Laboratory Notebook

All glycogen synthesis genes were successfully cloned into BioBricks.

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:

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:

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#.

• 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:

3x 20 µl reactions, 20 ng template DNA

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:

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.

The PCR products are stored in #18BT#.

Purification of the PCR products:

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

• 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

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:

• 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

• 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'

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

• master plates of glgA Ligation/CPEC clones

15-05-30

• cryos of glgB clones

• colony PCR

15-05-31

• overnight cultures

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

• pipette B0034.glgC in pSB1K3 for sequencing

15-06-17

Digestions with 40' digest and 20' inactivation

Ligations and transformations

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

Cryos

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

15-06-29

Ligation and transformation

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

• 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:

PfuS PCR

conditions for 30 cycles:

15-07-08

PCR 2 to subclone glgC into pSB1C3

Insert amplification:

15-07-09

PCR product purification

CPEC assembly of glgC in pSB1C3

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

15-07-13

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

15-07-15

• analysing sequencing results (see table above)

References