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=Laboratory Notebook=
 
=Laboratory Notebook=
  

Revision as of 00:38, 18 September 2015



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
  • Manual/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

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

PCR 2 to subclone glgC into pSB1C3

Insert amplification:

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

15-07-09

PCR product purification

PCR product purified PCR product
#MRVW#:I #XNHT#
#MRVW#:II #ZV3C#

CPEC assembly of glgC in pSB1C3

component volume [µl]
water 5.9
Q5 buffer 5
DMSO 0.75
dNTPs (2 mM) 5
Q5 polymerase 0.5
pSB1C3 fragment (#ZV3C#) 2.7
glgC with prefix/suffix #XNHT# 5.2
elongation time 1'42" (1'47")
product stored in #SWMB#:I

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

15-07-10

  • make masterplates (LB+C) and overnights of glgC in pSB1C3

15-07-11

  • cryo cultures
  • freeze culture
gene / clone cryo ID plasmid ID sequencing result
B0034.glgC in pSB1C3 #1 #ESAY# #QXBD# good, but not perfect
B0034.glgC in pSB1C3 #2 #DFKW# #OP1N# perfect
B0034.glgC in pSB1C3 #3 #VW9S# - -
B0034.glgC in pSB1C3 #4 #L81A# #MCM6# -
B0034.glgC in pSB1C3 #5 #SOVA# #NPCT# contaminated chromatogram
B0034.glgC in pSB1C3 #6 #Q9NL# - -

15-07-13

  • plasmid prep of glgC in pSB1C3 (see table above)

15-07-15

  • analysing sequencing results (see table above)

References