Team:Aachen/Notebook/Documentation/Methanol Polycistronic Expression Plasmid


Laboratory Notebook

Building the Polycistronic Plasmid via RDP Assembly

Building BioBricks out of polycistronic plasmid



Building the Polycistronic Plasmid via RDP Assembly

15-07-14

gradient PCR for following RDP part: X-mdh-Z'

  • gradient PCR products formerly stored in falcon #SY3M# (but now discarded)
  • check on agarose gel after amplification
  • PCR program:
step time T [C°]
denature 00:30 98
denature 00:30 98
anneal 00:30 60-67
elongate 00:40 72
elongate 05:00 72
store forever 16
  • pipeting volumes:
component volume per tube [µl]
H2O 15.55
F-Primer #KLF1# 0.8
R-Primer #DD84# 0.8
10x PCR buffer B2 2
dNTPs (10mM) O5 0.4
PfuS 0.2
Template #YSVQ# 0.25
  • results: 63.7 °C is the best temperature for this primer combination

Q5 amplification of RDP part X-mdh-Z'

  • amplification product stored in falcon #K913# (previosly in falcon #SY3M#)
  • PCR program:
step time T [C°]
denature 03:00 98
denature 00:15 98
anneal 00:15 63.7
elongate 00:40 72
elongate 02:00 72
store forever 16
  • pipeting volumes:
component volume per tube [µl]
H2O 20.75
FPrimer #KLF1# 2
RPrimer #DD84# 2
2x Q5 MM 25
Template #YSVQ# 0.25

15-07-28

Gradient PCRs for RDP parts

  • Gradient PCR products stored in: #964L# and #LPPT# (now discarded)
  • Z-xpk.B0015-X' gradient PCR products stored in #K1MB#

Z-hps-X'

  • Polymerase: PfuS
  • Reaction Volume: 25 µl
  • FW Primer: #ODYN#
  • RV Primer: #PWVZ#
  • Template: #H4M9#
  • result: 57.8 °C

X-phi-Z'

  • Polymerase: PfuS
  • Reaction Volume: 25 µl
  • FW Primer: #S6WV#
  • RV Primer: #1XBB#
  • Template: #ANNA#
  • result: 61.9 °C

Z-xpk.B0015-X'

  • Polymerase: PfuS
  • Reaction Volume: 25 µl
  • FW Primer: #RKW3#
  • RV Primer: #CNAT#
  • Template: #8ZRY#
  • result: 58.0 °C

15-07-29

RDP part Q5 amplification

  • Q5 amplification products stored in falcon #K913#

Z-hps-X'

  • FW Primer: #ODYN#
  • RV Primer: #PWVZ#
  • Template: #H4M9# (0.5 µl)
  • Elongation: 0'23
  • Annealing: 57.8 °C

X-phi-Z'

  • FW Primer: #S6WV#
  • RV Primer: #1XBB#
  • Template: #ANNA# (0.5 µl)
  • Elongation: 0'27
  • Annealing: 61.9 °C

Z-xpk.B0015-X'

  • FW Primer: #RKW3#
  • RV Primer: #CNAT#
  • Template: #8ZRY# (0.2 µl)
  • Elongation: 1'23
  • Annealing: 58.0 °C

15-07-30

Summary

construct name Q5 PCR clean-up ID minimal required ammount [µg]
Z-hps-X' #ZDFQ# 15
X-phi-Z' #XL1B# 15
X-mdh-Z' #KLV4# 15

Further Q5 amplifications for higher DNA amount

  • stored in falcon #64M1#

1: X-mdh-Z'

  • 6*50 µl reactions
component volume per tube [µl]
H2O 20.75
FPrimer #KLF1# 2
RPrimer #DD84# 2
2x Q5 MM 25
Template #YSVQ# 0.25
  • PCR program:
step time T [C°]
denature 03:00 98
denature 00:15 98
anneal 00:15 63.7
elongate 00:40 72
elongate 02:00 72
store forever 16

Stored in #BOS6#; DNA concentration: 153,8 [ng/µl]

2: Z-hps-X'

  • 6*50 µl reactions
component volume per tube [µl]
H2O 20.5
FPrimer #ODYN# 2
RPrimer #PWVZ# 2
2x Q5 MM 25
Template #H4M9# 0.5
  • PCR program:
step time T [C°]
denature 03:00 98
denature 00:15 98
anneal 00:15 57.8
elongate 00:23 72
elongate 02:00 72
store forever 16

Stored in #NXRA#; DNA concentration 105,6 [ng/µl]

3: X-phi-Z'

  • 6*50 µl reactions
component volume per tube [µl]
H2O 20.5
FPrimer #S6WV# 2
RPrimer #1XBB# 2
2x Q5 MM 25
Template #ANNA# 0.5
  • PCR program:
step time T [C°]
denature 03:00 98
denature 00:15 98
anneal 00:15 61.9
elongate 00:27 72
elongate 02:00 72
store forever 16

Stored in #PRTC#; DNA concentration: 138,6 [ng/µl]

4: Z-xpk.B0015-X'

  • 4*50 µl reactions
component volume per tube [µl]
H2O 20.8
FPrimer #RKW3# 2
RPrimer #CNAT# 2
2x Q5 MM 25
Template #8ZRY# 0.2
  • PCR program:
step time T [C°]
denature 03:00 98
denature 00:15 98
anneal 00:15 61.9
elongate 01:23 72
elongate 02:00 72
store forever 16

Stored in #XMO9# DNA concentration: 186,9 [ng/µl]

15-08-06

BsaI digest of RDP Parts 10x overdigest

X-mdh-Z'

Tube 'W'

component volume per tube [µl]
ddH2O 75
BsaI 2
NEB 10x CutSmart Buffer 10
#BOS6# 13

Concentration after clean-up: 18.7 [ng/µl]



Z-hps-X'

Tube 'X'

component volume per tube [µl]
ddH2O 69.1
BsaI 2
NEB 10x CutSmart Buffer 10
#NXRA# 18.9

Concentration after clean-up: 12.7 [ng/µl]



X-phi-Z'

Tube 'Y'

component volume per tube [µl]
ddH2O 73.6
BsaI 2
NEB 10x CutSmart Buffer 10
#PRTC# 14.4

Concentration after clean-up: 11.8 [ng/µl]



Z-xpk.B0015X'

Tube 'Z'

component volume per tube [µl]
ddH2O 77.3
BsaI 2
NEB 10x CutSmart Buffer 10
#XMO9# 10.7

Concentration after clean-up: 22.0 [ng/µl]


Store digestions in Falcon #TRYE#

15-08-07

We did the digestion twice because of non sufficient concentrations

First Digest:
Construct conc. [ng/µl]
"W" / X-mdh-Z' 18,7
"X" / Z-hps-X' 12,7
"Y" / X-phi-Z' 11,8
"Z" /Z-xpk.B0015-X' 22,0
Second digest:

BsaI digestion of RDP Parts 5x overdigest

X-mdh-Z

Tube 'I'

component volume per tube [µl]
ddH2O 62
BsaI 2
NEB 10x CutSmart Buffer 10
#BOS6# 26

Concentration after clean-up: 49.0 [ng/µl]


Z-hps-X'

Tube 'II'

component volume per tube [µl]
ddH2O 50.1
BsaI 2
NEB 10x CutSmart Buffer 10
#NXRA# 37.9

Concentration after clean-up: 66.5 [ng/µl]


X-phi-Z'

Tube 'III'

component volume per tube [µl]
ddH2O 59.1
BsaI 2
NEB 10x CutSmart Buffer 10
#PRTC# 28.9


Z-xpk.B0015-X'

Concentration after clean-up: 22.4 [ng/µl]

Tube 'IV'

component volume per tube [µl]
ddH2O 66.6
BsaI 2
NEB 10x CutSmart Buffer 10
#XMO9# 21.4


Results of second digest


Construct conc. [ng/µl]
"I" / X-mdh-Z' 49.0
"II" /Z-hps-X' 66.5
"III" /X-phi-Z' 22.4
"IV" / Z-xpk.B0015-X' 25.3

RDP parts summary

Assembly-ready RDP parts:

Part ID Part description c [pmol/µl] volume [µl]
#PRDW# X-mdh-Z' 0.04 44.7
#VO4C# Z-hps-X' 0.03 49
#ZALV# X-phi-Z' 0.04 78
#ZR1Q# Z-xpk.B0015.X' 0.04 22

Resuspending RDP parts from kitplate

Part ID Part description volume [µl]
#PLTB# Z-Ter-X' 110
#RYPQ# dA20-ChlR-Z' 110
#XOXM# dA20-ChlR-X' 110
#HNXW# dA20-AmpR-Z' 110
#FO4B# dA20-KanR-Z' 110
#PVFL# Z-CAP-Ori.3-dT20 110
#OZD1# X-CAP-Ori.3-dT20 110
#RAXA# X-Blk 220
#BWD6# Z-Blk 220

Assembling oligos

  • oligos had to be lyophilized to be resuspended in TE buffer
  • Gel control of the 43 bp oligos: looked good
  • oligo assembly: BBa_B1002-RDP (#HTSR#), BBa_B1006-RDP (#8AMS#), AP19-RDP (#MBWS#)
    • heated on 90 °C, then linear cool down in 45 min to room temperature

15-08-12

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:

  • masterplates of T7.poly
  • masterplates of AP19.poly

15-08-14

  • Colony PCR on AP19.Poly and T7.Poly

Results table:

construct Primers elongation time expected length [bp] tested clones positive clones
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


15-08-16

Tasks:

  • purify plasmid of AP19 polycistronic clone #1
    • Cryo #Q1Q3#
    • purified plasmid #POLY#
  • make masterplates of yesterdays trafos
  • do/make colony PCR/overnight cultures of yesterdays masterplates
  • test KAPA 2G Mix
  • do test PCR with purified plasmid of AP19 poly
    • RESULT: POSITIVE!!

15-08-17

Again, do:

  • AP19 Poly plasmid purification + cryo:
    • cryo: #LHZV#
    • pasmid: #WVDR#

15-08-18

Morning

Plasmid purifications and sequencing

  • elute all 6 AP19.Poly overnight cultures (all from #Q1Q3#) into one target container named #C3NS# for sufficient DNA concentration

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

Noon

Purify 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

Check-PCR of purified T7.POLY plasmids

  • with 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# (AP19.Poly, positive control) 16 #W6WP# + #HSP2# 1927 positive
17 #HSP1# + #XSP3# 1939 positive
18 #XSP2# + #94LB# 2182 positive

RDP Assembly

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

Transformation of plasmids in BL21 Gold (DE3) and plate on M9 + K + 40mM Glucose as well as LB+K

construct transformed plasmid BL21 Gold (DE3) cryo
AP19.POLY in pSB1KRDP #POLY# #AW9K#
pSB1KRDP #QWRC# #....#

15-08-19

  • make master plates and overnights of BL21 Gold (DE3) transformants (M9+K+40mM Glucose)
    • pSB1KDP from #QWRC# on/in LB+K
    • pSB1KDP from #QWRC# on/in M9+K
    • AP19.Poly from #POLY# on/in LB+K
    • AP19.Poly from #POLY# on/in M9+K
  • analyze sequencing results of polycistronic construct
    • Sequencing of #C3NS# did not work, so check with PCR and test digest
      • Test PCR: three primer pairs (#W6WP# + #HSP2#; #HSP1# + #XSP3#; #XSP2# + #94LB#)
      • #POLY# as positive control
    • if positive: send in for sequencing


Sequencing for tomorrow:

construct & clone number cryo ID plasmid ID sequencing sample numbers sequencing result
AP19.POLY in pSB1KRDP #1 #Q1Q3# #LHZV# #W3NB# #XULU# 1-8 positive (result after several sequencings because of problems with sequencing service)

sequencing prouven polycistronic construct!!!!

Sequencing Result of AP19.Poly construct

Aachen Poly with seq chromatogram.JPG
Successful Sequencing of Polycistronic Construct

Building BioBricks out of polycistronic plasmid

15-08-19

Next steps

  • build RuMP pathway (AP19.mdh.hps.phi, like MCC but without xpk, for comparison)
  • bring the polycistronic construct into pSB1C3 (to provide it as a BioBrick) and into pSB1K3 (to characterize it further) METHODS:
    • CPEC
    • restriction with XbaI and SpeI and ligation
    • restiction with EcoRI and PstI and ligations
    • Gibson Assembly
    • digestion with XbaI and SpeI, then digestion with alkaline phosphatase, then ligation

15-08-17 polycistronic expression test

SDS-PAGE of AP19 Polycistronic construct derived from cryo #Q1Q3#

Results:

  • mdh: (expected 40,7 kDa) is visible as a big band in Q1Q3
  • phi+hps: (expected 20 & 22,6 kDa) there is a big band between 17 & 26 kDa
  • xpk: (expected 92 kDa) is visible as a big band in #Q1Q3#

15-08-25

RuMP pathway

  • for comparison of MCC pathway, we built the RuMP pathway in a polycistronic plasmid
  • #FO4B# + #XYD9# + #PRDW# + #VO4C# + #ZALV# + #PLTB# + #OZ1D#
  • AP19.MDH.HPS.PHI.Ter in pSB1KRDP, assembly product stored in #BMDS#
  • Hps RDP part is empty now

Cloning AP19 Poly into Biobrick backbone via CPEC

  • fragment amplification products in:
    • #OX3R#
    • #PQMO#
  • CPEC products stored in Falcon #ZTYD#

Strategy: Polycistronic construct into Biobrick backbone

Fragment amlifications
  • amplify #XULU# with primers #HSTL# and #8XOZ#

Programme:

step time T [C°]
denature 00:30 98
denature 00:15 98
anneal 00:15 66
elongate 02:40 72
elongate 02:00 72
store forever 16

PCR mix:

components Volume [µl]
H2O 19
2x Q5 MM 25
#HSTL# 2.5
#8XOZ# 2.5
#XULU# 1
  • amplification product stored in #OX3R#
  • Amplify Biocbrick backbones with #FYTO# and #TSTV#
    • pSB1C3 amplification product stored in #6WCN#
    • use #QLE4# as template for pSB1K3 backbone amplification

Programme:

step time T [C°]
denature 00:30 98
denature 00:15 98
anneal 00:15 68
elongate 01:10 72
elongate 02:00 72
store forever 16

PCR mix:

components Volume [µl]
H2O 21.5
2x Q5 MM 25
#TSTV# 1.25
#FYTO# 1.25
#QLE4# 1
  • pSB1K3 amplification product stored in #PQMO#

CPEC

  • use #OX3R# and [#6WCN# / #PQMO#] in a CPEC to build the AP19 Poly construct in pSB1[C/K]3 backbone
  • Components (total reaction volume=25 µl)
components amount
H2O 11.5 µl
Q5 Buffer 5 µl
DMSO 0.75 µl
dNTPs 5 µl
Q5 Polymerase 0.5 µl
Backbone (#6WCN# / #PQMO#) 100 ng
fragment (#OX3R#) 0.073 pmol
  • CPEC Programme:
step time T [C°]
denature 00:30 98
denature 00:10 98
anneal 00:30 55
elongate 03:45 72
elongate 10:00 72
store forever 8
  • products stored in Falcon #ZTYD#

15-08-26

RuMP Pathway

  • not all of the 10 overnights grew
clone cryo ID plasmid ID conc. [ng/µl] result
#1 #QM6C# #VK8F# 19,1 neg
#2 #KYAV# #HYNT# 21,3 nge
#4 #YROE# #VLVZ# 51,7 neg
#6 #F3KA# #OD9H# 47,2 neg
#7 #KFYB# #YDBL# 34,8 neg
#8 #8OCO# #861V# 30,8 neg
#9 #1H9P# #HH6E# 51,7 neg
  • SDS page will show which one expresses the genes
  • staining seems to be difficult (used DNA loading dye instead of SDS loading dye)

Test PCR

  • Primers W6WP and 94LB, expected lenght 2926 bp, 0'44 elongation time

Sequencing

Plasmid #YDBL# was send to sequencing with #W6WP# & #94LB#

15-08-28

  • screening for good constructs: ALL CLONES NEGATIVE!!
construct clone Cryo ID Plasmid ID Plasmid conc. [ng/µl] result
AP19.Poly in pSB1K3 in DH5alpha #1 #NBBB# #34ZR# neg
#2 #NLCA# #DHLZ# neg
#3 #DAAW# #VSHE# neg
#4 #XTDS# #Z9YB# neg
AP19.Poly in pSB1K3 in BL21 #1 #K9V1# #F3MF# neg
#2 #L4DS# #BLPR# neg
#3 #MH8O# #RLRA# neg
#4 #FWZW# #O31X# neg
AP19.Poly in pSB1C3 in BL21 #1 #H1YN# #3M83# neg
AP19.Poly in pSB1C3 in DH5alpha #1 #B1WF# #DZLQ# neg
#2 #ZEFM# #4YSX# neg
#3 #ERWH# #D1ZS# neg
#4 #NLZR# #ADHH# neg
#5 #D4H3# #NRZ9# neg
#6 #4A4L# #A4BS# neg
#7 #3VMY# #6XKM# neg
#8 #VFB3# #MM4R# neg
#9 #F9DQ# #LZWV# neg
#10 #OL63# #CMME# neg
#11 #AA4O# #6RFK# neg
#12 #8ZX6# #QLS1# neg
#13 #L3PR# #E6XR# neg
#14 #FS19# #HLVZ# neg
#15 #CVEX# #QY8T# neg
#16 #DDO6# #WXRA# neg
#17 #LRPO# #YQZ8# neg
#18 #DDQQ# #BLOA# neg
  • cultures of clones with kanamycin resistance in BL21 were prepared for SDS-PAGEs

15-08-29

  • results of check PCR (AP19 Poly in BioBrick backbones): NO PLASMID WITH CONSTRUCT
  • again do transformation of:
    • AP19 Poly in pSB1C3 in DH5alpha (Ligation and Trafo ID: 3)
    • AP19 Poly in pSB1K3 in BL21 Gold DE3 (Ligation and Trafo ID: 4)
  • digests and ligation products stored in Falcon #OLAE#

digest:

  • 30 µl reaction volume
  • enzyme and buffer volumes acording to cheat sheet
# plasmid Enzymes Water added [µl]
#OLAE#:A #CD8B# E + P 21.6
#OLAE#:D #CD8B# X + S 21.6
#OLAE#:E #AQY3# X + S 21.4
#OLAE#:F #OX3R# X + S 24.0


ligation:

  • 20 µl reaction volume
  • 4 µl part digest
  • 2 µl backbone digest
# part digest backbone digest
#OLAE#:3 #OLAE#:F #OLAE#:D
#OLAE#:4 #OLAE#:F #OLAE#:E


transformation:

# transformed plasmid or ligation product ID strain description
3 #OLAE#:3 DH5alpha AP19 Poly (from #OX3R#) in pSB1C3
4 #OLAE#:4 BL21 Gold DE3 AP19 Poly (from #OX3R#) in pSB1K3

Tasks for following days

  • masterplates and overnight cultures of transformations !!!red-white screening!!! (see above) (sunday, 15-08-30) done
  • plasmid purification and cryos of overnights cultures (monday, 15-08-31)
  • test digest for screening (monday, 15-08-31)
  • strategic decission on what to send in for sequencing (monday, 15-08-31)
  • is it possible to have all required BioBrick and expression plasmids sequenced and validated on wednesday? If yes, then we' ll have to concentrate on generating relieable data. If not, a small group has to focus on cloning, until it is finished. (monday, 15-08-31)

15-08-31

restriction ligation of AP19.Poly Biobrick parts

construct clone Cryo ID Plasmid ID Plasmid conc. [ng/µl] result
AP19.Poly in pSB1C3; DH5alpha #1 #BL11# #ATYH# 155,5 neg
#2 #AR3W# #PEQT# 114,0 neg
#3 #C8QO# #ZBZT# 103,0 neg
#4 #AKYK# #11AZ# 200,5 neg
#5 #KAKW# #OWF9# 192,5 neg
#6 #AWSE# #M1DW# 144,5 neg
#7 #XS6A# #ZCZN# 194,5 neg
#8 #ZW99# #4CCM# 154,0 neg
AP19.Poly in pSB1K3; BL21 Gold #1 #8PL8# #81KE# 286,5 neg
#2 #L6PR# #WARS# 201,0 neg
#3 #LM4Y# #ZW1Y# 207,0 neg
#4 #KOKW# #DLNH# 237,0 neg
#5 #Y8NN# #S98X# 182,0 neg
#6 #9VS1# #Q49D# 231,0 neg
#7 #FYOP# #FXHO# 221,5 neg
#8 #FM6X# #RF3B# 207,5 neg
  • for screening of construct, do test digest with SpeI and XbaI
  • just negative clones of Poly and Mdh construct

Just negative results in test digest never just cut X,S for cloning

new restriction ligation

digest:

# plasmid Enzymes Water added [µl]
#FCBA#:8 #OX3R# E + P 24.0
#FCBA#:7 #AQY3# E + P 21.4
  • also use digest #OLAE#:A (J04450 in pSB1C3 #CD8B# digest with E + P)
  • check digest on gel

Ligation:

  • 20 µl reaction volume
  • 6 µl part digest
  • 2 µl backbone digest
# part digest backbone digest
Y #FCBA#:8 #OLAE#:A
Z #FCBA#:8 #FCBA#:7
  • ligation products in #FCBA#

15-09-01

Summary

Our aim is to get AP19.Poly in the BioBrick backbones pSB1C3 and pSB1K3. Later this day we can make masterplates and overnight cultures of the AP19 clones in both backbones. Tomorrow plasmid purification and test digest will follow.

In the evening of wednesday we will prepare sequencing. We need positive clones of:

  • AP19.Poly in pSB1C3
  • AP19.Poly in pSB1K3

TODO for today

  • Night: AP19.Poly in K3 and AP19.Poly in C3 Masterplates and overnights

15-09-02

TODO

  • Plasmid purification of all constructs
  • screening of all constructs with test digest
  • prepare sequencing for thursday morning

Constructs for screening

construct clone Cryo ID Plasmid ID Plasmid conc. [ng/µl] result
AP19.Poly in pSB1C3; DH5alpha #1 ## ## neg
#2 ## ## neg
#3 ## ## neg
#4 ## ## neg
#5 ## ## neg
#6 ## ## neg
#7 ## ## neg
#8 ## ## neg
#9 ## ## neg
#10 ## ## neg
#11 ## ## neg
#12 ## ## neg
#13 ## ## neg
#14 ## ## neg
#15 ## ## neg
AP19.Poly in pSB1K3; BL21 #1 #SCDM# #LX9X# no succesfull overnight
#2 #4EW8# #PAFZ# no succesfull overnight
#3 #8XQ9# #C6NZ# no succesfull overnight
#4 #XH9V# #16DW# no succesfull overnight
#5 #8WRB# #4P1T# no succesfull overnight
#6 #QMKT# #SBOC# test via SDS
#7 #8NWN# #VLZ1# test via SDS
#8 #VA9S# #SFKE# no succesfull overnight
#9 #48BC# #C3OX# no succesfull overnight
#10 #BOSP# #VC9Y# no succesfull overnight
#11 #B6ML# #VQBD# no succesfull overnight
#12 #CKFD# #EQBA# no succesfull overnight
#13 #YHQT# #QD9R# no succesfull overnight
#14 #QAMS# #43DT# no succesfull overnight
#15 #R9D1# #KZ6P# no succesfull overnight
  • overnights of AP19.Poly in pSB1K3 didn't grow the night, so they were made again at 15:30
  • all purified plasmids of AP19.Poly in K3 are sceened by sequencing with one primer
  • ONE succesfull overnight of AP19.Poly in K3

15-09-03

  • from the AP19.Poly in pSB1K3 constructs just the overnight of #6 grew, these will be prepared for plasmid purification and tests (test digest/PCR)
  • as restriction ligation strategy for biobrick constructs seems to be not reliable, we try Gibson Assembly for AP19.Poly in C3/K3

Gibson Assembly

Reaction Tube Description H2O Backbone [µl] Insert [µl] Gibson MM transformed in result
A Poly in K3 neg control 12,4 0,8 (#PQMO#) 1,9 (#OX3R#) 0 BL21 Gold DE3
B Poly in K3 4,9 0,8 (#PQMO#) 1,9 (#OX3R#) 7,5 BL21 Gold DE3
C Poly in C3 neg control 12,1 1,0 (#6WCN#) 1,9 (#OX3R#) 0 DH5alpha
D Poly in C3 4,6 1,0 (#6WCN#) 1,9 (#OX3R#) 7,5 DH5alpha
  • products stored in #D6LK#

15-09-04

Gibson Assembly transformation

  • The Gibson Assembly masterplates show also clones when no Gibson MM was used (just transformation of linar [?] DNA parts), so we can't trust the colonies on the plates with mastermix
    • DpnI digest seems not to work, so the ligation product was digested with DpnI again (1 µl of Schwaneberg DpnI, 1 µl of S2 DpnI, all afternoon at 37 °C)
    • after 9 h of incubation, constructs were transformed in BL21 (AP19.Poly in pSB1K3-constructs) and DH5alpha (AP19.Poly in pSB1C3-constructs)

15-09-05

AP19.POLY in pSB1K3 check PCR

  • Templates:
    • 1: #SBOC#
    • 2: #VLZ1#
  • KAPA 2G Mastermix
Mastermix Primerpair expected length [bp]
A #A9W9# and #HSP2# 1804
B #HSP1# and #XSP3# 1939
C #XSP2# and #XE3D# 2157

PCR program:

step T [°C] time
denature 95 0:30
denature 95 0:15
anneal 61 0:15
elongate 72 0:35
final elongation 72 2:00
store 16 forever

Summary and results:

ID Primerpair template expected length [bp] result
1A #A9W9# and #HSP2# #SBOC# 1804 wrong band
1B #HSP1# and #XSP3# #SBOC# 1939 wrong band
1C #XSP2# and #XE3D# #SBOC# 2157 wrong band
2A #A9W9# and #HSP2# #VLZ1# 1804 no bands
2B #HSP1# and #XSP3# #VLZ1# 1939 no bands
2C #XSP2# and #XE3D# #VLZ1# 2157 no bands

Gibson Assembly

  • trafo plates looks better after long incubation with DpnI, only very few red colonies
  • 15 overnights of each construct and more clones on trafo plates
construct clones result
AP19.Poly in pSB1K3 #1 -
#2 -
#3 -
#4 will be sequenced
#5 will be sequenced
#6 will be sequenced
#7 will be sequenced
#8 -
#9 -
#10 will be sequenced
#11 -
#12 -
#13 -
#14 -
#15 -
AP19.Poly in pSB1C3 #1 -
#2 will be sequenced
#3 -
#4 will be sequenced
#5 -
#6 will be sequenced
#7 -
#8 -
#9 -
#10 -
#11 -
#12 -
#13 will be sequenced
#14 -
#15 will be sequenced
  • in general: many clones that look good on gel, 5 of each construct will be sequenced (two 5 ml overnights)

15-09-06

plasmid purification and test digests:

  • only use 1.5 ml of culture
    • ID distribution of temporary prepped plasmids for analytic purposes:
      • AP19 Poly in K3: PK 1-15
      • AP19 Poly in C3: PC 1-15
  • store rest of the cultures in 4 °C room
  • do test digest with EcoRI and PstI of:
    • AP19 Poly in K3 "minipreps" of overnigths from Gibson Assembly
    • AP19 Poly in C3 "minipreps" of overnights from Gibson Assembly
  • wait for results, then purify remaining culture volume (this time with plasmid purification kit) of positive clones and prepare for sequencing.
  • use masterplates to inoculate new liquid cultures
    • maybe we need more volume to purifiy

test digest

  • Master mix for 34 reactions
  • calculated DNA conc.: 25 ng/µl (new plasmid purification protocol, can't measure concentrations)
Component amount [µl]
H2O 170,2
EcoRI 7,4
PstI 7,4
10x NEB 2.1 37
  • to each tube, 6µl mastermix and 4 µl of DNA were added

plasmid purification with qiagen kit

  • AP19 Poly in K3: 4, 5, 6, 7, 10
  • AP19 Poly in C3: 2, 6, 12, 13, 15
ID construct plasmid ID cryo ID
PK 4 AP19 Poly in pSB1K3 from Gibson Assembly #FRNW# #MEPN#
PK 5 #QVDN# #BCCB#
PK 6 #Q3CN# #OHMA#
PK 7 #9V18# #O9HS#
PK 10 #FO14# #B4KL#
PC 2 AP19 Poly in pSB1C3 from Gibson Assembly #N9H9# #6PEY#
PC 6 #91RC# #HT1M#
PC 12 #P6AM# #XX6C#
PC 13 #1DWH# #TC4A#
PC 15 #PZHC# #RMNC#

15-09-07

  • all plasmids of polycistronic in K3 and C3 purified into 60 µl elution buffer (10 ml cell culture)
  • send in sequencing of #QVDN#, #9V18#; #91RC#, #1DWH# ALL NEGATIVE
  • again, overnights of Poly in K3 4, 5, 6, 7, and Poly in C3 2, 6, 12, 13, and 15 were made to put them on SDS (check if all genes are is expressed)

15-09-08

SDS Gel of AP19.Poly in pSB1K3 and pSB1C3

Construct Result on SDS Gel
Poly in C3 #2 -
Poly in C3 #6 -
Poly in C3 #12 -
Poly in C3 #13 -
Poly in K3 #5 -
Poly in K3 #6 -
Poly in K3 #7 -
  • Gel staining didn't work properly, so no reliable results can be made

15-09-09

  • Sequencing of #QVDN#, #9V18#; #91RC#, #1DWH# is all negative)

Test PCR

for screening of all purified plasmids of AP19.Poly in pSB1C3 and pSB1K3

  • make 2 mastermixes with two different primer pairs
    • Primers A: #A9W9# + #PSP2# ; expected lenght 3164 bp
    • Primers B: #HSP1# + #XE3D# ; expected lenght 3990 bp
  • KAPA 2G Mastermix PCR with 0,5 µl of purified plasmid as template

Programme

Time Temp
3:00 95
0:25 95
0:20 61
1:00 72
2:00 72

Constructs

Template #FRNW# #CVDN# #Q3CN# #9V18# #FO14# #N9H9# #91RC# #P6AM# #1DWH# #PZHC#
PCR tubes 1A, 1B 2A, 2B 3A, 3B 4A, 4B 5A, 5B 6A, 6B 7A, 7B 8A, 8B 9A, 9B 10A, 10B
result no bands

alkaline phosphatase digestion

Last try (!!!) to get polycistronic construct into a BioBrick vector just as much work as absulutely necessary will be invested in this workflow

Added 1 µl of phosphatase to #OLAE#:D and #OLAE#:E. Incubation for 20' at 37 °C.

Ligation with #OLAE#:F. Product: #9VCN#: I (in pSB1C3) + II (in pSB1K3)

Transformation of 5 µl into BL21 Gold (DE3)

15-09-11

  • new purified plasmids of #9PBD# (MDH in pSB1C3), #H4M9# (HPS in pSB1C3) and #KFEY# (XPK in pSB1C3)
  • overnights of AP19.Poly in pSB1C3 that grew were all red, in these in pSB1K3 did not grow
  • new overnights were made from master plate at 10:00 am

15-09-12

  • even though there were colonies on the LB plate from the transformation, the clones didn't grow in a liquid LB overnight cultures!
  • all 15 clones didn't grow in overnight culture, even after several trials!
SEEMS LIKE SUCCESSFULL TRANSFORMATIONS OF POLYCISTRONIC CONSTRUCT IN BIOBRICK BACKBONE CAN'T GROW


NO POLYCISTRONIC CONSTRUCT IN BIOBRICK BACKBONE WILL BE SEND TO THE REGISTRY!

References




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