===plasmids and cryos of MDH backups===
 +
 +
{|class="wikitable"
 +
! construct !! Cryo ID !! Plasmid ID !! Plasmid conc. [ng/µl]
 +
|-
 +
| MDH AP00 #16 || #Q6XM# || #CZ83# || 125,5
 +
|-
 +
| MDH AP00 #18 || #6F1N# || #XNTZ#|| 171,5
 +
|-
 +
| MDH AP04 #15 || #848Y# || #SRYN#|| 338
 +
|-
 +
| MDH AP04 #17  || #FCKK# || #TK9X#|| 134
 +
|-
 +
| MDH AP04 #18 || #3C1N# || #C8EB#|| 147,5
 +
|-
 +
| MDH AP19 #12|| #XYM1# || #VMBV#|| 72
 +
|-
 +
| MDH AP19 #14 || #RB99# || #4P6A#|| 101,5
 +
|}
 +
 +
===BsaI clean-up===
 +
* elution with H2O
 +
{|class="wikitable"
 +
! RDP part !! volume [µl] !! n [pmol] !! c [ng/µl]
 +
|-
 +
| Z-XPK.B0015-X' (1) || 98 || 1.6 || 29
 +
|-
 +
| Z-XPK.B0015-X' (2) || 98 || 1.44 || 26
 +
|}
 +
* evaporation of H2O
 +
* resuspend with TE buffer to 0.04 pmol/µl
 +
 +
stored in #NDMC# & #Z6QT#
 +
 +
===Colony PCRs===
 +
'''Program:'''
 +
{|class="wikitable"
 +
! step !! time !! T [°C]
 +
|-
 +
| denature || 5'00'' || 94
 +
|-
 +
| denature || 0'30'' || 94
 +
|-
 +
| annealing || 0'30'' || 55
 +
|-
 +
| elongation || varying || 68
 +
|-
 +
| final elongation || 2'00'' || 68
 +
|-
 +
| store || forever || 16
 +
|}
 +
 +
'''Results table:'''
 +
{|class="wikitable"
 +
! construct !! Primers !! elongation time !! expected length [bp] !! tested clones !! positive clones
 +
|-
 +
| MDH.AP10 precursor || #MSP2#, #94LB# || 1'00'' || 920 || 1, 2, 3, 4, 5, 19, 20, 21 || -
 +
|-
 +
| HPS.AP00 precursor || #W6WP#, #94LB# || 1'13'' || 1160 || 9-16 || 15
 +
|-
 +
| HPS.AP04 precursor || #W6WP#, #94LB# || 1'13'' || 1160 || 9-16 || 9
 +
|-
 +
| HPS.AP10 precursor || #W6WP#, #94LB# || 1'13'' || 1160 || 9-14 || 11, 13, 14
 +
|-
 +
| HPS.AP19 precursor || #W6WP#, #94LB# || 1'13'' || 1160 || 9-16 || 9, 13, 14 , 15, 16
 +
|-
 +
| PHI.AP00 precursor || #W6WP#, #94LB# || 1'13'' || 1074 || 1-8 || -
 +
|-
 +
| PHI.AP04 precursor || #W6WP#, #94LB# || 1'13'' || 1074 || 1-8 || -
 +
|-
 +
| PHI.AP10 precursor || #W6WP#, #94LB# || 1'13'' || 1074 || 1-8 || 2, 3, 4, 10
 +
|-
 +
| PHI.AP19 precursor || #W6WP#, #94LB# || 1'13'' || 1074 || 1-6 || 5
 +
|-
 +
| AP19 polycistronic construct || varying || 2'15'' || varying || 0, 1, 3, 4 || 1
 +
|-
 +
|  || #W6WP#, #HSP2# ||  || 1878 || 0, 1, 3, 4 ||
 +
|-
 +
|  || #HSP1#, #XSP3# ||  || 1939 || 0, 1, 3, 4 ||
 +
|-
 +
|  || #XSP2#, #94LB# ||  || 2182 || 0, 1, 3, 4 ||
 +
|-
 +
| T7 polycistronic construct || varying || 2'15'' || varying || 1, 2, 7, 8 || -
 +
|-
 +
|  || #W6WP#, #HSP2# ||  || 1927 || 1, 2, 7, 8 ||
 +
|-
 +
|  || #HSP1#, #XSP3# ||  || 1939 || 1, 2, 7, 8 ||
 +
|-
 +
|  || #XSP2#, #94LB# ||  || 2182 || 1, 2, 7, 8 ||
 +
|}
 +
* colony PCR of polycistronic constructs was repeated by Michael
 +
 +
===Assembly of MDH precursors===
 +
* this time with blockers (find protocol in sciebo)
 +
**Assembly product MDH.AP00: #N1O1#
 +
**Assembly product MDH.AP04: #VYM6#
 +
**Assembly product MDH.AP10: #Q9QQ#
 +
**Assembly product MDH.AP19: #ONWS#
 +
 +
==15-08-15==
 +
===Tasks:===
 +
* prep plasmid of yesterdays positive clones
 +
* (repeat colony PCR of T7 polycistronic clones)
 +
* '''make masterplates of MDH precursors (new assembly, this time with blockers)'''
 +
* assembly of HPS and PHI precursors (with blockers)
 +
* tidy up fridge
 +
* plate trafos of HPS and PHI precursors
 +
* plate new trafos of MDH.AP04 and MDH.AP10 precursors
 +
* assemble MDH expression plasmid (FO4B.XYD9.PRDW.PLTB.OZ1D)
 +
* trafo of MDH expression plasmid
 +
* plate trafo on LB+K plates
 +
 +
===Plasmid prep of Hps and Phi precursors===
 +
 +
{|class="wikitable"
 +
! construct !! Cryo ID !! Plasmid ID !! Plasmid conc. [ng/µl]
 +
|-
 +
| HPS AP19 #14 || #FOF4# || #B3PQ# || 170
 +
|-
 +
| HPS AP10 #14 || #638V# || #P4SB#|| 148
 +
|-
 +
| HPS AP10 #11 || #4ZSB# || #P89F#|| 143,5
 +
|-
 +
| HPS AP04 #9  || #NY1C# || #1TMT#|| 190,5
 +
|-
 +
| HPS AP19 #15 || #SM3L# || #NFOS#|| 204
 +
|-
 +
| HPS AP00 #15|| #HAKR# || #6ACO#|| 126,5
 +
|-
 +
| HPS AP19 #13 || #COMY# || #VSWC#|| 261
 +
|-
 +
| HPS AP19 #9  || #OZ4O# || #L8Y4#|| 171,5
 +
|-
 +
| HPS AP10 #13 || #8P4S# || #4QLE#|| 238,5
 +
|-
 +
| HPS AP19 #16|| #CSLZ# || #6P68#|| 121,5
 +
|-
 +
| PHI AP10 #3 || #BV3E# || #L9E9#|| 161
 +
|-
 +
| PHI AP10 #4 || #OFHN# || #FVVW#|| 171,5
 +
|-
 +
| PHI AP19 #5 || #E6HX# || #HA1W#|| 192,5
 +
|}
 +
 +
===Assembly of HPS precursors===
 +
* this time with blockers (find protocol in sciebo)
 +
**Assembly product HPS.AP00: #WQPR#
 +
**Assembly product HPS.AP04: #6WOE#
 +
**Assembly product HPS.AP10: #XHXQ#
 +
**Assembly product HPS.AP19: #VM1V#
 +
 +
===Assembly of PHI precursors===
 +
* this time with blockers (find protocol in sciebo)
 +
**Assembly product PHI.AP00: #AFA4#
 +
**Assembly product PHI.AP04: #8FXO#
 +
**Assembly product PHI.AP10: #KCDE#
 +
**Assembly product PHI.AP19: #QHM6#
 +
 +
===Assembly of MDH expression plamid===
 +
* find protocol in sciebo
 +
** assembly product AP19.MDH.TER in #DK1F#
 +
 +
===Transformations===
 +
{|class="wikitable"
 +
! # !! description !! assembly product
 +
|-
 +
| 1 || HPS.AP00 precursor || #WQPR#
 +
|-
 +
| 2 || HPS.AP04 precursor || #6WOE#
 +
|-
 +
| 3 || HPS.AP10 precursor || #XHXQ#
 +
|-
 +
| 4 || HPS.AP19 precursor || #VM1V#
 +
|-
 +
| 5 || PHI.AP00 precursor || #AFA4#
 +
|-
 +
| 6 || PHI.AP04 precursor || #8FXO#
 +
|-
 +
| 7 || PHI.AP10 precursor || #KCDE#
 +
|-
 +
| 8 || PHI.AP19 precursor || #QHM6#
 +
|-
 +
| 9 || MDH.AP04 precursor || #VYM6#
 +
|-
 +
| 10 || MDH.AP10 precursor || #Q9QQ#
 +
|-
 +
| 11 || AP19.MDH.TER expression plasmid || #DK1F#
 +
|}
 +
 +
==15-08-16==
 +
===Tasks:===
 +
* prep plasmid of polycistronic clone #1
 +
**Cryo #Q1Q3#
 +
**purified plasmid #POLY#
 +
* make masterplates of yesterdays trafos
 +
* do/make colony PCR/overnights of yesterdays masterplates
 +
* test KAPA 2G Mix
 +
* do 'colony PCR' with purified plasmid of AP19 poly
 +
 +
==15-08-17==
 +
===Precursor plasmids:===
 +
{|class="wikitable"
 +
! construct !! cryo IDs !! plasmid IDs !! screening result
 +
|-
 +
| MDH00 precursor || #L99Q#, #VLO3#, #EHQC#, #OD19#, #FHEL#, #MA9W# || #HR4E#, #ZZEE#, #3HPD#, #M1VB#, #QDVN#, #AHRM# || look at table below
 +
|-
 +
| MDH04 precursor || #OB4C#, #TVND#, #1S1V#, #COXQ#, #3NZQ#, #YZXD# || #NEKS#, #OX44#, #P9HA#, #9CBA#, #SYMS#, #YY6O# ||
 +
|-
 +
| MDH19 precursor || #HBWZ#, #K3V6#, #CYF6#, #S1HT#, #TKO4#, #4MS8# || #BY9L#, #DNRY#, #KV18#, #1KEA#, #SPHL#, #N1BM# ||
 +
|-
 +
| HPS00 precursor || #AVVO#, #X9PR#, #M4H8#, #44WP#, #DE4A#, #QMRB# || #XVYK#, #CMC1#, #ERZK#, #WXO8#, #D8PY#, #8AFD# ||
 +
|-
 +
| HPS04 precursor || #8SW4#, #RSH9#, #1C8L#, #E1OQ#, #Y3TR#, #3BLV# || #YE8A#, #DERR#, #DVRP#, #FKNX#, #N3E4#, #HNV4# ||
 +
|-
 +
| HPS10 precursor || #R8YT#, #QRZW#, #KVYA#, #NEFO#, #QN64#, #NSSM# || #1EQ3#, #PA13#, #FAES#, #Z64V#, #WSAD# ||
 +
|-
 +
| PHI00 precursor || #TNBW#, #VSQN#, #YPFO#, #EELW#, #9H4T#, #VLHL# || #D6QY#, #931O#, #QWQP#, #AERW#, #ORX1#, #ZCTY# ||
 +
|-
 +
| PHI04 precursor || #4MNP#, #DE96#, #YKZT#, #AQF8#, #ETTS#, #8COE# || #FXSC#, #P1O1#, #8CZD#, #EZYZ#, #VPP4#, #3EZP# ||
 +
|-
 +
| PHI10 precursor || #SQ9N#, #AO6C#, #9RZL#, #48P3#, #PPWW#, #DB3R# || #FTX4#, #VM6Z#, #V4CA#, #XW1Y#, #AZND#, #WCF3# ||
 +
|-
 +
| PHI19 precursor || #3WKA#, #94OY#, #TVM1#, #KPCQ#, #FB9L#, #HWM4# || #THQA#, #BND4#, #6H3Z#, #ME9O#, #39CF#, #RABS# ||
 +
|}
 +
 +
===further plasmids:===
 +
* AP19 Poly
 +
** cryo: #LHZV#
 +
** pasmid: #WVDR#
 +
* MDH expression plasmid
 +
** cryo: #MSX3#
 +
** plasmid: #WDXZ#
 +
* HPS19 precursor
 +
** cryo: #ABCE#
 +
** plasmid: #TTC4#
 +
 +
'''AP19.MDH.Ter in pSB1KRDP'''
 +
 +
The MDH clones 2-19 were screened with a KAPA2G colony PCR. Controls are clone #1 and the plasmid #WDXZ#. Initial denaturation was set to 8'00" and the primer concentration was increased by 30 % to 6.5 mM. 0'15" denaturation and 0'15" annealing at 65-0.4°C per cycle. Elongation time was 0'20" for an expected product of 1205 bp (#W6WP# and #MSP3#). The PCR was not successful - only a band of plasmid template was visible.
 +
 +
Overnight cultures were prepared for all clones.
 +
 +
==15-08-18==
 +
===Morning===
 +
Plasmid preps and sequencing. All '''BOLD''' IDs do not exist yet. Prepping instructions:
 +
* prep each of the 6 POLY cultures, but elute all into one target container named #C3NS#
 +
* make '''cryos''' of the AP19.MDH cultures of clone #2+3 and then prep them. (both IDs in the table below)
 +
 +
Pipetting calculation table is ready at: '''\WetLab\Documentation\Methanol\15-08-18 Sequencings\'''
 +
 +
{|class="wikitable"
 +
! construct & clone number !! cryo ID !! plasmid ID !! sequencing sample numbers !! sequencing result !! further procedure
 +
|-
 +
| AP19.POLY in pSB1KRDP #1 || #W3NB# || '''#C3NS#''' || 1-8 || NO CONTIGS FOUND || Repeat Sequencing
 +
|-
 +
| AP19.MDH.Ter in pSB1KRDP #1 || above || #WDXZ# || 9+10 || empty backbone || Discard
 +
|-
 +
| AP19.MDH.Ter in pSB1KRDP #2 || '''#MSX3#''' || '''#FDX4#''' || 11+12 || mutation in promoter || Discard
 +
|-
 +
| AP19.MDH.Ter in pSB1KRDP #3 || '''#FZ8Q#''' || '''#MOMX#''' || 13+14 || empty backbone || Discard
 +
|-
 +
| MDH.TER.AP00 in pSB1CRDP #1 || #L99Q# || #HR4E# || 15 || MUTATION || Discard
 +
|-
 +
| MDH.TER.AP00 in pSB1CRDP #2 || #OD19# || #M1VB# || 16 || MUTATION || Discard
 +
|-
 +
| MDH.TER.AP00 in pSB1CRDP #6 || #MA9W# || #AHRM# || 17 || MUTATION || Discard
 +
|-
 +
| MDH.TER.AP04 in pSB1CRDP #1 || #OB4C# || #NEKS# || 18 || SPACER MUTATION || Discard
 +
|-
 +
| MDH.TER.AP04 in pSB1CRDP #2 || #TVND# || #OX44# || 19 || VALID || Keep
 +
|-
 +
| MDH.TER.AP04 in pSB1CRDP #3 || #1S1V# || #P9HA# || 20 || VALID || Discard
 +
|-
 +
| MDH.TER.AP19 in pSB1CRDP #1 || #HBWZ# || #BY9L# || 21 || VALID || Discard
 +
|-
 +
| MDH.TER.AP19 in pSB1CRDP #2 || #K3V6# || #DNRY# || 22 || VALID || Discard
 +
|-
 +
| MDH.TER.AP19 in pSB1CRDP #3 || #CYF6# || #KV18# || 23 || VALID || Keep
 +
|-
 +
| HPS.B1006.AP00 in pSB1CRDP #3 || #M4H8# || #ERZK# || 24 || VALID || Discard
 +
|-
 +
| HPS.B1006.AP00 in pSB1CRDP #4 || #44WP# || #WXO8# || 25 || VALID || Keep
 +
|-
 +
| HPS.B1006.AP00 in pSB1CRDP #5 || #DE4A# || #D8PY# || 26 || NO CONTIGS FOUND || Discard
 +
|-
 +
| HPS.B1006.AP04 in pSB1CRDP #2 || #RSH9# || #DERR# || 27 || MUTATION || Discard
 +
|-
 +
| HPS.B1006.AP04 in pSB1CRDP #3 || #1C8L# || #DVRP# || 28 || MUTATION || Discard
 +
|-
 +
| HPS.B1006.AP04 in pSB1CRDP #6 || #3BLV# || #HNV4# || 29 || MUTATION || Discard
 +
|-
 +
| HPS.B1006.AP10 in pSB1CRDP #2 || #QRZW# || #1EQ3# || 30 || NO CONTIGS FOUND || Discard
 +
|-
 +
| HPS.B1006.AP10 in pSB1CRDP #4 || #NEFO# || #FAES# || 31 || NO CONTIGS FOUND|| Discard
 +
|-
 +
| HPS.B1006.AP19 in pSB1CRDP #1 || #ABCE# || #TTC4# || 32 || MUTATION || Discard
 +
|-
 +
| PHI.B1002.AP00 in pSB1CRDP #2 || #VSQN# || #931O# || 33 || VALID || Keep
 +
|-
 +
| PHI.B1002.AP00 in pSB1CRDP #3 || #YPFO# || #QWQP# || 34 || NO CONTIGS FOUND || Discard
 +
|-
 +
| PHI.B1002.AP00 in pSB1CRDP #4 || #EELW# || #AERW# || 35 || MUTATION || Discard
 +
|-
 +
| PHI.B1002.AP04 in pSB1CRDP #1 || #4MNP# || #FXSC# || 36 || NO CONTIGS FOUND || Discard
 +
|-
 +
| PHI.B1002.AP04 in pSB1CRDP #3 || #YKZT# || #8CZD# || 37 || PROMOTER OK || Discard
 +
|-
 +
| PHI.B1002.AP04 in pSB1CRDP #4 || #AQF8# || #EZYZ# || 38 || VALID || Keep
 +
|-
 +
| PHI.B1002.AP10 in pSB1CRDP #3 || #9RZL# || #V4CA# || 39 || VALID || Keep
 +
|-
 +
| PHI.B1002.AP10 in pSB1CRDP #4 || #48P3# || #XW1Y# || 40 || VALID || Discard
 +
|-
 +
| PHI.B1002.AP10 in pSB1CRDP #6 || #DB3R# || #WCF3# || 41 || VALID || Discard
 +
|-
 +
| PHI.B1002.AP19 in pSB1CRDP #2 || #94OY# || #BND4# || 42 || NO CONTIGS FOUND || Discard
 +
|-
 +
| PHI.B1002.AP19 in pSB1CRDP #5 || #FB9L# || #39CF# || 43 || MUTATION || Discard
 +
|-
 +
| PHI.B1002.AP19 in pSB1CRDP #6 || #HWM4# || #RABS# || 44 || VALID || Keep
 +
|}
 +
 +
===Noon===
 +
Prep plasmids of T7.POLY in BL21 (DE3)
 +
 +
{|class="wikitable"
 +
! construct & clone number !! cryo ID !! plasmid ID !! check PCR result
 +
|-
 +
| T7.lacO.POLY in pSB1KRDP #1 || #LHBN# || #VYCO# || discard
 +
|-
 +
| T7.lacO.POLY in pSB1KRDP #5 || #HB93# || #ANPV# || discard
 +
|-
 +
| T7.lacO.POLY in pSB1KRDP #6 || #6LOZ# || #96TS# || discard
 +
|-
 +
| T7.lacO.POLY in pSB1KRDP #7 || #KYOQ# || #XVE3# || discard
 +
|-
 +
| T7.lacO.POLY in pSB1KRDP #8 || #MCMR# || #BSLA# || discard
 +
|}
 +
 +
* RDP-Assembly of T7.lacO.MDH.Ter in pSB1KRDP (product: #....#)
 +
* transform #....# into BL21 Gold (DE3) AND DH5a
 +
====Check-PCR the prepped T7.POLY plasmids====
 +
*with the three primer pairs, to find a complete construct
 +
*elongation time 0'33''
 +
* #POLY# as positive control (fitting bands already 15-08-16)
 +
{|class="wikitable"
 +
! Part ID !! Sample !! Primers !! expected length [bp] !! result
 +
|-
 +
| #VYCO# || 1 || #W6WP# + #HSP2# || 1927 || negative
 +
|-
 +
|  || 2 || #HSP1# + #XSP3#|| 1939 || negative
 +
|-
 +
|  || 3 || #XSP2# + #94LB#|| 2182 || negative
 +
|-
 +
| #ANPV# || 4 || #W6WP# + #HSP2#||1927 || negative
 +
|-
 +
|  || 5 || #HSP1# + #XSP3#||1939 || negative
 +
|-
 +
|  || 6 || #XSP2# + #94LB#||2182 || negative
 +
|-
 +
| #96TS# || 7 || #W6WP# + #HSP2#||1927 || negative
 +
|-
 +
|  || 8 || #HSP1# + #XSP3#||1939 || negative
 +
|-
 +
|  || 9 || #XSP2# + #94LB#||2182 || negative
 +
|-
 +
| #XVE3# || 10 || #W6WP# + #HSP2#||1927 || negative
 +
|-
 +
|  || 11 || #HSP1# + #XSP3#||1939 || negative
 +
|-
 +
|  || 12 || #XSP2# + #94LB#||2182 || negative
 +
|-
 +
| #BSLA# || 13 || #W6WP# + #HSP2#||1927 || negative
 +
|-
 +
|  || 14 || #HSP1# + #XSP3#||1939 || negative
 +
|-
 +
|  || 15 || #XSP2# + #94LB#||2182 || negative
 +
|-
 +
| #POLY# || 16 || #W6WP# + #HSP2#||1927 || positive
 +
|-
 +
|  || 17 || #HSP1# + #XSP3#||1939 || positive
 +
|-
 +
|  || 18 || #XSP2# + #94LB#||2182 || positive
 +
|}
 +
* Label yesterdays SDS gel and upload the results to the internal wiki.
 +
 +
===Afternoon===
 +
* make M9+K+Glucose40mM overnight cultures of
 +
** B0015 in pSB1K3 in DH5a
 +
** AP19.POLY in pSB1KRDP in DH5a clone #1
 +
** AP19.MDH.Ter in pSB1KRDP in DH5a clone #1-6
 +
* make two LB+K overnight cultures of T7.POLY on LB+K
 +
 +
====RDP Assembly====
 +
* Assembly of #FO4B# and #PVFL# (Kanamycin Cap with high copy cap) the product is #QWRC# -> we need this strain as a negative control that carries the plasmid but does not express anything
 +
 +
Transform important plasmids into BL21 Gold (DE3) and plate on M9+K+40mMGlucose as well as LB+K
 +
{|class="wikitable"
 +
! construct !! transformed plasmid !! BL21 Gold (DE3) cryo
 +
|-
 +
| AP19.POLY in pSB1KRDP || #POLY# || #....#
 +
|-
 +
| AP19.MDH.Ter in pSB1KRDP || #WDXZ#, #FDX4#, #MOMX# || #....#
 +
|-
 +
| pSB1KRDP || #QWRC#  || #....#
 +
|}
  
 
=References=
 
=References=

Revision as of 10:57, 18 September 2015


Laboratory Notebook

15-08-08

Constructing Precursor Plasmids

gene RDP parts product in expected length / result plasmid ID cryo ID
Mdh (mdh.TER.AP00) #XOXM# + #PRDW# + #PLTB# + #PQT6# + #PVFL# #K3YV# ; empty 3497 bp / no evaluable result #CDPA#, #ZYBC# #SF63#, #MBRR#
Mdh (mdh.TER.AP04) #XOXM# + #PRDW# + #PLTB# + #WPW6# + #PVFL# #AVQ6#; empty 3497 bp / no evaluable result #YNOB#, #1KSW# #DCN9#, #WHBF#
Mdh (mdh.TER.AP10) #XOXM# + #PRDW# + #PLTB# + #3S8V# + #PVFL# #ZE1A#; empty 3497 bp / no evaluable result #EBWO#, #936C# #8KAA#, #EB88#
Mdh (mdh.TER.AP19) #XOXM# + #PRDW# + #PLTB# + #MBWS# + #PVFL# #NDKS#; empty 3497 bp / no evaluable result #1MC6#, #ESQD# #48C8#, #8BZ9#
  • heat shocked in DH5alpha with 5 µl of DNA

15-08-09

Gel control of MDH precursor plasmids:

  • no good picture could be taken because the UV light destroyed the DNA; picture of it will be taken tomorrow in the Schwaneberg lab
  • master plates and overnights of the backup plasmids
  • plasmid prep and cryos of MDH precursor plasmids

15-08-10

  • make cryos and prepp plasmids of MDH precursor plasmids
  • Measure concentrations of prepped MDH precursor plasmids
  • Gel control of MDH precursor plasmids in Schwaneberg Lab
  • Measure concentrations of prepped backup plasmids
  • Pipett sequencing of backup plasmids and MDH precursor plasmids
  • test digest of backup and MDH precursor plasmids:
digest ID template ID expected lengths [bp] result
A #CDPA# 2226 / 1279 2226 / -
B #ZYBC# 2226 / 1279 2226 / 1700
C #YNOB# 2226 / 1279 2226 / 1700
D #1KSW# 2226 / 1279 2226 / 1700
E #EBWO# 2226 / 1279 2226 / -
F #936C# 2226 / 1279 2226 / -
G #1MC6# 2226 / 1279 2226 / 1279
H #ESQD# 2226 / 1279 2226 / 1279
I #LWDR# 2226 / 1187 2226 / 1187 + one long band
J #4TSQ# 2226 / 1187 2226 / 1187
K #KTHK# 2226 / 665 2226 / 665
L #O9ZE# 2226 / 584 2226 / 584
M #6QE3# 2226 / 2644 2226 / 2644 + one short band
N #KVEY# 2226 / 2644 2226 / 2644 + two long bands

digests stored in falcon #PSHF#


15-08-09

Gel control of MDH precursor plasmids:

  • no good picture could be taken because the UV light destroyed the DNA; picture of it will be taken tomorrow in the Schwaneberg lab
  • master plates and overnights of the backup plasmids
  • plasmid purification and cryos of MDH precursor plasmids

15-08-10

  • make cryos and purify plasmids of MDH precursor plasmids
  • Measure concentrations of purified MDH precursor plasmids
  • Gel control of MDH precursor plasmids
  • Pipett sequencing of mdh precursor plasmids
  • test digest of MDH precursor plasmids:
digest ID template ID expected lengths [bp] result
A #CDPA# 2226 / 1279 2226 / -
B #ZYBC# 2226 / 1279 2226 / 1700
C #YNOB# 2226 / 1279 2226 / 1700
D #1KSW# 2226 / 1279 2226 / 1700
E #EBWO# 2226 / 1279 2226 / -
F #936C# 2226 / 1279 2226 / -
G #1MC6# 2226 / 1279 2226 / 1279
H #ESQD# 2226 / 1279 2226 / 1279
I #LWDR# 2226 / 1187 2226 / 1187 + one long band
J #4TSQ# 2226 / 1187 2226 / 1187
K #KTHK# 2226 / 665 2226 / 665
L #O9ZE# 2226 / 584 2226 / 584
M #6QE3# 2226 / 2644 2226 / 2644 + one short band
N #KVEY# 2226 / 2644 2226 / 2644 + two long bands

digests stored in falcon #PSHF#

15-08-11

  • sequencing of following samples: #1MC6#, #ESQD#, #4TSQ#, #KTHK#, #O9ZE#
  • oligo assembly: BBa_B1002-RDP (#HTSR#), BBa_B1006-RDP (#8AMS#), AP19-RDP (#MBWS#)
    • heated on 90 °C, then linear cool down in 45 minutes to room temperature

assembly of HPS Precursors

gene RDP parts product in
Hps (HPS.B1006.AP00) #RYPQ# + #VO4C# + #8AMS# + #M1ZY# + #OZD1# #V9H9#
Hps (HPS.B1006.AP04) #RYPQ# + #VO4C# + #8AMS# + #HB96# + #OZD1# #XKX6#
Hps (HPS.B1006.AP10) #RYPQ# + #VO4C# + #8AMS# + #4T9A# + #OZD1# #SNDK#
Hps (HPS.B1006.AP19) #RYPQ# + #VO4C# + #8AMS# + #XYD9# + #OZD1# #BC4V#
  • there is the very small probability that #SNDK# and #BC4V# were mixed up. Seqencing will show
  • heat shocked in DH5alpha with 5 µl of DNA

15-08-12

  • there were colonies on the trafo plates of the HPS precursors
  • Oligos from 15-08-11: put them on 2% agarose:

(good results: 0,8µl DNA and more than 3,5 µl 50bp Ladder)

part expected lenght result
BBa_B1002-RDP (#HTSR#) 45 bp fragments of ca. 50bp
BBa_B1006-RDP (#8AMS#) 50 bp fragments of ca. 50bp
AP19-RDP(#MBWS#) 47 bp fragments of ca. 50bp

Assembly of PHI precursors

gene RDP parts product in
Phi (PHI.B1002.AP00) #VVSC#
Phi (PHI.B1002.AP04) #BNHW#
Phi (PHI.B1002.AP10) #D1LL#
Phi (PHI.B1002.AP19) #EB8T#
  • each 5µl transformed in DH5alpha
  • masterplates and colony PCR will follow

colony PCR of MDH precursor clones

  • primers: #MSP2# and #94LB#
  • elongation time: 1'00
  • annealing temperature: 58°C
  • expected length: 920 bp
ID description positive clones
A 1-12 MDH.Ter.AP00
B 1-15 MDH.Ter.AP04
C 1-12 MDH.Ter.AP10
D 1-11 MDH.Ter.AP19

assembly of polycistronic versions

T7 polycistronic version assembly product: #DSHM#
AP19 polycistronic version assembly product: #MCLC#
  • each 5 µl transformed in DH5 alpha and additionally, T7 version in BL21

15-08-13

tasks:

  • colony PCR of HPS precursors
  • colony PCR of MDH precursors (again)
  • masterplates of T7 poly
  • masterplates of AP19 poly
  • masterplates of PHI precursors
  • overnight cultures of positive colony PCR clones

Q5 amplification of Z-XPK.B0015-X'

  • 6 reactions á 100µl
Mastermix:
component amount [µl]
ddH2O 250,8
F-Primer #RKW3# 24
R-Primer #CNAT# 24
2x Q5 Mastermix 300
Template #8ZRY# 1,2
Programme:
Time Temp [°C]
3:00 98
cyc. 0:25 98
cyc. 0:20 58
cyc. 1:23 72
2:00 72
store 8

30 cycles

  • all amplifications look good on the gel

Colony PCRs

MDH colony PCR:

  • Primers: #MSP2#, #94LB#
step time Temp [°C]
denature 5'00 94
denature 0'30 94
anneal 0'30 55
elongate 1'00 68
elongate 2'00 68
store forever 16

HPS colony PCR:

  • Primers: #HSP1#, #94LB#
step time Temp [°C]
denature 5'00 94
denature 0'30 94
anneal 0'30 55
elongate 0'40 68
elongate 2'00 68
store forever 16

Results:

construct ID construct description expected length [bp] positive clones
A 1-8 MDH 00 precursor 920 bp (A16), A18
B 1-8 MDH 04 precursor 920 bp B15, (B17), (B18)
C 1-8 MDH 10 precursor 920 bp -
D 1-8 MDH 19 precursor 920 bp D12, D14
E 1-8 HPS 00 precursor 579 bp -
F 1-8 HPS 04 precursor 579 bp -
G 1-8 HPS 10 precursor 579 bp -
H 1-8 HPS 19 precursor 579 bp -

15-08-14

tasks:

  • colony PCR of all PHI precursors
  • colony PCR of all HPS precursors
  • colony PCR of MDH AP10 precursor
  • colony PCR of AP19 polycistronic constructs (A)
  • colony PCR of T7 polycistronic constructs (G)
  • prep plasmid of:
    • MDH AP00 precursors (A16, A18)
    • MDH AP04 precursors (B15, B17, B18)
    • MDH AP19 precursors (D12, D14)
  • label gels of yesterday
  • BsaI clean-up


plasmids and cryos of MDH backups

construct Cryo ID Plasmid ID Plasmid conc. [ng/µl]
MDH AP00 #16 #Q6XM# #CZ83# 125,5
MDH AP00 #18 #6F1N# #XNTZ# 171,5
MDH AP04 #15 #848Y# #SRYN# 338
MDH AP04 #17 #FCKK# #TK9X# 134
MDH AP04 #18 #3C1N# #C8EB# 147,5
MDH AP19 #12 #XYM1# #VMBV# 72
MDH AP19 #14 #RB99# #4P6A# 101,5

BsaI clean-up

  • elution with H2O
RDP part volume [µl] n [pmol] c [ng/µl]
Z-XPK.B0015-X' (1) 98 1.6 29
Z-XPK.B0015-X' (2) 98 1.44 26
  • evaporation of H2O
  • resuspend with TE buffer to 0.04 pmol/µl

stored in #NDMC# & #Z6QT#

Colony PCRs

Program:

step time T [°C]
denature 5'00 94
denature 0'30 94
annealing 0'30 55
elongation varying 68
final elongation 2'00 68
store forever 16

Results table:

construct Primers elongation time expected length [bp] tested clones positive clones
MDH.AP10 precursor #MSP2#, #94LB# 1'00 920 1, 2, 3, 4, 5, 19, 20, 21 -
HPS.AP00 precursor #W6WP#, #94LB# 1'13 1160 9-16 15
HPS.AP04 precursor #W6WP#, #94LB# 1'13 1160 9-16 9
HPS.AP10 precursor #W6WP#, #94LB# 1'13 1160 9-14 11, 13, 14
HPS.AP19 precursor #W6WP#, #94LB# 1'13 1160 9-16 9, 13, 14 , 15, 16
PHI.AP00 precursor #W6WP#, #94LB# 1'13 1074 1-8 -
PHI.AP04 precursor #W6WP#, #94LB# 1'13 1074 1-8 -
PHI.AP10 precursor #W6WP#, #94LB# 1'13 1074 1-8 2, 3, 4, 10
PHI.AP19 precursor #W6WP#, #94LB# 1'13 1074 1-6 5
AP19 polycistronic construct varying 2'15 varying 0, 1, 3, 4 1
#W6WP#, #HSP2# 1878 0, 1, 3, 4
#HSP1#, #XSP3# 1939 0, 1, 3, 4
#XSP2#, #94LB# 2182 0, 1, 3, 4
T7 polycistronic construct varying 2'15 varying 1, 2, 7, 8 -
#W6WP#, #HSP2# 1927 1, 2, 7, 8
#HSP1#, #XSP3# 1939 1, 2, 7, 8
#XSP2#, #94LB# 2182 1, 2, 7, 8
  • colony PCR of polycistronic constructs was repeated by Michael

Assembly of MDH precursors

  • this time with blockers (find protocol in sciebo)
    • Assembly product MDH.AP00: #N1O1#
    • Assembly product MDH.AP04: #VYM6#
    • Assembly product MDH.AP10: #Q9QQ#
    • Assembly product MDH.AP19: #ONWS#

15-08-15

Tasks:

  • prep plasmid of yesterdays positive clones
  • (repeat colony PCR of T7 polycistronic clones)
  • make masterplates of MDH precursors (new assembly, this time with blockers)
  • assembly of HPS and PHI precursors (with blockers)
  • tidy up fridge
  • plate trafos of HPS and PHI precursors
  • plate new trafos of MDH.AP04 and MDH.AP10 precursors
  • assemble MDH expression plasmid (FO4B.XYD9.PRDW.PLTB.OZ1D)
  • trafo of MDH expression plasmid
  • plate trafo on LB+K plates

Plasmid prep of Hps and Phi precursors

construct Cryo ID Plasmid ID Plasmid conc. [ng/µl]
HPS AP19 #14 #FOF4# #B3PQ# 170
HPS AP10 #14 #638V# #P4SB# 148
HPS AP10 #11 #4ZSB# #P89F# 143,5
HPS AP04 #9 #NY1C# #1TMT# 190,5
HPS AP19 #15 #SM3L# #NFOS# 204
HPS AP00 #15 #HAKR# #6ACO# 126,5
HPS AP19 #13 #COMY# #VSWC# 261
HPS AP19 #9 #OZ4O# #L8Y4# 171,5
HPS AP10 #13 #8P4S# #4QLE# 238,5
HPS AP19 #16 #CSLZ# #6P68# 121,5
PHI AP10 #3 #BV3E# #L9E9# 161
PHI AP10 #4 #OFHN# #FVVW# 171,5
PHI AP19 #5 #E6HX# #HA1W# 192,5

Assembly of HPS precursors

  • this time with blockers (find protocol in sciebo)
    • Assembly product HPS.AP00: #WQPR#
    • Assembly product HPS.AP04: #6WOE#
    • Assembly product HPS.AP10: #XHXQ#
    • Assembly product HPS.AP19: #VM1V#

Assembly of PHI precursors

  • this time with blockers (find protocol in sciebo)
    • Assembly product PHI.AP00: #AFA4#
    • Assembly product PHI.AP04: #8FXO#
    • Assembly product PHI.AP10: #KCDE#
    • Assembly product PHI.AP19: #QHM6#

Assembly of MDH expression plamid

  • find protocol in sciebo
    • assembly product AP19.MDH.TER in #DK1F#

Transformations

# description assembly product
1 HPS.AP00 precursor #WQPR#
2 HPS.AP04 precursor #6WOE#
3 HPS.AP10 precursor #XHXQ#
4 HPS.AP19 precursor #VM1V#
5 PHI.AP00 precursor #AFA4#
6 PHI.AP04 precursor #8FXO#
7 PHI.AP10 precursor #KCDE#
8 PHI.AP19 precursor #QHM6#
9 MDH.AP04 precursor #VYM6#
10 MDH.AP10 precursor #Q9QQ#
11 AP19.MDH.TER expression plasmid #DK1F#

15-08-16

Tasks:

  • prep plasmid of polycistronic clone #1
    • Cryo #Q1Q3#
    • purified plasmid #POLY#
  • make masterplates of yesterdays trafos
  • do/make colony PCR/overnights of yesterdays masterplates
  • test KAPA 2G Mix
  • do 'colony PCR' with purified plasmid of AP19 poly

15-08-17

Precursor plasmids:

construct cryo IDs plasmid IDs screening result
MDH00 precursor #L99Q#, #VLO3#, #EHQC#, #OD19#, #FHEL#, #MA9W# #HR4E#, #ZZEE#, #3HPD#, #M1VB#, #QDVN#, #AHRM# look at table below
MDH04 precursor #OB4C#, #TVND#, #1S1V#, #COXQ#, #3NZQ#, #YZXD# #NEKS#, #OX44#, #P9HA#, #9CBA#, #SYMS#, #YY6O#
MDH19 precursor #HBWZ#, #K3V6#, #CYF6#, #S1HT#, #TKO4#, #4MS8# #BY9L#, #DNRY#, #KV18#, #1KEA#, #SPHL#, #N1BM#
HPS00 precursor #AVVO#, #X9PR#, #M4H8#, #44WP#, #DE4A#, #QMRB# #XVYK#, #CMC1#, #ERZK#, #WXO8#, #D8PY#, #8AFD#
HPS04 precursor #8SW4#, #RSH9#, #1C8L#, #E1OQ#, #Y3TR#, #3BLV# #YE8A#, #DERR#, #DVRP#, #FKNX#, #N3E4#, #HNV4#
HPS10 precursor #R8YT#, #QRZW#, #KVYA#, #NEFO#, #QN64#, #NSSM# #1EQ3#, #PA13#, #FAES#, #Z64V#, #WSAD#
PHI00 precursor #TNBW#, #VSQN#, #YPFO#, #EELW#, #9H4T#, #VLHL# #D6QY#, #931O#, #QWQP#, #AERW#, #ORX1#, #ZCTY#
PHI04 precursor #4MNP#, #DE96#, #YKZT#, #AQF8#, #ETTS#, #8COE# #FXSC#, #P1O1#, #8CZD#, #EZYZ#, #VPP4#, #3EZP#
PHI10 precursor #SQ9N#, #AO6C#, #9RZL#, #48P3#, #PPWW#, #DB3R# #FTX4#, #VM6Z#, #V4CA#, #XW1Y#, #AZND#, #WCF3#
PHI19 precursor #3WKA#, #94OY#, #TVM1#, #KPCQ#, #FB9L#, #HWM4# #THQA#, #BND4#, #6H3Z#, #ME9O#, #39CF#, #RABS#

further plasmids:

  • AP19 Poly
    • cryo: #LHZV#
    • pasmid: #WVDR#
  • MDH expression plasmid
    • cryo: #MSX3#
    • plasmid: #WDXZ#
  • HPS19 precursor
    • cryo: #ABCE#
    • plasmid: #TTC4#

AP19.MDH.Ter in pSB1KRDP

The MDH clones 2-19 were screened with a KAPA2G colony PCR. Controls are clone #1 and the plasmid #WDXZ#. Initial denaturation was set to 8'00" and the primer concentration was increased by 30 % to 6.5 mM. 0'15" denaturation and 0'15" annealing at 65-0.4°C per cycle. Elongation time was 0'20" for an expected product of 1205 bp (#W6WP# and #MSP3#). The PCR was not successful - only a band of plasmid template was visible.

Overnight cultures were prepared for all clones.

15-08-18

Morning

Plasmid preps and sequencing. All BOLD IDs do not exist yet. Prepping instructions:

  • prep each of the 6 POLY cultures, but elute all into one target container named #C3NS#
  • make cryos of the AP19.MDH cultures of clone #2+3 and then prep them. (both IDs in the table below)

Pipetting calculation table is ready at: \WetLab\Documentation\Methanol\15-08-18 Sequencings\

construct & clone number cryo ID plasmid ID sequencing sample numbers sequencing result further procedure
AP19.POLY in pSB1KRDP #1 #W3NB# #C3NS# 1-8 NO CONTIGS FOUND Repeat Sequencing
AP19.MDH.Ter in pSB1KRDP #1 above #WDXZ# 9+10 empty backbone Discard
AP19.MDH.Ter in pSB1KRDP #2 #MSX3# #FDX4# 11+12 mutation in promoter Discard
AP19.MDH.Ter in pSB1KRDP #3 #FZ8Q# #MOMX# 13+14 empty backbone Discard
MDH.TER.AP00 in pSB1CRDP #1 #L99Q# #HR4E# 15 MUTATION Discard
MDH.TER.AP00 in pSB1CRDP #2 #OD19# #M1VB# 16 MUTATION Discard
MDH.TER.AP00 in pSB1CRDP #6 #MA9W# #AHRM# 17 MUTATION Discard
MDH.TER.AP04 in pSB1CRDP #1 #OB4C# #NEKS# 18 SPACER MUTATION Discard
MDH.TER.AP04 in pSB1CRDP #2 #TVND# #OX44# 19 VALID Keep
MDH.TER.AP04 in pSB1CRDP #3 #1S1V# #P9HA# 20 VALID Discard
MDH.TER.AP19 in pSB1CRDP #1 #HBWZ# #BY9L# 21 VALID Discard
MDH.TER.AP19 in pSB1CRDP #2 #K3V6# #DNRY# 22 VALID Discard
MDH.TER.AP19 in pSB1CRDP #3 #CYF6# #KV18# 23 VALID Keep
HPS.B1006.AP00 in pSB1CRDP #3 #M4H8# #ERZK# 24 VALID Discard
HPS.B1006.AP00 in pSB1CRDP #4 #44WP# #WXO8# 25 VALID Keep
HPS.B1006.AP00 in pSB1CRDP #5 #DE4A# #D8PY# 26 NO CONTIGS FOUND Discard
HPS.B1006.AP04 in pSB1CRDP #2 #RSH9# #DERR# 27 MUTATION Discard
HPS.B1006.AP04 in pSB1CRDP #3 #1C8L# #DVRP# 28 MUTATION Discard
HPS.B1006.AP04 in pSB1CRDP #6 #3BLV# #HNV4# 29 MUTATION Discard
HPS.B1006.AP10 in pSB1CRDP #2 #QRZW# #1EQ3# 30 NO CONTIGS FOUND Discard
HPS.B1006.AP10 in pSB1CRDP #4 #NEFO# #FAES# 31 NO CONTIGS FOUND Discard
HPS.B1006.AP19 in pSB1CRDP #1 #ABCE# #TTC4# 32 MUTATION Discard
PHI.B1002.AP00 in pSB1CRDP #2 #VSQN# #931O# 33 VALID Keep
PHI.B1002.AP00 in pSB1CRDP #3 #YPFO# #QWQP# 34 NO CONTIGS FOUND Discard
PHI.B1002.AP00 in pSB1CRDP #4 #EELW# #AERW# 35 MUTATION Discard
PHI.B1002.AP04 in pSB1CRDP #1 #4MNP# #FXSC# 36 NO CONTIGS FOUND Discard
PHI.B1002.AP04 in pSB1CRDP #3 #YKZT# #8CZD# 37 PROMOTER OK Discard
PHI.B1002.AP04 in pSB1CRDP #4 #AQF8# #EZYZ# 38 VALID Keep
PHI.B1002.AP10 in pSB1CRDP #3 #9RZL# #V4CA# 39 VALID Keep
PHI.B1002.AP10 in pSB1CRDP #4 #48P3# #XW1Y# 40 VALID Discard
PHI.B1002.AP10 in pSB1CRDP #6 #DB3R# #WCF3# 41 VALID Discard
PHI.B1002.AP19 in pSB1CRDP #2 #94OY# #BND4# 42 NO CONTIGS FOUND Discard
PHI.B1002.AP19 in pSB1CRDP #5 #FB9L# #39CF# 43 MUTATION Discard
PHI.B1002.AP19 in pSB1CRDP #6 #HWM4# #RABS# 44 VALID Keep

Noon

Prep plasmids of T7.POLY in BL21 (DE3)

construct & clone number cryo ID plasmid ID check PCR result
T7.lacO.POLY in pSB1KRDP #1 #LHBN# #VYCO# discard
T7.lacO.POLY in pSB1KRDP #5 #HB93# #ANPV# discard
T7.lacO.POLY in pSB1KRDP #6 #6LOZ# #96TS# discard
T7.lacO.POLY in pSB1KRDP #7 #KYOQ# #XVE3# discard
T7.lacO.POLY in pSB1KRDP #8 #MCMR# #BSLA# discard
  • RDP-Assembly of T7.lacO.MDH.Ter in pSB1KRDP (product: #....#)
  • transform #....# into BL21 Gold (DE3) AND DH5a

Check-PCR the prepped T7.POLY plasmids

  • with the three primer pairs, to find a complete construct
  • elongation time 0'33
  • #POLY# as positive control (fitting bands already 15-08-16)
Part ID Sample Primers expected length [bp] result
#VYCO# 1 #W6WP# + #HSP2# 1927 negative
2 #HSP1# + #XSP3# 1939 negative
3 #XSP2# + #94LB# 2182 negative
#ANPV# 4 #W6WP# + #HSP2# 1927 negative
5 #HSP1# + #XSP3# 1939 negative
6 #XSP2# + #94LB# 2182 negative
#96TS# 7 #W6WP# + #HSP2# 1927 negative
8 #HSP1# + #XSP3# 1939 negative
9 #XSP2# + #94LB# 2182 negative
#XVE3# 10 #W6WP# + #HSP2# 1927 negative
11 #HSP1# + #XSP3# 1939 negative
12 #XSP2# + #94LB# 2182 negative
#BSLA# 13 #W6WP# + #HSP2# 1927 negative
14 #HSP1# + #XSP3# 1939 negative
15 #XSP2# + #94LB# 2182 negative
#POLY# 16 #W6WP# + #HSP2# 1927 positive
17 #HSP1# + #XSP3# 1939 positive
18 #XSP2# + #94LB# 2182 positive
  • Label yesterdays SDS gel and upload the results to the internal wiki.

Afternoon

  • make M9+K+Glucose40mM overnight cultures of
    • B0015 in pSB1K3 in DH5a
    • AP19.POLY in pSB1KRDP in DH5a clone #1
    • AP19.MDH.Ter in pSB1KRDP in DH5a clone #1-6
  • make two LB+K overnight cultures of T7.POLY on LB+K

RDP Assembly

  • Assembly of #FO4B# and #PVFL# (Kanamycin Cap with high copy cap) the product is #QWRC# -> we need this strain as a negative control that carries the plasmid but does not express anything

Transform important plasmids into BL21 Gold (DE3) and plate on M9+K+40mMGlucose as well as LB+K

construct transformed plasmid BL21 Gold (DE3) cryo
AP19.POLY in pSB1KRDP #POLY# #....#
AP19.MDH.Ter in pSB1KRDP #WDXZ#, #FDX4#, #MOMX# #....#
pSB1KRDP #QWRC# #....#

References

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