Single Expression

Laboratory Notebook

15-06-09

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

name	plasmid ID	enzyme 1	enzyme 2
A2 (glgA)	#M1M6#	EcoRI	PstI
B2 (glgB)	#YHT3#	EcoRI	PstI
pSB1K30	#PCFX#	EcoRI	PstI

15-06-10

after digest glg A and B:
- A and B will be ligated in pSB1K30 (ligation tubes: A2 and B2)
- stored in #QHHR#
transformation into DH5α

15-06-11

master plates of glgA, glgB in pSB1K30 (LB+K)

15-06-12

colony PCR on glgA/B constructs to identify positive clones
make overnight cultures of glgA/B constructs

clone number	cryo ID	plasmid ID	sequencing result
glgA in pSB1K30 #2	#3DPV#	#D6VB#	mRFP
glgA in pSB1K30 #3	#4LCM#	#RLDN#	mRFP
glgB in pSB1K30 #5	#6YBE#	#ATE6#	pT7 is missing

15-06-13

make cryos of glgA/B constructs (table above)
plasmid prep of glgA, glgB (table above)

15-06-15

measured concentrations of purified glgA/glgB constructs
heat shock of glgA (#D6VB#, #RLDN#) and glgB (#ATE6#) in pSB1K30 into BL21(DE3)

15-06-16

BL21 transformation master plates (glgB transformation was not successful)

15-06-17

Digestions with 40' digest and 20' inactivation

(previous digests A2, B2, pSB1K30 were discarded from #SZKD# because agarose gels were really ugly)

gene	enzymes	expectation	observation	products
glgA (#M1M6#)	EcoRI + PstI	...	confirmed	#SZKD#:A2
glgB (#YHT3#)	EcoRI + PstI	...	confirmed	#SZKD#:B2
pSB1K30 (#PCFX#)	EcoRI + PstI	...	confirmed but weak	#SZKD#:K30

Ligations and transformations

construct	part I	part II	backbone	product	host
glgA in pSB1K30	#SZKD#:A2	-	#SZKD#:K30	#99MC#:LA	BL21
glgB in pSB1K30	#SZKD#:B2	-	#SZKD#:K30	#99MC#:LB	BL21

15-06-18

make master plates and liquid cultures of transformations (glgA in pSB1K30, glgB in pSB1K30; 6 liquid, 12 plate)

15-06-19

Colony PCR on glgA,B,C constructs

gene / clones	tube number	expected length	machine	elongation time	result
glgA in pSB1K30 clones 1-12	49-60	1770	2	1'50"	three okay
glgB in pSB1K30 clones 1-12	61-72	2523	3	3'05"	all negative

Cryos

gene / clone	cryo ID	plasmid ID	sequencing result
glgA in pSB1K30 #2	#VWSS#	#FHTH#	-
glgA in pSB1K30 #3	#W8VB#	#K8DM#	perfect
glgA in pSB1K30 #6	#4TPL#	#K3EO#	good, but possibly one (unlikely) insertion

Master plates of glgA, glgB clones that were white under green light.

15-06-22

prep good plasmids of glgA (table above)
pipette glgA in pSB1K30 for sequencing (#A9W9# and #XE3D#)
colony PCR on glgA, glgB plates from Saturday (#A9W9# and #XE3D#)

gene / clones	tube number	expected length	machine	elongation time	result
glgA in pSB1K30 clones 13-15	12-14	1821	2	1'50"	all three are good
glgB in pSB1K30 clones 13-15	15-17	2572	3	3'05"	clone 14 is good

overnight cultures of good clones from the glgB colony PCR (LB+K)

15-06-23

cryos and plasmid prep from overnight cultures of glgB
the plasmid prep went wrong and resulted in concentrations < 10 ng/µl

gene / clone	cryo ID	plasmid ID	sequencing result
glgB in pSB1K30 #14	#Q6Z1#	#KK8K#	confirmed ligation sites

overnight cultures of glgB in pSB1K30 from the master plate

15-06-24

in the morning: freeze overnight culture of glgB
in the afternoon: plasmid prep of glgB (re-used the existing ID from the table of Monday)
pipette glgB for sequencing

15-06-26

Digestions

BioBrick in Backbone	gene	template ID	enzymes	product	purpose
K1585312 in pSB1A2	glgC	#F8Y8#	EcoRI, PstI	#LVVK#:1	subcloning into pSB1C3, pSB1K30
pSB1K30	mRFP	#1K30#	EcoRI, PstI	#LVVK#:4	target backbone for expression

15-06-29

Ligation and Transformation (first try)

construct	gene	Part I	Part II	Backbone	products	host	purpose
K1585312 in pSB1K30	glgC	#LVVK#:1	-	#LVVK#:4	#RRCV#:2	BL21 Gold (DE3)	for expression tests

>> #LVVK#:4 (pSB1K30) looked bad on agarose gel! Do the ligation again with pSB1K30 from a different tube!

Ligation (second try, only 1 µl of K30)

construct	gene	Part I	Part II	Backbone	products	host	purpose
K1585312 in pSB1K30	glgC	#LVVK#:1	-	#SZKD#:K30	#RRCV#:2	BL21 Gold (DE3)	for expression tests

transformation is planned for the day after

15-06-30

heat shock of K1585312 in pSB1K30 ligation product (5 µl #RRCV#:2) into 40 µl BL21 Gold (DE3)

15-07-01

master plate of K1585312 in pSB1K30 (BL21 Gold DE3)

15-07-02

colony PCR of K1585312 in pSB1K30 (desired: 1683, negative: 1227)
~~overnights of good clones~~

colony PCR with KAPA 2G

conditions for 30 cycles:

step	temperature [°C]	duration
initial denaturation	95	3'00"
denaturation	95	0'15"
annealing	58	0'15"
elongation	72	0'25"
final elongation	72	2'00"

Digestions

BioBrick in Backbone	gene	template ID	enzymes	product	purpose
K1585312 in pSB1A2	glgC	#F8Y8#	EcoRI, PstI	#LVVK#:1	subcloning into pSB1K30
pSB1K30	mRFP	#4K19#	EcoRI, PstI	#LVVK#:4	target backbone for expression

Ligation

construct	gene	Part I	Part II	Backbone	products	host	purpose
K1585312 in pSB1K30	glgC	#LVVK#:1	-	#LVVK#:4	#RRCV#:2B	BL21 Gold (DE3)	for expression tests

15-07-03

~~cryo cultures~~
~~plasmid prep~~
~~pipette for sequencing~~

15-07-06

~~transformation of #RRCV#:2B into BL21 Gold (DE3)~~
decided to use pSB1A30 because the difference between induced and non-induced pSB1K30 could not be seen on SDS gels

PCR 1 to subclone glgC into pSB1A30: backbone amplification

component	template	forward primer	reverse primer	product length	purified PCR product ID	product ID	tube #
I13507 in pSB1A30	#LYZH#	#TSTV#	#FYTO#	2206	#T6X9#	#MRVW#:III	3+4

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

~~master plate (LB+K+IPTG) of glgC in pSB1K30 transformations~~

PCR2 to subclone glgC into pSB1A30: glgC amplification

component	template	forward primer	reverse primer	product length	product ID
glgC in pSB1A2	#F8Y8#	#O3M6#	#NBZZ#	1360	#MRVW#: I

PfuS PCR

conditions for 30 cycles:

step	temperature [°C]	duration
initial denaturation	98	3'00"
denaturation	98	0'30"
annealing	58	0'30"
elongation	72	0'45"
final elongation	72	5'00"

15-07-09

PCR product purification

PCR product	purified PCR product
#MRVW#:I	#XNHT#
#MRVW#:III	#T6X9#

CPEC assembly of glgC in pSB1A30

component	volume [µl]
water	5.1
Q5 buffer	5
DMSO	0.75
dNTPs (2 mM)	5
Q5 polymerase	0.5
pSB1A30 fragment (#T6X9#)	3.9
glgC with prefix/suffix #XNHT#	4.8
elongation time	1'47"
product stored in	#SWMB#:II

5 µl #SWMB#:II were heat shocked into BL21 Gold (DE3) and plated on LB+A

15-07-10

make masterplates (LB+A) and overnights of glgC in pSB1A30

15-07-11

cryo cultures
freeze culture

gene / clone	cryo ID	plasmid ID	sequencing result
B0034.glgC in pSB1A30 #1	#HXXD#	-	-
B0034.glgC in pSB1A30 #2	#D9TA#	-	-
B0034.glgC in pSB1A30 #3	#PX1E#	-	-
B0034.glgC in pSB1A30 #4	#KKYF#	-	-
B0034.glgC in pSB1A30 #5	#M9EY#	-	-
B0034.glgC in pSB1A30 #6	#OHES#	#VMN3#	looks perfect
B0034.glgC in pSB1A30 #9	#S6KX#	#YLS8#	no T7 promoter
B0034.glgC in pSB1A30 #10	#YNNY#	#8MKM#	no T7 promoter

all clones except #6 were red

15-07-13

plasmid prep of glgC in pSB1A30 clone #6 in #VMN3#
new overnights of white clones on masterplate: clone #9 and #10

15-07-14

plasmid prep of glgC in pSB1A30 clone #9+10 (table above)
all three glgC in pSB1A30 plasmids went into sequencing

15-07-15

analysis of sequencing results
2 precultures of glgC in pSB1A30 from clone #6, one of RFP in pSB1A30 #6DA3#

Expression

15-06-30

glgA Plated #W8VB# and #KK16# on LB+K

15-07-01

make overnight precultures from the plate (2x #W8VB#, 1x #KK16# -> A,B,C)
reserve 3x 500 ml shake flasks

15-07-02

08:50 inoculate 50 ml main cultures LB+K (labelled A,B,C) with 1 ml of culture

monitor the OD and induce cultures A+B with IPTG at OD=0.6

5 h after induction from each culture take 2x 2 ml sample (centrifuge, discard supernatant and freeze)

10 h after induction from each culture take 2x 2 ml sample (centrifuge, discard supernatant and freeze)

24 h after induction from each culture take 2x 2 ml sample (centrifuge, discard supernatant and freeze)

flask	combination	inoculated with (time)	IPTG induction (time)	6 h sample	18.5 h sample	pellet
A	K1585310 in pSB1K30 induced	1 ml #W8VB# preculture (08:50)	50 µl IPTG (15:10)	A6, 20:45 #9E9N#	#TX14#	#F3Y6#
B	K1585310 in pSB1K30 uninduced	1 ml #W8VB# preculture (08:50)	not induced	B6, 20:45 #EL3O#	#X4KT#	#KBPA#
C	I13507 in pSB1K30 induced	1 ml #KK16# preculture (08:50)	50 µl IPTG (15:10)	C6, 20:45 #9KA9#	#81RD#	#ZCWT#

OD monitoring (numbers show time in hours)

flask/ OD	1	2	3.167	5.333	6.333	7.5	8.5
A	0.052	0.069	0.084	0.261	1.07	1.728	1.689
B	0.056	0.077	0.094	0.314	1.241	1.834	1.811
C	0.024	0.027	0.036	0.116	0.406	1.178	1.225

15-07-03

take the final sample
prepare SDS-PAGE (cooked samples in #B13F#)
run SDS-PAGE

SDS-PAGE of GlgA expression
GlgA was expressed in pSB1K30 and compared to mRFP expression. The small arrows point to the GlgA bands of 52.4 kDa. No difference between induced and uninduced was observed. After overnight expression the strong bands were still present.

15-07-06

glgB Plated #Q6Z1# on LB+K

15-07-07

make overnight precultures from the plate (2x Q6Z1, (1x KK16) -> A,B,C)
reserve 3x 500 ml shake flasks

15-07-08

08:55 inoculate 50 ml main cultures LB+K (labelled A,B,C) with preculture to an OD of 0.1

monitor the OD and induce cultures A+B with IPTG at OD=0.6

5 h after induction from each culture take 2x 2 ml sample (centrifuge, discard supernatant and freeze)

10 h after induction from each culture take 2x 2 ml sample (centrifuge, discard supernatant and freeze)

24 h after induction from each culture take 2x 2 ml sample (centrifuge, discard supernatant and freeze)

flask	combination	inoculated with (time)	IPTG induction (time)	5 h sample	19.5 h sample	pellet
A	K1585311 in pSB1K30 induced	2.5 ml #Q6Z1# preculture (08:50)	50 µl IPTG (13:40)	A1, 18:50 #B9CL#	#6W3P#	#WP6P#
B	K1585311 in pSB1K30 uninduced	2.5 ml #Q6Z1# preculture (08:50)	not induced	B1, 18:50 #SKMW#	#NM6Q#	#ASLE#
C	I13507 in pSB1K30 induced	4.5 ml #KK16# preculture (08:50)	50 µl IPTG (13:40)	C1, 18:50 #9SZH#	#QEHO#	#1CAP#

OD monitoring (numbers show time in hours)

flask/ OD	1	2.25	3.2	4.2	5	8.33	9	24.2
A	0.147	0.190	0.285	0.481	0.666	2.95	2.15	2.5
B	0.147	0.198	0.311	0.552	0.785	2.9	2.42	2.6
C	0.100	0.108	0.109	0.115	0.111	0.64	1.41	2.0

15-07-09

cooked SDS samples in #6SOX#
run SDS page

SDS-PAGE of GlgB expression
GlgB was expressed in pSB1K30 and compared to mRFP expression. The small arrows point to the glgB bands of 84 kDa. No difference between induced and uninduced was observed.

15-07-15

glgC overnight of clone # from Masterplate of glgC in pSB1A30

15-07-16

09:15 inoculate 25 ml main cultures LB+A (labelled A,B,C) with preculture to an OD of 0.1

monitor the OD and induce cultures A+B with IPTG at OD=0.6

5 h after induction from each culture take 2x 2 ml sample (centrifuge, discard supernatant and freeze)

10 h after induction from each culture take 2x 2 ml sample (centrifuge, discard supernatant and freeze)

24 h after induction from each culture take 2x 2 ml sample (centrifuge, discard supernatant and freeze)

flask	combination	inoculated with (time)	IPTG induction (time)	5 h sample	19.5 h sample	pellet
A	K1585312 in pSB1A30 induced	0.65 ml #OHES# (from master plate) preculture A (09:15)	25 µl IPTG (13:05)	A1, 18:10 #NMHL#	A19, 09:00 #ZXSL#	#AQND#
B	K1585312 in pSB1A30 uninduced	0.65 ml #OHES# (from master plate) preculture A (09:15)	not induced	B1, 18:10 #WDQZ#	B19, 09:00 #BSVL#	#XROO#
C	I13507 in pSB1A30 induced	0.37 ml #6DA3# preculture (09:15)	25 µl IPTG (11:30)	C1, 18:10 #H4XD#	C19, 09:00 #EQMY#	#LDVR#

OD monitoring (numbers show time in hours)

flask/ OD	1	2.25	3	4	6	9	24
A	0.132	0.199	0.293	0.469	2.8	4.21, 4.21, 4.21	5.72, 6.03, 5.65
B	0.126	0.202	0.291	0.463	3.6	4.34, 4.32, 4.35	5.12, 4.91, 5.05
C	0.254	1.031	1.612	4.1	7.9	5.39, 5.41, 5.42	5.38, 5.20, 5.12

15-07-17

cooked SDS samples of glgC expressio are in #FWCT#
SDS PAGE of glgC expression tests

SDS PAGE of glgC expression
glgC was expressed in pSB1A30 and compared to mRFP expression. The small arrows point to the GlgC bands of 48 kDa. No difference between induced and uninduced was observed. After overnight expression the strong bands were no longer present.

References

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Team:Aachen/Notebook/Documentation/Glycogen Single Expression

Laboratory Notebook

15-06-09

15-06-10

15-06-11

15-06-12

15-06-13

15-06-15

15-06-16

15-06-17

15-06-18

15-06-19

15-06-22

15-06-23

15-06-24

15-06-26

15-06-29

15-06-30

15-07-01

15-07-02

15-07-03

15-07-06

15-07-08

15-07-09

15-07-10

15-07-11

15-07-13

15-07-14

15-07-15

Expression

15-06-30

15-07-01

15-07-02

15-07-03

15-07-06

15-07-07

15-07-08

15-07-09

15-07-15

15-07-16

15-07-17

References