Knockout Generation

Laboratory Notebook

15-06-01

plate pCas containing NEB10β (#YPKO#) on LB+K and grow at 30 °C

15-06-02

make steril-filtered arabinose (10 ml, 1 mol/l)
make chloramphenicol (10 ml)

15-06-03

make electrocompetent cells with pCas
- 500 µl arabinose added at OD 0.27 (to final concentration of 10 mM)
- calculation of cell density [http://www.genomics.agilent.com/biocalculators/calcODBacterial.jsp?_requestid=14961 Calculator]

15-06-08

test electrocompetent cells with pUC19 (amp resistance)
transformed and incubated

15-06-09

designed and ordered primers #T9T9#, #CT4E#, #6V6P#, #WAO8# and new #A9W9# and #XE3D# from IDT
transformation of glgX (#1DEN#) and glgP (#C1QW#, #DC6X#, #HCTL#, #4MCW#, pUC19 control) into electrocompetent pCas carrying cells
- plating at 30 °C on LB+K+A plates

15-06-10

analysis of sequencing results
overnights in 2 ml LB+K of anti glgX and anti glgP with 10 µl of 100 mM IPTG (0.5 mM final concentration)
master plates without IPTG on LB+K

15-06-11

dilution plating of glgX and glgP knockout candidates on LB+K
plate controls: pCas carrying cells (#YPKO#, LB+K), uncured clones from the trafo plates (antiX/P on LB+K+A)
analysis of anti glgP sequencings: N20 is intact and the sequences look good

15-06-12

colony PCR using #A9W9# and #XE3D# to verify plasmid curing
overnight cultures of good looking clones
overnights from #YPKO# to get enough sequencing template

15-06-13

make cryos of glgX and glgP knockout candidates

construct & clone	cryo ID	genomic DNA ID	PCR product	purified PCR product	sequencing result
glgX knockout candidate #2	#PH3R#	#OM6C#	#LA33#, purified 11+12	#FP1F#	point mutation in the multi stop, but the gene is disrupted
glgX knockout candidate #4	#SKKT#	#1WKE#	purified	#1Y4Z#	-
glgX knockout candidate #5	#F93Y#	#1DOS#	purified	#CLR4#	-
glgP knockout candidate from #HCTL# #1	#6RPW#	#VZD3#	bad	-	-
glgP knockout candidate from #HCTL# #3	#XC4Y#	#TWZ1#	-	-	-
glgP knockout candidate from #HCTL# #4	#CKT3#	#MMLP#	-	-	-
glgP knockout candidate from #HCTL# #5	#NN38#	#NKOA#	-	-	-
glgP knockout candidate from #4MCW# #2	#1SHE#	#1RM3#	#LOFF#:P1	#NP3B#	no chromatograms
glgP knockout candidate from #4MCW# #3	#TSBH#	#O1FO#	-	-	-
glgP knockout candidate from #4MCW# #4	#BH9D#	#E3E9#	-	-	-
glgP knockout candidate from #4MCW# #5	#HHTX#	#1QAT#	-	-	-
glgP knockout candidate from #C1QW# #2	#BWXX#	#FFA9#	#LOFF#:P2	#PY96#	no chromatograms
glgP knockout candidate from #C1QW# #3	#Z1E3#	#CAAV#	-	-	-
glgP knockout candidate from #C1QW# #4	#RS8L#	#SRKE#	-	-	-
glgP knockout candidate from #C1QW# #5	#FQ6W#	#A6OB#	-	-	-

extraction of genomic DNA
plasmid prep pCas from the frozen overnights (#APMK#, #63ZY#)

15-06-15

measure extracted DNA concentrations
dilute genomic DNA to 500 ng/µl
PCR-amplify the genomic regions of glgX and glgP knockouts with 1000 ng/50 µl reaction
- glgX #OM6C# with #6V6P# and #WAO8# 55-64 °C (expected: 1024, maybe: 1006) > products in #LA33#, 63.5 is the best annealing temperature
- glgP #VZD3# with #T9T9# and #CT4E# 55-64 °C (expected: 1015, maybe: 3351) > products in #DOMW#

Aachen 15-06-16 genomic gradient glgX.png

Gradient PCR on genomic region of glgX
PCR worked at all temperatures.

Gradient PCR on genomic region of glgP
PCR worked at all temperatures.

30 cycles

step	temperature [°C]	duration
initial denaturation	98	0'30"
denaturation	98	0'30"
gradient annealing	55-64	0'30"
elongation	72	0'33" (glgX) or 1'43" (glgP)
final elongation	72	5'00"

amplify the genomic regions of all remaining candidates
- annealing temperature for glgX 63.5 °C
- annealing temperature for glgP 63.9 °C (product from #1RM3# is #LOFF#:P1 and from #FFA9# is #LOFF#:P2)
PCR product purification
pipette purified PCR products for sequencing
pipette pCas plasmids for sequencing with 5 of 32 primers, because we don't have enough template DNA...

15-06-16

PCR product purification of P1 and P2 from #LOFF#
agarose gel of 2.5 µl P1/P2 and 0.5 µl loading dye (the gel is wrapped in foil in our 4° fridge) -> no visible bands except ladder
sequencing of genomic glgX and glgP regions
- bring the sequencing samples to the Fraunhofer IME because you are too late ;-)
colony PCR on glgP (from plate of #C1QW# and cured) and glgX (from plate #1DEN# and cured) knockout candidates

parameter	glgP	glgX genomic (1024 bp)	glgX edit (531 bp)
forward primer	#T9T9#	#6V6P#	#SNYO#
reverse primer	#CT4E#	#WAO8#	#WAO8#
denaturation	0'15"	0'15"	0'15"
annealing [°C]	62.0	63.5	63.5
annealing time	0'15"	0'15"	0'15"
elongation time	1'43"	0'33"	0'33"
picking instructions	colony > PCR tube > master plate	colony > 5 µl water > (master plate, 1 µl into PCR tube)

15-06-17

electroporation of anti glgP plasmid into 25 µl electrocompetent pCas cells
- arabinose added to every 500 µl of SOC medium (to final concentration of 10 mM)
- 2 µl of plasmid DNA (#C1QW#, #HCTL#, #4MCW#)
colony PCR of negative control (cryo #YPKO#) for glgX genomic PCR with #SNYO# and #WAO8#

time	temperature
10:00	94°C
00:15	94°C
00:15	58°C
00:33	68°C
02:00	68°C

sequencing results: #PH3R# is an almost perfect knockout of glgX

15-06-18

colony PCR on many glgP colonies (controls!)
- resuspended the cells in 5 µl LB+K
- primers #T9T9# and #CT4E#
- 1-11: knockout candidate from #C1QW#
- 13-23: knockout candidates from #HCTK#
- 24-35: knockout candidates from #4MCW#
master plate in parallel with 400 µl IPTG
overnight cultures (2ml) of good looking colonies with 40 µl IPTG

15-06-19

Cryos and genomic DNA extraction of the glgP knockout candidates

candidate	cryo ID	genomic DNA	PfuS PCR product	purified ID	sequencing result
from #C1QW# clone #7	#DK14#	#FQEL#	#YWMH#:P7	#YOCW#	bad sequencing
from #C1QW# clone #9	#XHEK#	#QQFH#	#YWMH#:P9	#KFX4#	probably perfect (selected)
from #C1QW# clone #10	#FEHT#	#CS83#	#YWMH#:P10	#4ZXT#	probably perfect

PfuS PCR to amplify the genomic region. The products are in #YWMH#.

30 cycles

step	temperature [°C]	duration
initial denaturation	98	10'00"
denaturation	98	0'30"
annealing #T9T9# and #CT4E#	62	0'30"
elongation	72	1'43"
final elongation	72	5'00"

15-06-22

heat shock transformation of 4 µl pCas (#EANB#) into 100 µl BL21 Gold (DE3) from the Schwaneberg group (plated on LB+K at 30 °C)
PCR product purifications from #YWMH# (see table above)
pipetted purified PCR products for sequencing with #T9T9#, #CT4E# and #WQRS#

15-06-23

plate glgP knockout candidates #DK14#, #XHEK# and #FEHT# on LB+K+IPTG and grow at 30 °C (preparation for competent cells on Thursday)
master plate of pCas in BL21(DE3) and grow at 30 °C
make an overnight culture of a pCas in BL21(DE3) clone and grow at 30 °C
aliquot buffers into numbered falcons (like we did the first time)

15-06-24

cryo of pCas in BL21 Gold (DE3) -> #RMBC#
analyze sequencing results
from masterplates make overnight precultures (LB+K) of pCas in BL21(DE3) and one good glgP knockout strain (sequencing #KFX4#, corresponding cryo #XHEK#)

15-06-25

make electrocompetent cells (see manual: genome editing)
- inoculated main culture at 10:00
- arabinose 500 µL in 50 mL
- pCas in NEB10β-ΔglgP (induced at OD=0.27)
- pCas in BL21(DE3) (induced at OD=0.38)
- at 13:20 pCas= OD 0.781 anti glgP= OD 0.512: cool down!
- measure OD before aliquoting: NEB10β-ΔglgP: OD=20 BL21(DE3): OD=28.2
- aliquot 25 µl into PCR tubes and freeze them in big falcons!

15-06-26

have a beer

15-06-29

Transformations to be plated on LB+K+A

strain	transformed with	result
NEB10β-ΔglgP-pCas-cells	nothing	no colonies
NEB10β-ΔglgP-pCas-cells	#1DEN#	small colonies
BL21 Gold (DE3)-pCas	#C1QW#	no colonies

15-06-30

colony PCR on glgPX knockout candidates (5 µl LB+K in PCR tube, master plate LB+K of 1, 3, 4, 5, 6, 7, 8, 9, overnights+IPTG of clones 3, 4, 5, 7)

template	tube	forward primer	reverse primer	purpose	expected length
NEB10β-ΔglgP glgX knockout candidate 1-12	1-12	#SNYO#	#WAO8#	find edited clones	531 bp
NEB10β-ΔglgP glgX knockout candidate 13	13	#SNYO#	#K4TH#	to confirm that #SNYO# binds on the plasmid	463 bp
NEB10β-ΔglgP	14	#SNYO#	#WAO8#	negative control	no bands
NEB10β-ΔglgP	15	#6V6P#	#WAO8#	to see if the PCR worked	1024 bp
picking instructions for 1-13	colony > 5µl LB+K > (master plate, 1 µl into PCR tube)
elongation time	1'10"

30 cycles

step	temperature [°C]	duration
initial denaturation	94	10'00"
denaturation	94	0'30"
annealing	63.5	0'30"
elongation	72	1'10"
final elongation	72	5'00"

overnight cultures of good knockout candidates

Electroporations

strain	plasmid	purpose
BL21 Gold (DE3)-pCas	#LYZH#	testing the electrocompetence
BL21 Gold (DE3)-pCas	#C1QW#	glgP knockout

15-07-01

cryo + genomic DNA extraction

clone	cryo ID	genomic DNA ID	purified PfuS PCR product	sequencing result
NEB10β ΔglgP ΔglgX candidate #3	#WVKQ#	#HMYH#	#AT9O#	perfect glgX knockout
NEB10β ΔglgP ΔglgX candidate #4	#LNL4#	#L4P1#	#MMCH#	perfect glgX knockout
NEB10β ΔglgP ΔglgX candidate #5	#39Y9#	#DHOL#	#DMNT# PCR did not work	-
NEB10β ΔglgP ΔglgX candidate #7	#ELM1#	#WWTY#	#APMY# PCR did not work	-

PfuS PCR to amplify the genomic region. The products are in #SHYS#.

30 cycles

parameter	value	duration
initial denaturation	98	10'00"
denaturation	98	0'30"
annealing #6V6P# and #WAO8#	63.5	0'30"
elongation	72	0'33" (1024 bp)
final elongation	72	2'00"
reaction volume	50 µl
template DNA	2 µl per reaction

PCR product purification
pipette for sequencing
colony PCR to check for pSB1A3 curing
overnight of pSB1A30 in BL21 clones + IPTG (gave them to iSK/iLG/iTS)

PCR on glgP knockout in BL21
- primers #T9T9# and #CT4E#
- knockout candidate from #C1QW#
- resuspend glgP knockout candidates in 5 µl LB medium

colony PCR on BL21 Gold DE3 glgP knockout candidates

template	tube	forward primer	reverse primer	purpose	expected length
BL21 Gold DE3 glgP knockout candidate 1-24	1-24	#T9T9#	#CT4E#	find edited clones	1015/3351 bp
NEB10β-ΔglgP	25	#T9T9#	#CT4E#	positive control with desired length	1015 bp
BL21 Gold DE3-pCas-pSB1A30	26	#T9T9#	#CT4E#	negative control with no knockout	3351 bp
LB+K	27	#T9T9#	#CT4E#	negative control	no bands
picking instructions for 1-13	colony > 5µl LB+K > (master plate, 1 µl into PCR tube)
elongation time	1'10"

30 cycles with KAPA2G Fast Ready Mix

step	temperature [°C]	duration
initial denaturation	95	10'00"
denaturation	95	0'30"
annealing	62	0'30"
elongation	72	0'55" (1015/3351 bp)
final elongation	72	2'00"

Aachen 15-07-01 BL21 glgP colony PCR.png

Colony PCR on glgP knockout candidates
The Colony PCR was not very succesful. Nevertheless, three candidates (4, 18, 20) look good.

The colony PCR was not very successful. Many samples did not work, including the controls. Nevertheless, we have identified three candidates (4, 18, 20). Overnight cultures of good knockout candidates 4, 18, 20. 2 ml of LB+K+IPTG were inoculated with 2 µl of suspended cells

15-07-02

make cryos and genomic DNA isolations of BL21 Gold DE3 glgP knockout candidates

clone	cryo ID	genomic DNA ID	purified PfuS PCR product	sequencing result
BL21 Gold (DE3) ΔglgP candidate #4	#3RZ1#	#8V4S#	#9KST#	perfect (selected)
BL21 Gold (DE3) ΔglgP candidate #18	#ZTYF#	#4DTX#	#YSBT#	perfect
BL21 Gold (DE3) ΔglgP candidate #20	#MEWN#	#99LV#	#4BX6#	perfect

PfuS PCR to amplify the genomic region. The products are in #DO3L#. Positive control: #QQFH# (tube 7) Negative control: water (tube 8)

30 cycles

parameter	value	duration
initial denaturation	98	5'00"
denaturation	98	0'30"
annealing #T9T9# and #CT4E#	62.0	0'30"
elongation	72	1'43" (1015 bp)
final elongation	72	5'00"
reaction volume	50 µl
template DNA	2 µl per reaction

15-07-03

purified yesterdays PfuS PCR products of BL21 glgP knockout candidates

15-07-06

pipette for sequencing
plate anti glgX glgP double knockout candidate in NEB10β on LB- +IPTG and incubate first at 30 °C then at 37 °C to cure both plasmids
plate anti glgP in BL21 Gold (DE3) on LB+K+IPTG and incubate at 30 °C

15-07-07

plate double knockout candidate in NEB10β on LB+K to test if pCas is cured
overnights of cured double-knockout candidates for cryo cultures

15-07-08

overnights of ΔglgP in BL21 for electrocompetent cells
cryos of cured double-knockout candidates
- #CFF4#
- #RAMR#
- #WHPX#

test on LB+K+A showed that curing had worked

15-07-08

analysis of sequencing results
overnight culture of BL21 Gold (DE3) ΔglgP (from #3RZ1#) on LB+K at 30 °C

15-07-09

inoculated main culture at 9:20 am
prepare aliquots of buffers for electrocompetent cells

time	OD
1	0.068
2.33	0.245

induced at 12:00

make electrocompetent BL21 Gold (DE3) ΔglgP cells

transform by electroshock:
- #LYZH# into BL21 Gold (DE3) ΔglgP for testing electrocompetence
- #1DEN# into BL21 Gold (DE3) pCas for single knockout
- #1DEN# into BL21 Gold (DE3) ΔglgP for double knockout in BL21

15-07-10

none of the electroporations worked!
repeat transformations:
- #LYZH# into BL21 Gold (DE3) ΔglgP for testing electrocompetence
- #1DEN# into BL21 Gold (DE3) pCas for single knockout
- #1DEN# into BL21 Gold (DE3) ΔglgP for double knockout in BL21

15-07-11

put transformation plate in the fridge until Monday

Transformations plated on LB+K+A

strain	transformed with	result
BL21-ΔglgP-pCas-cells	#LYZH#	>100 colonies
BL21-pCas-cells	#1DEN#	2 colonies
BL21-ΔglgP-pCas-cells	#1DEN#	8 colonies

15-07-13

parallel masterplates and overnights of good clones

colony Taq PCR

parameter	glgX genomic (1024 bp)	glgX edit (531 bp)
forward primer	#6V6P#	#SNYO#
reverse primer	#WAO8#	#WAO8#
picking instructions	colony > 5 µl water > (master plate, 1 µl into PCR tube)

30 cycles

step	temperature [°C]	duration
initial denaturation	94	10'00"
denaturation	94	0'30"
annealing	63.5	0'30"
elongation	72	1'10"
final elongation	72	5'00"

15-07-14

glgXP double knockout and glgX single knockout in BL21
The primer SNYO binds specifically on the iGEM Aachen MultiSTOP and therefore indicates the insertion of this fragment. All double knockout candidates look good, but only #1 of the double knockout candidates has the correct length.

Aachen 15-07-14 glgX BL21 glgXP BL21 knockout genomic area.png

glgXP double knockout and glgX single knockout in BL21
The primer 6V6P binds in the genomic area, so this is a control beacuse a band is present whether the gene is disrupted or not.

gel shows that all glgX double-knockouts in BL21 have worked
just clone #1 of the single-knouckout in BL21 looks good
cryos
genomic DNA extraction

clone	genomic DNA stored in
glgX #1	#AABE#
glgXP #1	#9NMT#
glgXP #2	#XRQA#
glgXP #3	#XBRK#

PfuS PCR to amplify the genomic region. The products are in #9SBX#.

30 cycles

parameter	value	duration
initial denaturation	98	5'00"
denaturation	98	0'30"
annealing #6V6P# and #WAO8#	63.5	0'30"
elongation	72	0'35" (1024 bp)
final elongation	72	5'00"
reaction volume	50 µl
template DNA	2 µl per reaction

tube 1: glgX single-knockout BL21 #1
tube 2: glgXP double-knockout BL21 #1
tube 3: glgXP double-knockout BL21 #2
tube 4: glgXP double-knockout BL21 #3
tube 5: water control

PfuS PCR did not work!!! will be repeated

30 cycles

parameter	value	duration
initial denaturation	98	10'00"
denaturation	98	0'30"
annealing #6V6P# and #WAO8#	63.5	0'30"
elongation	72	0'35" (1024 bp)
final elongation	72	5'00"
reaction volume	50 µl
template DNA	2 µl per reaction

products stored in #9SBX#

15-07-15

Check PCR products on gel: all samples have correct length
PCR product purification

Aachen 15-07-16 PfuS pcr XP double and X single knockout in BL21.png

PfuS PCR to check the glgX and the double knockout candidates

clone	cryo ID	genomic DNA ID	purified PfuS PCR product	sequencing result
BL21 Gold (DE3) ΔglgX candidate #1	#ZPKP#	#AABE#	#M6DH#	perfect
BL21 Gold (DE3) ΔglgP ΔglgX candidate #1	#CK4H#	#9NMT#	#E8PP#	negative
BL21 Gold (DE3) ΔglgP ΔglgX candidate #2	#A3Y6#	#XRQA#	#COYB#	negative
BL21 Gold (DE3) ΔglgP ΔglgX candidate #3	#LMNN#	#XBRK#	#3DNC#	negative

pipette for sequencing

15-07-17

electroporation of #1DEN# into BL21 Gold (DE3) cells with pCas
IPTG (30 °C) and pCas curing (37°C) of #3RZ1#, #ZPKP#, #PH3R# and #XHEK#
overnight culture of BL21 Gold DE3

15-07-18

OneTaq PCR on edited genome:
- 4 clones, #3RZ1# as negative control, #ZPKP# as positive control
- no bands for #1, clones #3 and #4 look best

parameter	glgX genomic (1024 bp)	glgX edit (531 bp)
forward primer	#6V6P#	#SNYO#
reverse primer	#WAO8#	#WAO8#
picking instructions	colony > 5 µl water > (master plate, 1 µl into PCR tube)

30 cycles

step	temperature [°C]	duration
initial denaturation	94	10'00"
denaturation	94	0'30"
annealing	63.5	0'30"
elongation	68	1'10"
final elongation	68	5'00"

pipetting table (to be done for each master mix)

component	amount [µl]
H20	48
#WAO8# (#WAO8#)	6
#6V6P# (#SNYO#)	6
2xMM OneTaq	75

Aachen 15-07-18 glgXP knockout candidates colony PCR.png

PfuS PCR to check the double knockout candidates

overnight cultures with IPTG of good clones
plate cured #3RZ1#, #ZPKP#, #PH3R# and #XHEK# on LB+K and LB+A (control) and make an LB^- overnight
cryo of BL21 Gold DE3

15-07-19

Curing did not work 100%, so we re-plated the knockout strains on a new LB^- plate

Cryo cultures and genomic DNA preparation of glgXP double knockout candidates

strain	cryo	genomic DNA	purified PCR product	sequencing result
BL21 Gold DE3 glgXP candidate #1	#H49M#	#LW4W#	-
BL21 Gold DE3 glgXP candidate #2	#VENK#	#4ORE#	#CXQY#	did not work
BL21 Gold DE3 glgXP candidate #3	#KENF#	#SW4F#	#OSW1#	did not work
BL21 Gold DE3 glgXP candidate #4	#D8XW#	#KHRV#	#S9NT#	did not work

15-07-20

plated cells to cure from LB^- on LB+K and on LB+A again
measure and dilute genomic DNA (table above)
PfuS PCR to amplify genomic region of glgX knockout

30 cycles

parameter	value	duration
initial denaturation	98	10'00"
denaturation	98	0'30"
annealing #6V6P# and #WAO8#	63.5	0'30"
elongation	72	0'35" (1024 bp)
final elongation	72	5'00"
reaction volume	50 µl
template DNA	2 µl per reaction

products in #PVFC#

gel of PCR products: clones #2-4 look good and are purified (see table above)

Aachen 15-07-20 glgXP PfuS genomic region.png

PfuS PCR of glgXP knockout candidates
The genomic region of all clones was amplified with #6V6P# and #WAO8# for sequencing. Only clones 2-4 have the correct length.

pipette for sequencing

15-07-21

curing did not work again!
to test the LB+K plate, we plated:

template	ID	expectation	result
sfGFP in pSB1A3	#IBF4#	no growth	strong growth
glgXP double knockout NEB10β	#WHPX#	no growth	no growth
NEB10β	#ZEYD#	no growth	no growth
BL21 Gold (DE3)	#34CT#	no growth	no growth
BL21 Gold (DE3) ΔglgP	cryo: #3RZ1#	no growth	growth

15-07-22

we did not pick single colonies from LB- plate, so we repeat curing
three single colonies from each LB- plate (#3RZ1#, #ZPKP#, #PH3R# and #XHEK#) are picked and streaked on LB- and LB+K
none of the BL21 glgXP double knockouts worked!

strain	cryo	genomic DNA	purified PCR product	sequencing result
BL21 Gold DE3 glgXP candidate #1	#H49M#	#LW4W#	-
BL21 Gold DE3 glgXP candidate #2	#VENK#	#4ORE#	#CXQY#	did not work
BL21 Gold DE3 glgXP candidate #3	#KENF#	#SW4F#	#OSW1#	did not work
BL21 Gold DE3 glgXP candidate #4	#D8XW#	#KHRV#	#S9NT#	did not work

15-07-23

Curing:
- #ZPKP# -> all three clones were cured
- #XHEK# -> only the first clone was cured
- #3RZ1# and #PH3R#-> curing did not work for any clone

make overnights of the cured clones LB^-
for #3RZ1# and #PH3R#: pick 6 more colonies and test them on LB+K (also plate on LB^-)

double knockout did not work again!
- we will test if our electrocompetent BL21 (DE3) ΔglgP still have the pCas by plating them on LB+K (30 °C)

15-07-24

electrocompetent BL21 ΔglgP still have the pCas
curing just worked for one clone of #PH3R#
- make overnights of the cured clone #2 from #PH3R# in LB^-
#3RZ1# was not cured: pick more colonies to cure

15-07-25

cryo culture of cured #PH3R# (ID: #CL6W#)
put LB+K and LB^- of #3RZ1# into fridge

15-07-27

overnight of cured #3RZ1# clone #8 in LB^-

15-07-28

cryo of cured #3RZ1# clone #8 in #FVNV#

15-07-29

plate #WHPX# on LB^-

15-07-30

make overnight from plate
PfuS PCR to check BL21 knockouts again (NEB10β did not grow on M9)
for X knockout tubes 3+4:

30 cycles

parameter	value	duration
initial denaturation	98	10'00"
denaturation	98	0'30"
annealing #6V6P# and #WAO8#	63.5	0'30"
elongation	72	0'35" (1024 bp)
final elongation	72	5'00"
reaction volume	50 µl
template DNA #VBTL#	2 µl per reaction

for P knockout tubes 1+2

30 cycles

parameter	value	duration
initial denaturation	98	10'00"
denaturation	98	0'30"
annealing #T9T9# and #CT4E#	62	0'30"
elongation	72	1'43" (1015 bp)
final elongation	72	5'00"
reaction volume	50 µl
template DNA #C1BL#	2 µl per reaction

products are stored in #WWP8#

15-07-31

make new sterile filtered arabinose: for 10 ml of 1M solution: 1.5 g arabinose
make electrocompetent NEB10β-ΔglgX-ΔglgP cells and BL21-ΔglgX cells (see manual: genome editing)
- inoculated main culture at 09:45
- NEB10β-ΔglgP-ΔglgX
- BL21(DE3)-ΔglgX (induced at OD=0.288)
- at 12:10 BL21= OD 0.515: cool down!
- at NEB10β= OD 0.51: cool down!
- measure OD before aliquoting: NEB10β-ΔglgP-ΔglgX: OD=18 BL21(DE3)-ΔglgX: OD=25
- aliquot 25 µl into PCR tubes and freeze them in big falcons!

repeat PfuS PCR to check BL21 ΔglgX
- tube 5 and 6

30 cycles

parameter	value	duration
initial denaturation	98	10'00"
denaturation	98	0'30"
annealing #6V6P# and #WAO8#	63.5	0'30"
elongation	72	0'35" (1024 bp)
final elongation	72	5'00"
reaction volume	50 µl
template DNA #VBTL#	2 µl per reaction

PfuS did not work! No bands are on the gel

15-08-03

electroporation of #C1QW# into electrocompetent BL21 ΔglgX
control: #LYZH#
make an overnight culture of #XFWX#

15-08-04

masterplate of Bl21 (DE3) Gold anti-glgX-glgP +IPTG on LB+K
repeat PfuS PCR to check BL21 ΔglgX from #XFWX#: extracted genomic DNA: #WBKH#

30 cycles

parameter	value	duration
initial denaturation	98	10'00"
denaturation	98	0'30"
annealing #6V6P# and #WAO8#	63.5	0'30"
elongation	72	0'38" (1024 bp)
final elongation	72	5'00"
reaction volume	50 µl
template DNA #WBKH#	2 µl per reaction

gel looks good

Aachen 15-08-04 BL21 glgX knockout check, sequencing.png

BL21 Gold (DE3) ΔglgX knockout check
The genomic region of all clones was amplified with #6V6P# and #WAO8# for sequencing. Only clones 2-4 have the correct length.

PCR clean-up
product in #LRCC#
#LRCC# was sent into sequencing

15-08-05

no colonies on masterplate of Bl21 (DE3) Gold anti-glgX-glgP
make a gel to check if #C1QW# is okay
repeat transformation of #C1QW# in BL21 anti-glgX electrocompetent cells
- added 5 µl arabinose to SOC medium
#C1QW# is empty and discarded
- overnight of ##

15-08-06

colony PCR on colonies from trafo plate
double knockout in BL21 did not work again!

15-08-07

repeat electroporation of newly purified #C1QW# in Bl21 (DE3) Gold ΔglgX
no colonies visible on the plate!
repeat electroporation with different voltage

tube #	competent cells	transformed plasmid	purpose	result
1	BL21 Gold (DE3) ΔglgX	#C1QW#	BL21 double knockout	no colonies
2	BL21 Gold (DE3) ΔglgX	#LYZH#	control	no colonies
3	BL21 Gold (DE3) with pCas	#C1QW#	test #C1QW# (targeting plasmid)	no colonies
4	BL21 Gold (DE3) with pCas	#LYZH#	control	ok

15-08-08

transformation has not worked again

15-08-10

electroporation of #1DEN# in BL21 antiX and #C1QW# in antiP electrocompetent
- 10 µl arabinose in SOC medium
plate and overnight of #C81O# and #FVNV# to make electrocompetent cells

15-08-11

repeat electroporation because there were just a few colonies
- 10 µl arabinose in SOC medium
make electrocompetent cells of cured BL21 anti glgX #C81O# and BL21 anti glgP #FVNV#
- accidentally, cells were centrifuged at 1000 rpm, not 1000 g :-(((
colony Taq PCR on colonies with #1DEN# (no colonies on plate with #C1QW#)

parameter	glgX genomic (1024 bp)	glgX edit (531 bp)
forward primer	#6V6P#	#SNYO#
reverse primer	#WAO8#	#WAO8#
picking instructions	colony > 5 µl water > (master plate, 1 µl into PCR tube)

30 cycles

step	temperature [°C]	duration
initial denaturation	94	10'00"
denaturation	94	0'30"
annealing	63.5	0'30"
elongation	68	1'10"
final elongation	68	5'00"

tubes

no	template	primers
1	1DEN no 1	WAO8, 6V6P
2	1DEN no 2	WAO8, 6V6P
3	1DEN no 3	WAO8, 6V6P
4	1DEN no 4	WAO8, 6V6P
5	1DEN no 5	WAO8, 6V6P
6	AVQR negative control	WAO8, 6V6P
8	1DEN no 1	WAO8, SNYO
9	1DEN no 2	WAO8, SNYO
10	1DEN no 3	WAO8, SNYO
11	1DEN no 4	WAO8, SNYO
12	1DEN no 5	WAO8, SNYO
13	AVQR negative control	WAO8, SNYO
14	water control	WAO8, SNYO

make a master plate of double knockout candidates (LB+K+IPTG) in parallel
gel of anti XP knockout candidate Taq PCR
- clone number 1-4 look good
make overnights (LB+K+IPTG) of good knockout clones
make overnights of #C81O# and #FVNV# to make new electrocompetent cells
make an LB+A plate of #BAB3# to make new #1DEN#

15-08-12

no colonies on plate with #C1QW# in BL21 ΔglgX
a few colonies on plate with #1DEN# in BL21 ΔglgP
- made a master plate with six clones
many colonies on plate of #LYZH# (control)
made electrocompetent cells
- inoculate main culture of #C81O# and #FVNV# at 10:25
- OD of ΔglgX: 21.7
- OD of ΔglgP: 20.1
made cryo cultures of double knockout candidates and extracted genomic DNA

clone	cryo	extracted genomic DNA
1	#8Y3P#	#3996#
2	#SVSX#	#XCQO#
3	#QBVS#	#ZPDW#
4	#FNWA#	#WNTA#

PfuS PCR to check BL21 ΔglgXP knockout candidates

30 cycles

parameter	value	duration
initial denaturation	98	10'00"
denaturation	98	0'30"
annealing #6V6P# and #WAO8#	63.5	0'30"
elongation	72	0'38" (1024 bp)
final elongation	72	5'00"
reaction volume	50 µl
template DNA	2 µl per reaction

products stored in #938K#

PCR to check double knockout candidates

no 1 and 2 looked good on the gel
- PCR purification (1: #A6XM#, #C3B3#)

#BAB3# overnight culture (LB+A)

15-08-13

pipette #A6XM# and #C3B3# for sequencing
prep #BAB3# overnight culture for new #1DEN#
One Taq PCR on new #1DEN# in BL21 ΔglgP

parameter	glgX genomic (1024 bp)	glgX edit (531 bp)
forward primer	#6V6P#	#SNYO#
reverse primer	#WAO8#	#WAO8#
picking instructions	colony > 5 µl water > (master plate, 1 µl into PCR tube)

30 cycles

step	temperature [°C]	duration
initial denaturation	94	10'00"
denaturation	94	0'30"
annealing	63.5	0'30"
elongation	68	1'10"
final elongation	68	5'00"

Aachen 15-08-13 glgXP knockout candidates BL21.png

BL21 Gold (DE3) ΔglgX/P double knockouts check

run an agarose gel
- clone 3,4,6 look good on gel
- #SNYO# and #WAO8# also amplify regions of clones without iGEM MultiStop (with and without targeting plasmid)
make overnight cultures (LB+K+IPTG) of good clones

15-08-14

cryo cultures of BL21 ΔglgPΔglgX k.o. candidates

clone	cryo ID	purified genomic DNA ID	tube in PfuS PCR	purified PCR product	sequencing result
BL21 ΔglgXΔglgP #3	#EVAL#	#KNOF#	1+2	#ALPF#
BL21 ΔglgXΔglgP #4	#36VR#	#RBP3#	3+4	#WQ4L#
BL21 ΔglgXΔglgP #6	#YFH1#	#S8S1#	5+6	#HS4M#	concentration was too low

genomic DNA extraction
PfuS PCR on knockout candidates (tube 7: water control)

Aachen 15-08-14 bl21 glgxp double knockout pfuS PCR.png

PCR to check double knockout candidates

gel: tube 1,2,4 and 5 were good
PCR clean-up
pipette for sequencing

Results

strain	cryo with pCas	cryo without plasmids (LB)	cryo without plasmids (M9)
DH5a		#VD49#
NEB10β	#YPKO#	#ZEYD#
NEB10β ΔglgX (1 mutation in the Multi Stop)	#PH3R#	#CL6W#
NEB10β ΔglgP	#XHEK#	#BXR1#
NEB10β ΔglgX ΔglgP	#LNL4#	#WHPX#
BL21 Gold (DE3)	#RMBC#	#34CT#	#E4B3#
BL21 Gold (DE3) ΔglgX	#ZPKP#	#C81O#	#XFWX# confirmed again by sequencing
BL21 Gold (DE3) ΔglgP	#3RZ1#	#FVNV#	#4BW3#
BL21 Gold (DE3) ΔglgX ΔglgP with pCas

▲

Team:Aachen/Notebook/Documentation/Glycogen Knockout Generation

Laboratory Notebook

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Results