Team:Aachen/Notebook/Documentation/Glycogen Polycistronic Expression

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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

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#

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

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

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

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

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#

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