Team:Aachen/Notebook/Documentation/Methanol Monocistronic Diversity Library


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
  • plasmid purification and cryo cultures of mdh precursor plasmids

15-08-10

  • make cryo cultures 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

digests stored in falcon #PSHF#

15-08-11

  • sequencing of following samples: #1MC6#, #ESQD#,
  • 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#


  • heat shock transformation in DH5alpha with 5 µl of DNA

15-08-12

  • there were colonies on the transformation 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 no
B 1-15 mdh.Ter.AP04 5
C 1-12 mdh.Ter.AP10 no
D 1-11 mdh.Ter.AP19 2, 4

15-08-13

tasks:

  • colony PCR of hps precursors
  • colony PCR of mdh precursors (again)
  • masterplates of phi precursors
  • overnight cultures of positive colony PCR clones

Colony PCRs

mdh precursor 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 precursor 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 AP00 precursor 920 bp (A16), A18
B 1-8 mdh AP04 precursor 920 bp B15, (B17), (B18)
C 1-8 mdh AP10 precursor 920 bp -
D 1-8 mdh AP19 precursor 920 bp D12, D14
E 1-8 hps AP00 precursor 579 bp -
F 1-8 hps AP04 precursor 579 bp -
G 1-8 hps AP10 precursor 579 bp -
H 1-8 hps AP19 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
  • purify plasmid of:
    • mdh AP00 precursors (A16, A18)
    • mdh AP04 precursors (B15, B17, B18)
    • mdh AP19 precursors (D12, D14)
  • label gels of yesterday
  • BsaI digest 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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