Difference between revisions of "Team:Aachen/Notebook/Documentation/Glycogen Polycistronic Expression"

(15-07-17)
(15-07-20)
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** products are stored in #1W9S#
 
** products are stored in #1W9S#
  
{{Team:Aachen/Figure|Aachen_15-07-20 glgCAB test digest EcoRI.png|title=Test digest of glgCAB in pSB1C3|subtitle=#6BZC# and #EY9B# were test digested with EcoRI. #EY9B# looks very promising.|size=small}}
+
{{Team:Aachen/Figure|Aachen_15-07-20 glgCAB test digest EcoRI.png|title=Test digest of glgCAB in pSB1C3|subtitle=#6BZC# and #EY9B# were test digested with EcoRI. #EY9B# looks very promising.|size=medium}}
  
 
* '''clone 1 looks good, but clone 23 does not!'''
 
* '''clone 1 looks good, but clone 23 does not!'''

Revision as of 17:42, 18 September 2015


Laboratory Notebook

15-06-09

digest glgA/B plasmids (stored in #SZKD#)

name plasmid ID enzyme 1 enzyme 2
A1 (glgA) #M1M6# EcoRI SpeI
B1 (glgB) #YHT3# XbaI PstI
pSB1A3 #TRTN# EcoRI PstI


15-06-10

  • after digest glg A and B:
    • assemble A1 with B1 in pSB1A3 (ligation tube: A+B)
    • stored in #QHHR#
  • transformation into DH5α


15-06-11

  • master plates of glgA+B in pSB1A3 (LB+A)

15-06-12

  • colony PCR on glgAB construct to identify positive clones
  • make overnight cultures of glgAB construct
clone number cryo ID plasmid ID sequencing result
glgA.glgB in pSB1A3 #5 #Y4K4# #P6QM# glgB is missing

15-06-13

  • make cryos of glgAB construct (table above)
  • plasmid prep of glgAB (table above)

15-06-15

  • measured concentrations of purified glgAB constructs

15-06-16

  • digested glgAB plasmid (#P6QM#) with XbaI and PstI (product in #TZTD#) gel had three bands! -> new digest and ligation

15-06-17

Digestions with 40' digest and 20' inactivation

(previous digests A1, B1 were discarded from #SZKD# because agarose gels were really ugly)

gene enzymes expectation observation products
glgA (#M1M6#) EcoRI + SpeI 3533, 2051, 1482 confirmed #SZKD#:A
glgB (#YHT3#) XbaI + PstI 4286, 2234, 2052 confirmed #SZKD#:B

Ligations and transformations

construct part I part II backbone product host
glgAB in pSB1A3 #SZKD#:A #SZKD#:B #SZKD#:pSB1A3 #99MC#:LAB BL21


15-06-18

  • transformation of glgAB in pSB1A3 in BL21 did not work!
  • ligation of glgAB on a gel shows 3 independent bands
    • therefore, we cut pSB1A3 again with EcoRI and PstI

Digestions with 40' digest and 20' inactivation

(previous digests pSB1A3 was discarded from #SZKD# because we think it was cut with EcoRI and SpeI)

gene enzymes expectation observation products
pSB1A3 (#TRTN#) EcoRI + PstI ... ... #SZKD#:1A3

Ligations and transformations

construct part I part II backbone product host
glgAB in pSB1A3 #SZKD#:A #SZKD#:B #SZKD#:1A3 #99MC#:LAB DH5α

15-06-19

Master plates of glgAB clones that were white under green light.

15-06-22

  • colony PCR on glgAB plate from Saturday (#A9W9# and #XE3D#)
gene / clones tube number expected length machine elongation time result
glgAB in pSB1A2 clones 1-7 18-24 3986 3 3'59" clones 1-6 are good
  • overnight cultures of good clones from the colony PCR glgAB (LB+A)

15-06-23

  • cryos and plasmid prep from overnight culture of glgAB
  • the plasmid prep went wrong and resulted in a concentration of < 10 ng/µl
gene / clone cryo ID plasmid ID sequencing result
glgAB in pSB1A3 #5 #1Q88# #CT8H# confirmed ligation sites
glgAB in pSB1A3 #6 #YV86# #O3TQ# confirmed ligation sites
  • overnight cultures of the two clones above from the master plates

15-06-24

  • in the morning: freeze overnight cultures of glgB and glgAB
  • in the afternoon: plasmid prep of glgB and glgAB (re-use the existing IDs from the table of Monday)
  • pipette glgAB for sequencing #CT8H# and #O3TQ# were emptied during sequencing!


15-06-26

Digestions

BioBrick in Backbone gene template ID enzymes product purpose
K1585312 in pSB1A2 glgC #F8Y8# EcoRI, SpeI #LVVK#:2 construction of glgCAB
K1585320 in pSB1A2 glgAB #CT8H# XbaI, PstI #LVVK#:3 construction of glgCAB
  • overnight culture

15-06-29

  • Plasmid prep of glgAB in pSB1A3 because #CT8H# was empty (same ID)

Digestions

BioBrick in Backbone gene template ID enzymes product purpose
K1585320 in pSB1A2 glgAB #CT8H# XbaI, PstI #LVVK#:3 construction of glgCAB
  • run an agarose gel with the digestion products
    • gel showed that digest of glgAB and pSB1K30 has not worked!
    • we will use old pSB1K30 stored in #SZKD#

Ligation and Transformation

construct gene Part I Part II Backbone products host purpose
K1585321 in pSB1K30 glgCAB #LVVK#:2 #LVVK#:3 #LVVK#:4 #RRCV#:3 BL21 Gold (DE3) for polycistronic expression
  • new glgAB digestion because wrong concentration was used
BioBrick in Backbone gene template ID enzymes product purpose
K1585320 in pSB1A2 glgAB #CT8H# XbaI, PstI #LVVK#:3 construction of glgCAB
  • repeat ligation and freeze product for transformation

Ligation and Transformation

construct gene Part I Part II Backbone products host purpose
K1585321 in pSB1K30 glgCAB #LVVK#:2 #LVVK#:3neu #SZKD#:K30 #RRCV#:3 BL21 Gold (DE3) for polycistronic expression
  • glgAB digest did not work again!

PCR amplification of glgAB

  • 50 µl Q5 reaction with #A9W9# and #XE3D#
  • 2'00" elongation time, 58 °C annealing
  • products stored in #XMVF#

15-06-30

  • agarose gel of glgAB PCR product shows a very strong band at the expected length
  • the PCR product was purified into #PZ9B#

Digestion of glgAB PCR product

BioBrick in Backbone gene template ID enzymes product purpose
K1585320 in pSB1A2 glgAB #PZ9B# XbaI, PstI #LVVK#:3 construction of glgCAB

Ligation and Transformation

construct gene Part I Part II Backbone products host purpose
K1585321 in pSB1C3 glgCAB #LVVK#:2 #LVVK#:3 #CQ1V#:V 5 µl #RRCV#:3 40 µl BL21 Gold (DE3) for polycistronic expression

15-07-01

  • made a master plate of glgCAB in pSB1C3 with all available clones

15-07-02

  • colony PCR
  • 4 overnight cultures per clone (to have enough for sequencing)

conditions for 30 cycles:

step temperature [°C] duration
initial denaturation 95 3'00"
denaturation 95 0'15"
annealing #A9W9# and #XE3D# 58 0'15"
elongation 72 1'20"
final elongation 72 2'00"
  • repeat colony PCR
    • tubes are numbered 1-18 for colonies
    • colonies picked: 1,2,5,6,7,11,12,13,16-21,23-26
    • 19: water

conditions for 30 cycles:

step temperature [°C] duration
initial denaturation 95 10'00"
denaturation 95 0'15"
annealing #A9W9# and #XE3D# 58 0'15"
elongation 72 1'25"
final elongation 72 2'00"

15-07-03

  • plasmid prep of many different glgCAB clones
clone number cryo ID plasmid ID digest result
1 #LMCZ# #ZNMX# negative
2 #K49Y# #KLRW# negative
5 #FYOF# #ZKDX# negative
6 #K8MM# #AYWZ# negative
7 #LBRK# #M9BH# negative
11 #YBH1# #EAFT# negative
12 #99ER# #V6DM# negative
13 #L39B# #Z9BA# negative
16 #S3BW# #A4Y6# negative
17 #V3SS# #49S6# negative
18 #WOXE# #XO6W# negative
20 #KEMW# #4HTD# negative
  • (test) digestion of plasmids with EcoRI+PstI (products in #PCSW#)
  • agarose gel
  • overnight cultures of good clones from test digest

15-07-06

PCRs for Gibson/CPEC assembly

component template forward primer reverse primer product length product ID tube #
J04450 in pSB1C3 #CD8B# #TSTV# #FYTO# 2070 #MRVW#:II 1+2

PfuS PCR

conditions for 30 cycles:

step temperature [°C] duration
initial denaturation 98 2'00"
denaturation 98 0'30"
annealing 64 0'30"
elongation 72 1'10"
final elongation 72 5'00"

15-07-08

PCRs for Gibson/CPEC assembly

component template forward primer reverse primer product length product ID
glgC in pSB1A2 #F8Y8# #O3M6# #C3ZA# 1360 #MRVW#: IV
glgAB in pSB1A3 #CT8H# #93FZ# #NBZZ# 3713 #MRVW#:V

PfuS PCR

conditions for 30 cycles:

glgC

step temperature [°C] duration
initial denaturation 98 3'00"
denaturation 98 0'30"
annealing 59 0'30"
elongation 72 0'45"
final elongation 72 5'00"

glgAB

step temperature [°C] duration
initial denaturation 98 3'00"
denaturation 98 0'30"
annealing 57 0'30"
elongation 72 1'58"
final elongation 72 5'00"

The design of primer #93FZ# was flawed: it bound to the RBS.ATG section of glgB (within glgAB) with three mismatches and amplified a product of 2235 bp.

component template forward primer reverse primer product length product ID
glgA' #M1M6# #93FZ# #AVY6# 1157 #MRVW#: VI
glg'AB #CT8H# #EP9Z# #NBZZ# 2907 #MRVW#:VII

PfuS PCR

conditions for 30 cycles:

step temperature [°C] glgA' glg'AB
initial denaturation 98 3'00" 3'00"
denaturation 98 0'30" 0'30"
annealing 58 0'30" 0'30"
elongation 72 0'35" 1'27"
final elongation 72 5'00" 5'00"

15-07-09

  • 90'00" DpnI digest of #MRVW#:VI and #MRVW#:VII
  • the gel of #MRVW#:VII did not show any band - the old VII was discarded and the PCR was repeated with longer elongation time

PfuS PCR

conditions for 30 cycles:

step temperature [°C] glg'AB
initial denaturation 98 3'00"
denaturation 98 0'30"
annealing 56.5 0'30"
elongation 72 1'35"
final elongation 72 5'00"
  • expected length: 2907 bp


PCR product purification

PCR product purified PCR product
#MRVW#:II #ZV3C#
#MRVW#:III #T6X9#
#MRVW#:IV #QSWQ#
#MRVW#:V failed/obsolete
#MRVW#:VI #4TVW#
#MRVW#:VII #E9AQ#

Gibson Assembly of glgCAB in pSB1C3 (0.17 pmol total fragment amount, 30'00" incubation)

component volume [µl]
water 0
Gibson Master Mix 2x 12.5
linear pSB1C3 #ZV3C# 1.4
glgC for glgCAB #QSWQ# 5.3
glgA' for glgCAB #4TVW# 1.6
glg'AB for glgCAB #E9AQ# 4.3
product stored in #SWMB#:III

5 µl #SWMB#:III were heat shocked into DH5α and plated on LB+C

15-07-10

  • make masterplate (LB+C)


15-07-14

  • analyze master plate of additional glgCAB clones under green light, all clones were red!
  • repeat Gibson Assembly with now DpnI digested pSB1C3 backbone

Gibson Assembly of glgCAB in pSB1C3 (0.17 pmol total fragment amount, 60'00" incubation)

component volume [µl]
water 0
Gibson Master Mix 2x 12.5
linear pSB1C3 #ZV3C# 1.4
glgC for glgCAB #QSWQ# 5.3
glgA' for glgCAB #4TVW# 1.6
glg'AB for glgCAB #E9AQ# 4.3
product stored in #SWMB#:III

5 µl #SWMB#:III were heat shocked into DH5α and plated on LB+C

15-07-15

  • transformation was successful: under red-light 8 white clones were visible
  • overnights and masterplate of these good clones

15-07-16

  • cryos
  • plasmid prep
  • test digest with Xba and Pst (products in #NMTE#)
clone cryo plasmid ID test digest result
glgCAB in pSB1A30 #1 #9HZM# #QWNV# negative
glgCAB in pSB1A30 #2 #SR8W# #LTYR# negative
glgCAB in pSB1A30 #3 #VPTH# #QWBZ# negative
glgCAB in pSB1A30 #4 #NHYB# #6CEE# negative
glgCAB in pSB1A30 #5 #NHBT# #6KF4# could be something
glgCAB in pSB1A30 #6 #H443# #QOX1# negative
glgCAB in pSB1A30 #7 #YF88# #CHT1# could be something
glgCAB in pSB1A30 #8 #W4VO# #OQO3# negative
  • new overnights of clone#5 and #7 to have enough template for sequencing

15-07-17

CPEC assembly of glgCAB in pSB1C3

component volume [µl]
water 0
Q5 buffer 5
DMSO 0.75
dNTPs (O5) 1
Q5 polymerase 0.5
pSB1C3 fragment (#ZV3C#) 2.7
glgC for glgCAB #QSWQ# 9.8
glgA' for glgCAB #4TVW# 3.0
glg'AB for glgCAB #E9AQ# 8.0
elongation time 3'32"
product stored in #SWMB#:IV
  • agarose gel with glgA', Gibson product and CPEC product
  • heat shock transformation of #SWMB# :III and IV into DH5α
  • plasmid prep of potentially good glgCAB clones #5 in #FYH1# and #7 in #4RDQ#
  • test digest of #FYH1# and #4RDQ# with EcoRI (products in #4SK6#:1-2)
  • agarose gel comparing with undigested #FYH1# -> #FYH1# looks promising, but #4RDQ# is too short


Aachen 15-07-17 glgCAB test digestion.png
Test digest of glgCAB in pSB1C3
#FYH1# and #4RDQ# were test digested with EcoRI. #FYH1# looks very promising.

15-07-18

  • master plate of many white glgCAB clones on LB+C

15-07-19

  • 3x 5 ml LB+C overnight cultures of glgCAB clones #1 (CPEC) and #23 (Gibson)

15-07-20

  • Cryos and freezings of glgCAB clones #1 and #23
  • plasmid prep of glgCAB clones #1 and #23
strain cryo plasmid
glgCAB in pSB1C3 by CPEC in DH5α #1 #VAPH# #EY9B#
glgCAB in pSB1C3 by Gibson in DH5α clone #23 #PAZS# #6BZC#
  • pipette glgCAB in pSB1C3 #FYH1# for sequencing with all primers
  • test digest of glgCAB #1 and #23 with EcoRI
    • tube 1: #6BZC# (#23)
    • tube 2: #EY9B# (#1)
    • products are stored in #1W9S#
Aachen 15-07-20 glgCAB test digest EcoRI.png
Test digest of glgCAB in pSB1C3
#6BZC# and #EY9B# were test digested with EcoRI. #EY9B# looks very promising.
  • clone 1 looks good, but clone 23 does not!
  • prepare moving glgCAB over into a vector with promoter

15-07-21

  • digest of glgCAB clones, J23119 and K1585119 to combine CAB with the Anderson Promoter and pSB1A30
template ID enzymes tube number purpose
Anderson Promoter 19 #AHZ6# SpeI, PstI 1 to combine CAB and constitutive AP 19
K1585119 #OEVV# SpeI, PstI 2 to combine CAB and inducible AP 19
glgCAB #1 #EY9B# XbaI, PstI 3 to combine with both AP 19 versions
glgCAB #5 #FYH1# XbaI, PstI 4 to combine with both AP 19 versions
glgCAB #1 #EY9B# EcoRI, PstI 5 to combine with pSB1A30
glgCAB #5 #FYH1# EcoRI, PstI 6 to combine with pSB1A30
  • products stored in #TF4M#
  • ligation
name template 1 template 2 purpose
L1 tube 1 (const. AP 19) tube 3 (glgCAB #1 with X and P) glgCAB with constitutive AP 19
L2 tube 2 (induc. AP 19) tube 3 (glgCAB #1 with X and P) glgCAB with inducible AP 19
L3 tube 1 (const. AP 19) tube 4 (glgCAB #5 with X and P) glgCAB with constitutive AP 19
L4 tube 2 (induc. AP 19) tube 4 (glgCAB #5 with X and P) glgCAB with inducible AP 19
  • products are also stored in #TF4M#
  • all ligation products were transformed in NEB 10β

15-07-22

  • make a gel with all digests
Aachen 15-07-22 digest and ligation products of CAB and AP19.png
Digest and ligation of glgCAB and AP 19
For #AHZ6# there are two bands of the right length, one uncut and one cut. For #OEVV# there is only one weak band but it has the correct length. Both CAB digest products of #EY9B# have the correct length. There are no bands for the ligations.
strain cryo plasmid sequencing result
glgCAB in pSB1C3 by CPEC in DH5α #1 #VAPH# #EY9B# A>V mutation in glgB, glgC and A look perfect
glgCAB in pSB1C3 by Gibson in DH5α clone #23 #PAZS# #6BZC# sequencing did not work
glgCAB in pSB1C3 by Gibson in DH5α clone #5 #NHBT# #FYH1# glgA and B look perfect but glgC is missing
  • overnight cultures and master plates of transformations of ligations 1+2 (LB+A)
  • new overnights of clones #3-5 glgCAB in pSB1C3 by CPEC from masterplate

15-07-23

  • cryos and plasmid prep of glgCAB, AP19.glgCAB and AP19.lacIsite.glgCAB candidates
  • test digest with EcoRI (stored in #AMX6#)
    • 1: #4HB9#
    • 2: #6THO#
    • 3: #BB9Q#
    • C2: #HOEL#
construct cryo plasmid digest result sequencing result
K1585321 in pSB1C3 #2 #BA36# #NXOK# ... no chromatogram
K1585321 in pSB1C3 #3 #KRWH# #BB9Q# ... no chromatogram
K1585321 in pSB1C3 #4 #RHMM# #6THO# ... no chromatogram
J23119.K1585321 in J61002 #1 #K3K4# #4HB9# cloning did not work
J23119.K1585321 in J61002 #2 #W3QO# not prepped cloning did not work
K1585119.K1585321 in J61002 #1 #Y4N4# was red
K1585119.K1585321 in J61002 #2 #XFF9# was red
K1585119.K1585321 in J61002 #3 #KATB# was red
K1585119.K1585321 in J61002 #4 #MWVT# was red

15-07-24

  • sequencing results were bad, so we use parts from #FYH1# (just glgC missing) and #EY9B# (mutation in glgB) to construct the full plasmid again
  • these parts will be assembled in a Gibson Assembly

Q5 PCR with 27 cycles

PCR template primer 1 primer 2 annealing temperature elongation time product length
1 #FYH1# #EP9Z# #6ZOP# 71 °C 1'23" 2640 bp (parts of glgA and B)
2 #EY9B# #89VV# #AVY6# 71 °C 2'40" 5178 bp (all the rest)

PCR programm

step temperature time
initial denaturation 98 °C 0'30"
denaturation (cycle) 98 °C 0'10"
annealing (cycle) 71 °C 0'25"
elongation (cycle) 72 °C see above
final elongation 72 °C 2'00"

The products are temporarily stored in #94DS#.

Both products are digested with DpnI overnight at 37 °C and put back into #94DS#.

15-04-25

  • put DpnI digests back into #94DS# and freeze it

15-07-27

  • agarose gel of DpnI digests
  • PCR product purification
    • products stored in #MCWO# and #963L#
  • Gibson Assembly of #MCWO# and #963L# at 50 °C for 50 minutes
  • heat shock into DH5α

15-07-28

  • transformation was successful
  • masterplate of 30 clones from transformation plate of glgCAB in pSB1C3
  • overnight of 10 clones for test digest

15-07-29

  • plasmid prep and cryos of all overnights
construct cryo plasmid tube # in digest digest result sequencing result
K1585321 in pSB1C3 #1 #L8CN# #96EB#, #4RCD# 1 good point mutation
K1585321 in pSB1C3 #2 #NMTT# #MFQS#, #L6OH# 2 good point mutation
K1585321 in pSB1C3 #3 #MXKQ# #QAB9#, #8WE4# 3 good point mutation
K1585321 in pSB1C3 #4 #CKSK# #B316# 4 good -
K1585321 in pSB1C3 #5 #1TN1# #HOX9# 5 two bands instead of one -
K1585321 in pSB1C3 #6 #MKC3# #FNTZ# 6 good -
K1585321 in pSB1C3 #7 #99NB# #6DMQ# 7 too long (?) -
K1585321 in pSB1C3 #8 #P664# #6OT6# 8 good -
K1585321 in pSB1C3 #9 #P8NZ# #HF8O# 9 good -
K1585321 in pSB1C3 #10 #TO3R# #A1FM# 10 good -
  • test digest with EcoRI + agarose gel
  • new overnights of good glgCAB in C3 clones no.1-3 (3 per clone to have enough for seqencing)

15-07-30

  • plasmid prep (see table above)
  • pipette for sequencing

15-07-31

  • Sequencing showed that there still is a point mutation in glgB
  • decided to do a QuickChange with #EY9B#
  • QuickChange primers were designed and ordered

15-08-03

  • QuickChange with PfuS (E2)
  • tube 1:
component amount
plasmid template (1:100 dilution) 3.3 µl
deionized H2O 38.2 µl
10x PfuS buffer (B2) 5 µl
#BABZ# fwd_primer (10 µM) 2 µl
dNTP mix (10 mM) 1 µl
PfuS 0.5 µl
  • tube 2:
component amount
plasmid template (1:100 dilution) 3.3 µl
deionized H2O 38.2 µl
10x PfuS buffer (B2) 5 µl
#BOXR# rev_primer (10 µM) 2 µl
dNTP mix (10 mM) 1 µl
PfuS 0.5 µl
  • PCR program 1 (105 °C heated lid)

3 cyles

step temperature time
initial denaturation 98°C 0'30"
denaturation (cycle) 98°C 0'10"
annealing (cycle) 59°C 0'30"
extension (cycle) 72°C 4'15"
  • control
component amount
plasmid template (1:100 dilution) 3.3 µl
deionized H2O 40.2 µl
10x PfuS buffer (B2) 5 µl
dNTP mix (10 mM) 1µl
PfuS 0.5 µl
  • mix 25 µl of tube 1 and 25 µl of tube 2 and add 0.5 µl PfuS
  • PCR program 2 (105°C heated lid)

15 cycles

step temperature time
initial denaturation 98 °C 0'30"
denaturation (cycle) 98 °C 0'10"
annealing (cycle) 59 °C 0'30"
extension (cycle) 72 °C 4'15"
final elongation 72 °C 10'00"
  • products are in #189R#
  • make an agarose gel
  • PCR clean-up
  • DpnI digest over night

15-08-04

  • 20 minutes inactivation
  • PCR clean-up
  • transformation of PCR product
  • repeat the QuikChange with higher template and enzyme concentration because no bands were visible on the gel
  • QuickChange with PfuS (E2)
  • tube 1:
component amount
plasmid template #EY9B# (1:100 dilution) 6.6 µl
deionized H2O 34.9 µl
10x PfuS buffer (B2) 5 µl
#BABZ# fwd_primer (10 µM) 2 µl
dNTP mix (10 mM) 1 µl
PfuS 0.5 µl
  • tube 2:
component amount
plasmid template #EY9B# (1:100 dilution) 6.6 µl
deionized H2O 34.9 µl
10x PfuS buffer (B2) 5 µl
#BOXR# rev_primer (10 µM) 2 µl
dNTP mix (10 mM) 1 µl
PfuS 0.5 µl
  • PCR program 1 (105 °C heated lid)

3 cyles

step temperature time
initial denaturation 98 °C 0'30"
denaturation (cycle) 98 °C 0'15"
annealing (cycle) 59 °C 0'30"
extension (cycle) 72 °C 5'00"
  • control
component amount
plasmid template (1:100 dilution) 6.6 µl
deionized H2O 36.9 µl
10x PfuS buffer (B2) 5 µl
dNTP mix (10 mM) 1 µl
PfuS 0.5 µl
  • mix 25 µl of tube 1 and 25 µl of tube 2 and add 1 µl PfuS
  • PCR program 2 (105 °C heated lid)

15 cycles

step temperature time
initial denaturation 98 °C 0'30"
denaturation (cycle) 98 °C 0'15"
annealing (cycle) 59 °C 0'30"
extension (cycle) 72 °C 5'00"
final elongation 72 °C 10'00"
  • products are in #189R#
  • make an agarose gel
Aachen 15-08-05 glgCAB SDM.png
SDM of glgCAB
For tubes 1 and 2 there are faint bands at the correct length, but there are also strong undefined bands on top. For the control (#3) the 7066 bp band is faint as expected
    • correct bands were visible
  • DpnI digest over night

15-08-05

  • 20 minutes inactivation
  • PCR clean-up
  • agarose gel of purified PCR products
sample purified digest product ID gel result transformation result
number 1 #AZYS# correct bands visible ...
number 2 #ATM8# correct bands visible ...
control #SWEV# no 7000 bp bands ...

15-08-06

  • repeat DpnI digest of Gibson and SDM products
  • transformation into NEB10β

15-08-07

  • masterplate of clones from trafo plate

15-08-08

  • take out masterplates from 37 °C and put them in the fridge

15-08-09

  • overnights of 10 clones for test digest on monday

15-08-10

  • plasmid prep and cryos of clones
Clone cryo ID plasmid ID test digest result sequencing result
glgCAB in pSB1C3 #1 #XWFL# #A919# good bad
glgCAB in pSB1C3 #2 #ZNNL# #QOZV# good good
glgCAB in pSB1C3 #3 #41ZT# #RW33# good good
glgCAB in pSB1C3 #4 #6THN# #DTBM# good good
glgCAB in pSB1C3 #5 #VMV3# #COOX# 2 bands
glgCAB in pSB1C3 #6 #LRAT# #AO86# length not clear
glgCAB in pSB1C3 #7 #3W3Y# #TMXD# length not clear
glgCAB in pSB1C3 #8 #SHHS# #XB19# good point mutation
glgCAB in pSB1C3 #9 #VOBO# #ATH8# good point mutation
glgCAB in pSB1C3 #10 #33C1# #LY4Z# good point mutation
  • test digest

15-08-11

  • pipette for sequencing with one primer that covers point mutation (#RYZX#)
  • bring the samples to the Fraunhofer institute

15-08-14

  • overnights of #6THN#, #ZNNL# and #41ZT#for further sequencing of glgCAB clones

15-08-15

  • plasmid prep of CAB clones
    • products #QOZV# and #DTBM#

15-08-17

  • digest #EY9B# (tube1), #QOZV# (tube 2) and #DTBM# (tube 3) with EcoRI and PstI to transfer into pSB1A30
    • digest of #EY9B# did not work, so we use an digestion product from #TF4M# (tube 5)

ligation

tube no insert backbone purpose
2 #QOZV# #OHOZ# tube # 1 new glgCAB #2 in pSB1A30
3 #DTBM# #OHOZ# tube # 1 new glgCAB #4 in pSB1A30
  • products are stored in #3A9X#
  • transform in DH5α

15-08-18

  • sequencing results show that #QOZV# and #DTBM# are both missing glgC
  • all transformations were thrown away
  • we will test glgCAB with one point mutation

15-08-19

  • digest #8WE4# and #EY9B# with EcoRI and PstI to ligate it in pSB1A30 for expression
  • both look good on gel
Aachen 15-08-19 glgCAB cut with E+P.png
digest of glgCAB in pSB1C3
For tubes 1 and 2 there are strong bands at the correct length (2000 and 5000bp)
  • products stored in #WV93#

15-08-20

  • ligation of cut #8WE4# and #EY9B# in pSB1A30
  • heat shock in DH5α
tube no insert backbone purpose
2 #8WE4# #OHOZ# tube # 1 glgCAB with point mutation in pSB1A30
3 #EY9B# #OHOZ# tube # 1 glgCAB with point mutation in pSB1A30

15-08-21

  • overnight cultures and master plate of DH5α glgCAB clones

15-08-22

  • make cryo cultures of glgCAB in pSB1A30 DH5α overnight cultures
    • plasmid prep afterwards


no What? strain Cryo plasmid
1 glgCAB in A30 (cut from #EY9B#) DH5α #ADWX# #XTEN#
2 glgCAB in A30 (cut from #EY9B#) DH5α #ALXK# #CTMV#
3 glgCAB in A30 (cut from #EY9B#) DH5α #AR46# #Q13F#
4 glgCAB in A30 (cut from #EY9B#) DH5α #3MKZ# #YFXC#
2 glgCAB in A30 (cut from #8WE4#) DH5α #TNR8# #KSZQ#
3 glgCAB in A30 (cut from #8WE4#) DH5α #R4CZ# #KRYH#
4 glgCAB in A30 (cut from #8WE4#) DH5α #HMSM# #NPHP#
  • 3A assembly of glgAB (#CT8H#) and glgC (#4D6H#) in pSB1C3 (already cut: #CQ1V# tube V)
    • digest #4D6H# with EcoRI and SpeI, product in #KFA1#
    • digest #CT8H# with XbaI and PstI, looked bad ont the gel: concentration was too low
  • make an overnight culture of #1Q88# to produce new #CT8H#

15-08-23

  • plasmid prep of new #CT8H# , concentration measurement will be done tomorrow

15-08-24

  • test digest of glgCAB constructs with EcoRI
tube no plasmid result
1 #Q13F# bad
2 #YFXC# bad
3 #NPHP# bad
4 #KRYH# bad
5 #KSZQ# good
6 #XTEN# bad
7 #CTMV# good
  • transformation of #KSZQ# in BL21 Gold
  • digest of #CT8H# with XbaI and PstI (stored in #3649#)
    • gel looks good
  • ligation of glgAB (#3649#), glgC (#KFA1#) in pSB1C3 (#CQ1V# tube5)
    • tube L1 (also stored in #3649#)
  • transformation of new glgCAB into DH5α
  • electroporation of #KSZQ# in BL21 ΔX and BL21 ΔP electrocompetent

15-08-25

  • masterplate and overnight cultures of #KSZQ# in BL21 Gold, BL21 ΔX and BL21 ΔP in LB+A
  • masterplate and overnight of new glgCAB in DH5α in LB+C
  • new transformation of #KSZQ# in BL21 Gold


15-08-26

  • colony pcr of mutated GlgCAB (CAB')(in pSB1A30)
clone tube #
CAB' in pSB1A30 in BL21 ΔX clone #1-12 1-12
CAB' in pSB1A30 in BL21 ΔP clone #1-4 13-16
CAB' in pSB1A30 in BL21 ΔP clone #7-12 17-22
CAB' in pSB1A30 in BL21 ΔP clone #5+6 22-24
  • none of the clones had the expected length of 5362 bp on the gel
    • probably the fragment was too long
    • do 10 overnights of glgCAB in delta P each for test digest tomorrow, all clones in delta X were red
    • then new overnight of good one, main culture on friday, SDS on friday


  • new trafo of CAB' (mutated) in BL21 Gold wild type
  • plasmid prep and cryos of NEW GlgCAB in psB1C3
clone no cryo plasmid digestion tube result of digestion
1 #OR9C# #VCWE# 1 bad
3 #DYWY# #S9H8# 2 bad
5 #FQ69# #9CDT# 3 bad
7 #R4MF# #MDSB# 4 many bands, but also the correct one
8 #8MKC# #KZRZ# 5 many bands, but also the correct one
9 #NRSN# #PTRE# 6 (PTRE) bad
10 #3EHE# accidently discarded bad
A #KLH8# #RRPP# 7 bad
B #OC1P# accidently also #PTRE# 8 (PTRE*) bad
C #4EHF# #94RY# 9 bad
  • tubes 4+5 look good, products stored in#ZY4A#
  • digest of NEW GlgCAB
    • gel and inactivation
  • then ligate tube 4+5 (#MDSB# and #KZRZ#) into pSB1A30 (#OHOZ# tube 1), ligations also in #ZY4A#
  • transformation in DH5α
  • for sequencing: New GlgCAB in pSB1C3, clones that looked good in test digest
  • all clones from new CAB looked bad in test digest with EcoRI. We will not continue working with these ones..

15-08-27

  • plasmid prep of glgCAB' in delta P (clones # 1-10)
  • test digest with eco
    • all clones had the wrong length (ca. 3200 bp)
  • trafo of glgCAB in pSB1A30 in BL21 WT did not work again

We decided to sequence #KSZQ#, #MDSB# and #KZRZ# and and to digest pSB1A30 again (tube #T6X9#) and ligate CAB' into new pSB1A30

  • made a gel to test several constructs
Aachen 15-08-27 digests for CAB' assembly in A30.png
Cloning glgCAB'(mutated) in pSB1A30
All constructs that were used for glgCAB' assembly in A30 had the correct length. Also a fragment of the correct length (complete CAB in A30) can be seen in the coloumn for the ligation tube (7202bp).
  • CAB' (#8WE4#) was ligated with new digested pSB1A30 (#T6X9#) and cloned into Bl21 Gold DE3

15-08-28

  • a few clones were visible on the transformation plate of CAB' in pSB1A30
    • make overnight cultures and a master plate

15-08-29

  • plasmid prep and test digest of CAB' in pSB1A30 clone #2 (plasmid: #4XWH# cryo: #3OVK#)
    • gel looks bad, no correct bands visible for double digest
  • new overnight of CAB' in pSB1A30 clone #1 in order to repeat the above (it did not grow)

15-08-30

  • try to assemble glgCAB' in pSB1A30 via CPEC
    • use pSB1A30 from #T6X9#

PCR to amplify CAB'

component amount
ddH2O 23.6 µl
#O3M6# 0.6 µl
#NBZZ# 0.6 µl
B2 Buffer 3 µl
dNTPs 0.6 µl
PfuS (E2) 0.3 µl

PCR program

step temperature [°C] time
initial denaturation 98 3'00"
denaturation 98 0'30"
annealing 63 0'30"
elongation 72 2'37" (5039 bp)
final elongation 72 5'00"
  • heat shock of both BBa_C0012 from kit plate and ligation product "Lig iJR" (#WV93#) into BL21 Gold DE3
  • make three overnight cultures of #TNR8# to have enough plasmid for sequencing
  • do DpnI digest overnight (of PCR product for CPEC)

15-08-31

  • heat inactivate DpnI digest, purify and measure concentration (product in #N13D#), do CPEC with #T6X9#, transformation into BL21 Gold

CPEC program

step temperature time
initial denaturation 98 0'30"
denaturation (cycle) 98 0'10"
annealing (cycle) 55 0'30"
elongation (cycle) 72 1'52"
final elongation 72 10'00"
  • prep overnight cultures and prepare sequencing

15-09-01

  • do masterplates and overnights from Trafo plate (CAB' in A30 via CPEC)
  • digest glgAB (#CT8H#) and pSB1C30 (#MFNP#) with EcoRI and PstI
  • ligate glg AB into pSB1C3 (#OLAE# tube A) and pSB1C30 (#OQ81#)
    • ligation products stored in #OQ81#
    • heat shock into DH5α (pSB1C3) and BL21 (pSB1C30)

15-09-02

  • plasmid prep of overnights
  • test digest
    • gel showed that RFP is in all constructs of CAB
  • the backbone might be not digested correctly with DpnI after PCR
  • DpnI digest of #T6X9#
  • overnights and masterplates of glgAB in pSB1C3 and pSB1C30

15-09-03

  • sequencing shows that just glgC was in glgCAB ligation attempts via Biobrick assembly
  • sequencing showed that #KSZQ# (glgCAB' in pSB1A30) is PERFECT
  • cryo and plasmid prep of overnights of glgAB in C3 and C30
  • only clones 1,2,3,6,7 and glgAB in C30 were prepped becuase the other ones were red
    • test digest with EcoRI and PstI
    • only clones #3+6 of glgAB in pSB1C3 look good
    • subcloning into pSB1C30 did not work
clone cryo ID test digest result plasmid ID
glgAB in C3 clone #1 #XM94# bad -
glgAB in C3 clone #2 #SZW1# bad -
glgAB in C3 clone #3 #MQQ4# perfect #HAMP#
glgAB in C3 clone #6 #1KA4# perfect #YC8M#
glgAB in C3 clone #7 #B9ZD# bad -
glgAB in C30 clone #1 #AWWP# bad -
  • overnights of good glgAB clones #3+6 from masterplate
  • PCR purification of DpnI digested #T6X9#
  • transformation of 5 µl #KSZQ# into BL21 Gold DE3
  • M9 overnights of BL21 WT, BL21 glgA, BL21 glgB and BL21 ΔP for iodine staining
  • LB overnights cultures of good glgAB clones for growth experiments and SDS gels

15-09-04

  • master plates and overnights of #KSZQ# transformation clones
  • pipette sequencing of glgAB clones
  • for AB in C30: heat shock ligation product (in #OQ81#) into DH5α
    • for new C30 backbone: amplify #MFNP# with #TSTV# and #FYTO#,digest with 2µL DpnI (for 4 hours), heatinactivate, digest with E+P, ligate, transform into DH5α

15-09-05

  • cryos and plasmid prep of BL21 CAB' in A30, then test digest with EcoRI (Sciebo: 15-09-05 Test digest of CAB in A30)
Clone cryo ID plasmid ID test digest result sequencing result
glgCAB' in pSB1A30 #1 #WCQY# #YC3V# good
glgCAB' in pSB1A30 #2 #V4YZ# #3KZZ# bad
glgCAB' in pSB1A30 #3 #6AME# #XQ4R# bad
glgCAB' in pSB1A30 #4 #8HCP# #MKHV# bad
glgCAB' in pSB1A30 #5 #LFRD# #S3RW# good
glgCAB' in pSB1A30 #6 #B11R# #BBBF# bad
glgCAB' in pSB1A30 #7 #FNVQ# #QM4Y# bad


  • do new overnights of positive clones (#1+5(for SDS))
  • screen Trafo plates of AB in C30 in DH5α
    • all clones were white
    • do 5 overnights of each new and old ligation
    • parallel masterplates

15-09-06

  • do plasmid prep (see Manuals/Plasmid Prep!!!) and test digest with EcoRI of DH5α glgAB in pSB1C30
  • SDS-PAGE (Ladder-GlgCAB clone #1, GlgCAB #5,GlgA,GlgB,Wildtype for control)
    • glgC did not grow
  • HPLC samples
  • new 10 ml overnights (LB+20mM Glucose) of WT,A,B,deltaX,deltaP
  • overnights of GlgAB in pSB1C30 for CRYOS AND PREP
  • overnights of BL21 GlgCAB in pSB1A30 #1, #5 (LB+A+IPTG+20mM Glucose) and BL21 WT

15-09-07

  • cryos and prep DH5α glgAB in pSB1C30
construct Cryo purified plasmid
Old glgAB #2 #8ZZ4# #ZBVK#
Old glgAB #3 #8MCD# #14XB#
New glgAB #1 #N96D# #C9ZY#
New glgAB #2 #NDEF# #K8XD#
  • pipette DH5α glgAB in pSB1C30 and BL21 glgCAB' in pSB1A30 for sequencing, prepare sequencing sheet
  • transform four glgAB in pSB1C30 into BL21 Gold (see table above)
  • overnights of GlgCAB in pSB1A30 #1 and #5, GlgC in A30 (#OHES#) and WT Bl21 Gold for repeated iodine staining (incubate since 9 p.m.!)

15-09-08

  • Prep glgCAB in pSB1A30 #1 for remaining sequencing tubes
  • screen trafo of GlgAB in pSB1C30 BL21 Gold
    • do masterplates

15-09-09

  • sequencing did not work
  • make two overnights of both CAB clones #1 and #5 for new sequencing

15-09-10

  • pipette #S3RW# and #YC3V# again for sequencing

15-09-11

  • both #S3RW# and #YC3V# look perfect in sequencing!
    • confirmed glgCAB' in pSB1A30

Results

BioBrick gene backbone confirmed cryo confirmed plasmid
BBa_K1585320 glgAB pSB1A3 #1Q88# #CT8H# (ligation sites confirmed)
BBa_K1585321 glgCAB pSB1C3 #MXKQ# #8WE4#
BBa_K1585321 glgCAB' pSB1A30 #WCQY# #KSZQ#