Revision as of 10:30, 18 September 2015

BioBricks

Laboratory Notebook

All glycogen synthesis genes were successfully cloned into BioBricks.

BioBrick	gene	backbone	confirmed cryo	sequence-confirmed plasmid	submitted to registry
BBa_K118016	glgC	pSB1C3	#EK3S#	#4D6H#
BBa_K1585310	B0034.glgA	pSB1C3	#KMST#	#M1M6#	✓
BBa_K1585311	B0034.glgB	pSB1C3	#F4T6#	#YHT3#	✓
BBa_K1585312	B0034.glgC	pSB1C3	#DFKW#	#OP1N#	✓
BBa_K1585312	B0034.glgC	pSB1A2	#ZC8K#	#F8Y8#

15-05-11

order enzymes
Transformed BBa_K118016 from Kit Plate 2014 #3 well 20G into competent NEB10beta cells by heat shock (plated on LB+C)
gradient PCR using J04450 in pSB1C3 (#ZWDH#) as the template (diluted 1:10) primers are: #TSTV# and #FYTO# - the 10 products were put into #ES4X# and frozen

15-05-12

made master plates and liquid cultures (5 ml LB+C) of two BBa_K118016 clones
run the agarose gel of yesterdays gradient PCR products (#ES4X#) (expected length: 2070 bp)
- PCR did not work - Discussion of potential reasons: tubes were heated to long before cycler program was started -> primers could have been digested
pSB1C3 gradientPCR repeated. Products stored in #18FF#

15-05-13

PCR products from #18FF# were digested with DpnI
made cryos of BBa_K118016 in pSB1C3 in DH5α (#RKD1# and #EK3S#)
pPrep BBa_K118016 in pSB1C3 (#F3HE# and #4D6H#)
purify the good pSB1C3 backbone PCR products into #6MZP# and #WHYV#
diluted glgA (#OB8B#), glgB 1+2 (#3XZX# and #AV9S#), Phi (#8D18#) gBlocks to a concentration of 10 ng/µl using 20 µl of ddH₂O
Team:Aachen/Notebook/Protocols#CPEC assembly of #6MZP# or #WHYV# with synthesized linear DNA:

component	BBa_K1585310 (glgA)	BBa_K1585311 (glgB)
water [µl]	4	~~0.2~~
5x HF buffer [µl]	5	5
DMSO [µl]	0.75	0.75
dNTPs [µl]	5	5
Phusion polymerase	0.5	0.5
pSB1C3 backbone (#WHYV#)	2.3	2.3
fragment 1	7.4 µl #OB8B#	5.1 µl #3XZX#
fragment 2	none	6.1 µl #AV9S#
length [bp]	3525	4278
elongation time	53	1'04

The CPEC products were stored in #XY9Y#, electroporated (1.5 µl + 25 µl) into NEB10beta cells and plated on LB+C

Note: one week later we found out that there was an error in the Excel sheet - the elongation time should have been twice as long!

15-05-14

send BBa_K118016 in pSB1C3 (#F3HE# and #4D6H#) into sequencing using #A9W9# and #XE3D# at 65° annealing
CPEC results:

construct	clones	next steps
BBa_K1585310 (glgA)	0	run the CPEC product on an Agarose gel
BBa_K1585311 (glgB)	5	master plates and overnight cultures

15-05-15

all K1585311 in pSB1C3 constructs failed - the cultures and the master plates are dark red.
~~make cryo cultures and plasmid preps of 4xK1585311 in pSB1C3~~
~~check PCR the prepped plasmids of K1585311 in pSB1C3 using #A9W9# and #XE3D# at 65 °C annealing and 2523 bp expected product length~~

We can then try the CPEC again and for glgA do restriction with E+P and ligation into digested pSB1C3 in parallel - one of them will succeed.

For that amplified the gBlocks by Phusion PCR. (20 µl reactions, 2 ng template DNA, 0'30" initial denaturation, 0'30" denaturation, 0'30" annealing) The products are in #9CRS#.

template	Primer_F	Primer_R	Annealing Temperature [°C]	Product length [bp]	Elongation time	Result
glgA gBlock #OB8B#	#B8WH#	#3N6Z#	55	1535	0'46"	correct band + 3 unspecific products
glgB part 1 gBlock #3XZX#	#B8WH#	#BCAA#	55	1063	0'32"	perfect
glgB part 2 gBlock #AV9S#	#WWL1#	#3N6Z#	62	1265	0'38"	correct band + 1 unspecific product

~~prepare 4xK1585311 in pSB1C3 for sequencing with #A9W9# and #XE3D#~~
measured the concentrations of #F3HE# and #4D6H#
prepare K118016 in pSB1C3 (#F3HE# and #4D6H#) for sequencing with #A9W9# and #XE3D# at 65 °C

15-05-18

printed the K118016 sequencing sheet and send it in

Amplification of gBlocks by Phusion PCR:

conditions for 25 cycles:

step	temperature [°C]	duration
initial denaturation	98	0'30"
denaturation	98	0'30"
gradient annealing	55-67	0'30"
elongation	72	0'46"
final elongation	72	5'00"

column	1	2	3	4	5	6	7	8	9	10	11	12
temperature [°C]	54.9	55.1	55.9	57.0	58.4	59.9	61.5	63.1	64.6	65.8	66.6	67.0

3x 20 µl reactions, 20 ng template DNA

component	µl for mastermix
water	33.6
forward primer	1.5
reverse primer	1.5
HF buffer	12
dNTPs	4.8
Phusion polymerase	0.6
gBlock template	6

template	Primer_F	Primer_R	Annealing Temperature [°C]	Product length [bp]	Elongation time	Result
glgA gBlock #OB8B#	#B8WH#	#3N6Z#	55.1 (2) + 57.0 (4) + 59.9 (6)	1535	0'46"	weak band at 55.1 °C, medium bands at 57.0 and 59.9 °C, unspecific products in all
glgB part 2 gBlock #AV9S#	#WWL1#	#3N6Z#	59.9 (6) + 61.5 (7) + 63.1 (8)	1265	0'38"	medium bands at all temperatures, unspecific products of 700 bp in all

The PCR products are stored in #8K9Q#.

B0034 whole plasmid SDM of K118016 (#4D6H# and #F3HE#) to make K1585312 in pSB1C3. Q5 High-Fidelity Master Mix instead of Phusion. Products stored in #VVRL#.

15-05-19

annotate the gel pictures and fill in the results in the table of yesterdays documentation
transformation of potential K1585310 in pSB1C3 (#VVRL#) into DH5α
repeat yesterdays PCR with glgA and glgB part 2 gBlocks. This time with PfuS polymerase and higher annealing temperatures.

conditions for 25 cycles:

step	temperature [°C]	duration
initial denaturation	98	0'30"
denaturation	98	0'30"
gradient annealing	55-67	0'30"
elongation	72	0'46"
final elongation	72	5'00"

column	1	2	3	4	5	6	7	8	9	10	11	12
temperature [°C]	54.9	55.1	55.9	57.0	58.4	59.9	61.5	63.1	64.6	65.8	66.6	67.0

3x 50 µl reactions with PfuS polymerase according to the cheat sheet. We use 1 µl of gBlock template (10 ng/µl) per reaction. That's 10 ng per 50 µl reaction and completely sufficient! A slight delay was caused by using the wrong dNTP mix at first. Somehow we had more master mix volume than anticipated.

template	Primer_F	Primer_R	Annealing Temperature [°C]	Product length [bp]	Elongation time	Result
glgA gBlock #OB8B#	#B8WH#	#3N6Z#	59.9 (6) + 61.5 (7) + 63.1 (8)	1535	0'46"	two weak bands
glgB part 2 gBlock #AV9S#	#WWL1#	#3N6Z#	63.1 (8) + 64.6 (9) + 65.8 (10)	1265	0'38"	nothing

The PCR products are stored in #18BT#.

Purification of the PCR products:

fragment	from	tubes	prepped into
glgA	#18BT#	A1+A2	#BPTC#
glgB part 1	#9CRS#	B1	#KONM#
glgB part 2	#9CRS#	B2	#LO4N#
glgB part 2	#8K9Q#	B1+B2+B3	#TX1M#

15-05-20

measured the DNA concentrations in yesterdays purified PCR products (#BPTC#, #KONM#, #LO4N#, #TX1M#)
gradient PCR using #TSTV# and #FYTO# on 45 ng J04450 in pSB1C3 (#ZDWH#) per 50 µl PfuS reaction mix. Annealing temperatures 65-68 °C, 1'15" elongation time. Products stored in #YDTH# - on the gel it looks like 68 °C is really good, 67.3 has multiple unspecific products, but for an unknown reason the lower temperatures did not work.
pSB1C3 PCR product purification (#6WCN#)
Restrictions (2x the concentration of T4 Ligase due to human error.)
- digestion of amplified glgA (#BPTC#) with EcoRI and PstI -> product is I in #CQ1V#
- digestion of prepped pSB1C3 (#CD8B#) with EcoRI and PstI -> product is V in #CQ1V#
Ligation of 2 µl digested glgA and 2 µl digested pSB1C3
Q5-CPEC of glgB part 1 (#KONM#), glgB part 2 (#LO4N#) and linearized pSB1C3 (#6WCN#) -> product is B in #4A4P#
- 98:0'30", 15x(98:0'10", 55:0'30", 72:2'20"), 72:10'00"
transformation of 5 µl glgA ligation product into 50 µl home made chemically competent NEB10b + 200 µl SOC
transformation of 5 µl glgB CPEC product into 50 µl commercial chemically competent NEB5a + 200 µl SOC
after 60' of incubation at 37 °C the total volume of each transformation was plated on LB+C

master plates (four clones, one clone from each transformation plate) and overnight cultures (LB+C) of K1585312 in pSB1C3 (sequencing results: #F3HE# and #4D6H# are 100 % correct)

15-05-21

made cryos and pPrep of K1585312 in pSB1C3

clone number	cryo	purified plasmid
1	#A11C#	#OO3E#
2	#HNOX#	#DV8N#
3	#ENOA#	#P4DP#
4	#PM4P#	#X8TM#

sent K1585310 in pSB1C3 into sequencing with #A9W9# at 63 °C
master plates and overnight cultures LB+C of glgA and glgB clones

15-05-22

ordered sequencing primers
made LB+C plates and new LB medium
cryos of white glgA and glgB clones
plasmid prep of white glgA and glgB clones

Name / Clone Number	cryo	purified plasmid	sequencing result (27.05.)
glgA #1	#FYX1#	#6D83#	mRFP insert
glgB #1	#DVLR#	#4L6C#	ugly/contaminated
glgB #2	#F4T6#	#YHT3#	perfect
glgB #3	#CX98#	#3BQH#	non-silent C>T point mutation (P>S)
glgB #4	#Q8D6#	#AOMO#	non-silent G>T point mutation (G>C)
glgB #5	#4YEX#	not yet prepped	(not yet sequenced, but colony PCR looks good)

15-05-26

make new master plates of all remaining white colonies of glgA and some of glgB (for glgA also a few overnights?)

15-05-27

moved master plates of glgA and B into the fridge
cryos of glgA clones, freeze remaining culture for plasmid prep -> all glgA clones were negative on the colony PCR
analyze sequencing results (glgA is without the insert, glgB looks good for #YHT3#, but the sequencing did not reach the center - as expected)
prepared #YHT3# for sequencing with multiple new primers
added 1 µl to both PCR products in #VVRL# and put it into the 37 °C room for overnight digest

15-05-28

trash all existing glgA cryos and clones, because all of them are negative (sequencing/colony PCR)
the glgC DpnI digest (#VVRL#) is back in the freezer
re-do the amplification of glgA from existing gBlock DNA (#OB8B#) with the primers #B8WH# and #3N6Z# (iJR)
- 5x 50 µl PfuS PCR reaction
- expected: 1535 bp
- products in #64LF#

conditions for 30 cycles:

step	temperature [°C]	duration
initial denaturation	98	0'30"
denaturation	98	0'30"
gradient annealing	61-63.5	0'30"
elongation	72	0'50"
final elongation	72	5'00"

purify good glgA products into #F1VM# -> concentration is awful (19.5 ng/µl)
~~digest purified glgA product with EcoRI and PstI (product: #????#)~~
ligate #CQ1V#:I with pSB1C3 (#CQ1V#:V) (6 µl insert, 2 µl backbone) => product is #QZFH#:A

CPEC assembly of glgA

component	volume [µl]
water	10.7
Q5 buffer	5
DMSO	0.75
dNTPs (2 mM)	5
Q5 polymerase	0.5
pSB1C3 fragment (#6WCN#)	0.8
#BPTC#	2.3
elongation time	1'46"
product stored in	#PXR4#

Heat shock transformation of CPEC product (#PXR4#:A), ligation product (#QZFH#) and glgC digested ligation (#VVRL#) into DH5α cells
- trafo program in the cycler: 4 °C for 30', 42 °C for 60", 4 °C for 5'

number	description	id
1	glgA ligation	#OZFH#:A
2	glgA CPEC	#PXR4#:A
3	Hps ligation	#SEXY#:JR
4	glgC re-digested PCR product	#VVRL#:1

15-05-29

Testing last weeks clones of glgA and glgB

colony PCR of re-plated glgA clones (done by iJR and iCG) showed all negative
colony PCR of re-plated glgB clones (done by iJR and iCG) showed #15, #17, #18, #24 positive
overnight cultures of glgB clones #15, #17, #18, #24

Working with new clones

construct	colonies	red
glgA Ligation	11	45 %
glgA CPEC	1140	50 %
glgC	6	0 %

master plates of glgA Ligation/CPEC clones

15-05-30

cryos of glgB clones

gene	clone number	cryo-id	plasmid-id	sequencing result
glgB	15	#AWCC#	#ONXK#	S>Y mutation at position 2879
glgB	17	#BOO9#	#AC85#	part of the sequence looks contaminated
glgB	18	#ZKEA#	not prepped yet
glgB	24	#RVHD#	not prepped yet

colony PCR

template	clone numbers	expected	good clones
glgA ligated	1, 2, 3, 6, 7, 8	1770	2, 3, 6, 7
glgA CPEC	1-12	1770	4 (?)
glgC	1-6	1632	1, 2, 3, 5, 6
glgB	5	2523	yes

15-05-31

overnight cultures

construct	clone number	cryo-id	plasmid-id	sequencing result
glgA ligated	2	#KMST#	#M1M6#	perfect
glgA ligated	3	#8PFH#	#B64M#	perfect
glgA ligated	6	#FHPK#
glgA ligated	7	#4HKH#
glgA CPEC	4	#VWLO#	#6SHZ#	contaminated with two versions
glgC	1	#SOD9#	#PZNS#	negative
glgC	2	#YAPZ#	#6QC3#	negative
glgC	3	#XBNP#	#YWRR#	negative
glgC	5	#SDP4#	#AET8#	negative
glgC	6	#X8Q1#	#LQCS#	negative

15-06-03

sequencing results analyzed:
- glgA: #B64M# and #M1M6# are perfect
- glgB: #YHT3# is perfect
- glgC: did not work yet
heat shock of B0034 in pSB1A2 from KitPlate 2015.4 1N into NEB10b and plating on LB+A

15-06-04

4 overnight and master plate of B0034 in pSB1A2

15-06-05

~~cryos von B0034 in pSB1A2 in NEB10b~~ By accident the cultures were inoculated with two different clones.
~~freeze 4 cultures for later pPrep~~
colony PCR clones 1, 2, 3 from the master plate using #A9W9# and #XE3D#. Expected product length: 251 bp
gel picture looks perfect
~~plasmid prep of one good clone~~
~~pipet the plasmid for sequencing~~

15-06-07

~~inoculation of one LB+A overnight culture~~
new overnights from master plates (clone 1, 2 and 3)

15-06-08

cryos
plasmid prep from overnight #1
freeze #2 and #3

cut B0034 and glgC biobrick (BBa_K118016)
- glgC from #4D6H# mit Xba I + Pst
- B0034 from #4EX9# mit EcoRI + Spe
pSB1K3 from #AQY3# mit EcoRI + Pst

15-06-09

ligate B0034 and glgC in pSB1K3, product in #SY86#
trafo of B0034.glgC in pSB1K3 in DH5α per heat shock (#SY86#)

15-06-10

overnights of B0034.glgC in pSB1K3

15-06-11

cryos and plasmid prep of B0034.glgC in pSB1K3

clone number	cryo ID	plasmid ID	sequencing result
B0034.glgC in pSB1K3 #1	#66ZZ#	#WMVZ#	no RBS
B0034.glgC in pSB1K3 #2	#333V#	#YSVR#	no RBS
B0034.glgC in pSB1K3 #3	#C9TN#	was red	-
B0034.glgC in pSB1K3 #4	#SSOL#	#TDS1#	no RBS
B0034.glgC in pSB1K3 #5	#Z8BB#	#HNEA#	no RBS

pipette B0034.glgC in pSB1K3 for sequencing

15-06-17

Digestions with 40' digest and 20' inactivation

gene	enzymes	expectation	observation	products
glgC (#4D6H#)	XbaI + PstI	...	not run	#OHOZ#:C
B0034 (#4EX9#)	SpeI + PstI	...	not run	#OHOZ#:R

Ligations and transformations

construct	part I	part II	backbone	product	host
B0034.glgC in pSB1A2	#OHOZ#:R	#OHOZ#:C	-	#OKNR#:G	DH5α

15-06-18

make master plates and liquid cultures of transformation (B0034.glgC in pSB1A2; 6 liquid, 12 plate)

15-06-19

Colony PCR on glgC construct

gene / clones	tube number	expected length	machine	elongation time	result
glgC in pSB1A2 clones 1-12	25-36	1556	2	1'50"	all positive

Cryos

gene / clone	cryo ID	plasmid ID	sequencing result
glgC in pSB1A2 #1	#9F3M#	#SMTS#	perfect
glgC in pSB1A2 #2	#ZC8K#	#F8Y8#	perfect
glgC in pSB1A2 #3	#YV1C#	don't prep	-
glgC in pSB1A2 #4	#D3E6#	don't prep	-
glgC in pSB1A2 #5	#RTWO#	don't prep	-
glgC in pSB1A2 #6	#TN6K#	don't prep	-

15-06-22

prep good plasmids of glgA and glgC (table above)
pipette glgA in pSB1K30 and B0034.glgC for sequencing (#A9W9# and #XE3D#)

15-06-24

analysis of sequencing results

15-06-25

discard excess B0034.glgC in pSB1A2 cryos and plasmids

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

15-06-29

Ligation and transformation

construct	gene	Part I	Part II	Backbone	products	host	purpose
K1585312 in pSB1C3	glgC	#LVVK#:1	-	#CQ1V#:V	#RRCV#:1	DH5α	for part submission

15-06-30

we have clones of K1585312 into pSB1C3 (red-white screening)
made master plate (6 clones) and overnight cultures (3 cultures)

15-07-01

cryos and freezings of the three K1585312 in pSB1C3 cultures
colony PCR on all 6 clones > clone 1 and 2 look good

gene / clone	cryo ID	plasmid ID	sequencing result
B0034.glgC in pSB1C3 #1	#4XPZ#	#DRPT#	glgA instead of glgC
B0034.glgC in pSB1C3 #2	#1NZB#	#EZOB#	glgA instead of glgC
B0034.glgC in pSB1C3 #3	#FCSS#	no prep	-

pipetted for sequencing

15-07-03

overnight culture of glgC in pSB1A2 from #ZC8K# in LB+A

15-07-06

PCR 1 to subclone glgC into pSB1C3

Backbone amplification:

component	template	forward primer	reverse primer	product length	purified pcr product ID	product ID	tube #
J04450 in pSB1C3	#CD8B#	#TSTV#	#FYTO#	2070	#ZV3C#	#MRVW#:II	1+2

PfuS PCR