| Goettingen - iGEM 2022

Notebook

Overview

Labbook iGEM

28.02.
- Preparation of 500 mL TYA and 54b Clostridium sp. Medium

01.03.
- Preparation of 500 mL 135 acetobacterium medium and 879 Clostridium ljungdahlii (XY) medium
- Gassing of Hungates with N2 and autoclavation
- Preparation of 0.1 g/mL L-Cystein, 0.1 g/mL Na2Sx9H2O, 0.1 % Na-Resazurin
- Addition of Glucose to TYA and 54b Clostridium sp. Medium according to protocol

02.03.
- Preparation of vitamin solution and sterile filtration
- pH adjustment of 135 and 879 medium
- Inoculation of XY in XY medium, Cba in TYA medium, C.bei 8052 + C.bei A21 in 54b medium

03.03.
- Check of culture growth (Cba +, C.bei -, XY o)
- Microscopy of Cba and XY
- Gassing of hungates and anaerobic flasks

04.03.
- Check of culture growth (C.bei 🡪 fridge, XY inoculated until 07.03.)
Week 2 (07.03.-13.03.)

07.03.
- Inoculation of C.bei 8052 and A21 in TYA
- Inoculation of XY in XY medium
- Inoculation of Awo in 135 medium

08.03.
- Cryo Stocks of C.bei 8052, C.bei A21, Awo, Cba and XY prepared and stored @-80°C
- C.bei 8052, Awo, Cba 🡪 fridge; C.bei A21, XY 🡪 further incubation
- Cell pelleting of 1.5 mL C.bei 8052, C.bei A21, Awo, Cba and XY culture by centrifugation and removal of supernatant – stored @-20°C
- Inoculation of C.bei 8052, C.bei A21 and Cba in 135 XY Medium

09.03.
- Check of growth from 07./08.03. cultures
Growth: XY in XY medium
C.bei A21 in XY medium
C.bei 8052 in XY medium
Cba in XY medium
No Growth: Cba in 135 medium
C.bei A21 in TYA
C.bei 8052 in 135 medium
C.bei A21 in 135 medium
C. bei A21 in TYA
- gDNA isolation XY, Cba, C.bei A21, C.bei8052, Awo with the cell pellets from 08.03. – gDNA stored in fridge
- Preparation of anaerobic 6M HCl

10.03.
C.bei 8052, C.bei A21, Xy, Awo and Cba cultures
Primer: 08 forward and 1504 reverse 🡪 1496 bp product
Template: gDNA (09.03.) – only 50ng used
- 16S-rRNA Dream-Taq PCR for verification of clean C.bei 8052, C.bei A21, Xy, Awo and Cba cultures
Primer: 08 forward and 1504 reverse 🡪 1496 bp product
Template: gDNA (09.03.) – only 50ng used
- Gel electrophoresis for 16sRNA PCR verification

Week 3 (14.03.-20.03.)
14.03.
- Repetition of 16S-rRNA Dream-Taq PCR from 10.03. – this time 100 ng template according to protocol used
- Gel electrophoresis for 16sRNA PCR verification
- PCR clean-up of C.bei 8052, Xy, Awo and Cba 16s rRNA
- Sequencing of C.bei 8052, Xy, Awo and Cba 16s rRNA with 08 forward Primer

15.03.
- Verification of Sequencing results of C.bei 8052, Xy, Awo and Cba 16s rRNA 🡪 high identity with corresponding organism genome 🡪 right organism cultivated
- Repetition of 16S-rRNA Dream-Taq PCR for C.bei A21
- Gel electrophoresis for C.bei A21 16sRNA PCR verification
- PCR clean-up of C.bei A21 🡪 8 ng/µl
- Increasing concentration of C.bei A21 16s rRNA PCR sample via evaporator
- Sequencing of C.bei A21 16s rRNA with 08 forward Primer
- Inoculation of C.bei A21, C.bei 8052 and Cba in XY medium

16.03.
- Check of growth: No growth of 15.03. inoculated cultures, green precipitate is observable
- Re-inoculation of C.bei A21, C.bei 8052 and Cba in XY medium
- Verification of C.bei A21 16s rRNA sequencing 🡪 show possible contamination

17.03.
- Check of growth: C.bei 8052 and C.bei A21 grew, Cba grew only little 🡪 incubated for another day
- Preparation of TYA plates

18.03.
- Cba did not further grew 48h post-inoculation
- Re-inoculation of Cba from Cryo in XY medium

Week 4 (21.03.-27.03.)
22.03.
- Preparation of 0.1 g/mL Na2Sx9H2O
- N2-gassing and autoclavation of hungates
- Chimney gassing of TYA medium (28.02.) with N2

23.03.
- Shifting of XY, C.bei 8052, C.bei A21, Cba in fresh XY and TYA medium
- Preparation of TYA medium, XY medium without fructose, anaerobic 40% glucose, anaerobic 10% fructose until autoclavation

24.03.
- Check of growth
Growth: C.bei 8052 , C.bei A21, Cba, XY (slightly aerobic) in XY medium
Cba was further incubated
No Growth: All cultures in TYA medium 🡪 became aerobic
- TYA and XY medium were finished

Week 5 (28.03.-03.04.)
28.03.
- Na2S in TYA medium precipitated
- Check of growth: Cba inoculated 23.03. grew well
- Inoculation of C.bei A21, C.bei 8052 and Cba in TYA
- Inoculation of XY in XY
- Inoculation of XY in XY medium without fructose for growth on H2 and CO2 (Problem: only short gassing this day was possible)
- Plating C.bei A21, C.bei 8052 and Cba on TYA plates – 125 µL plated, incubation @35°C together

29.03.
- Check of growth:
Growth: Cba in XY and TYA medium
C.bei 8052 in TYA
Slight/No Growth: C.bei A21 in TYA and XY medium 🡪 became aerobic
C.bei A21, C.bei 8052 and Cba on TYA plates
- Inoculation of XY in XY medium without fructose, 450 µl 10% fructose added

30.03.
- Check of growth:
Growth: XY and Cba in XY medium with added fructose
C.bei A21 and Cba on TYA plates
Slight/No growth: XY in old XY medium
C.bei A21 in TYA 🡪 further incubation
C.bei 8052 on TYA plate 🡪 further incubation
- Picking of one C.bei A21 colony from TYA plate and inoculation in XY medium
- 60 mL XY Medium with added fructose adjusted to pH 8.28 for Awo test cultivation

31.03.
- Check of growth:
Growth: picked C.bei A21 in XY medium
Slight/no growth: C.bei A21 in TYA
Awo in XY medium pH 8.28 🡪 further incubation
XY on H2, CO2 🡪 further incubation
C.bei 8052 on TYA plate 🡪 further incubation
- Preparation of sterile 1 L LB medium and 100 mL 10% glycerin
- Preparation of 10x buffer for XY medium
- pelleting picked C.bei A21 culture from 30.03.
Week 6 (04.04.-10.04.)

04.04.
- Inoculation of picked C.bei A21 in TYA and XY medium, C. bei 8052 and Cba in TYA, XY in XY medium
- New Inoculation of XY for growth with H2, CO2
- No Growth of Awo in XY Medium pH 8.28 and C.bei 8052 on TYA plate
- Preparation of XY medium medium with pH adjusted to 8.0 and new inoculation of Awo out of Cryo in XY medium pH 8.0
- Gassing and autoclavation of hungates

05.04.
- Preparation of sterile 30mg/µl Kanamycin stock
- gDNA isolation of picked C.bei A21 according to Tims Gram Positive DNA Purification Protocol
- Inoculation of E.coli XL1-blue MRF’Kan in 2x 5 mL LB
- All cultures inoculated 04.04. were further incubated

06.04.
- Preparation of sterile, N2 gassed anaerobic flasks
- E.coli XL1-blue MRF’Kan culture was used to prepare electrocompetent E.coli cells – Problem: Cell pellet during centrifugation steps was loose

07.04.
- 16S-rRNA Dream-Taq PCR for verification of clean picked C.bei A21
- Gel electrophoresis for 16sRNA PCR verification
- PCR clean-up of picked C.bei A21 16sRNA PCR
- Sequencing of picked C.bei A21 16sRNA PCR with 08 forward Primer
- Check of growth:
Growth: C.bei 8052 and C.bei A21 in TYA
No growth: Cba in TYA 🡪 further incubation
Awo in XY medium pH 8.0 🡪 further incubation
XY in XY medium 🡪 further incubation

Week 7 (11.04.-17.04.)
11.04.
- Test-Trafo for determination of efficiency of prepared electrocompetent E.coli XL1-blue MRF’Kan
Plasmid used: 1 µl pMTL83151_pthlA-gusA (Pthla Promoter) c=193ng/µl
Plating: 100 µl undiluted + 10-1, 10-2, 10-3 and 10-4 dilution on LB-Chloramphenicol
- Check of growth:
Growth: Awo in XY medium pH 8.0
No growth: Cba in TYA 🡪 became aerobic
XY in XY medium
XY in XY medium without fructose with H2, CO2 🡪 further incubation
- Shifting of Awo in fresh XY medium pH 8.0

12.04.
- Check of growth: Awo in XY medium pH 8.0 grew and was stored in fridge
- Evaluation of electrocompetent E.coli XL1-blue MRF’Kan transformation efficiency
Undiluted: 370 colonies
10-1: 9 colonies
🡪 Efficiency= 9x101x(1000ng/100ng)x(1000µl/100µl)=9x10x10x10= 9x103 cells/µg DNA
10-2: 3 colonies
10-3 &10-4: no colonies

13.04.
- PCR for amplification of acsB gene of XY&Awo according to Fusion protocol (50µL)
Primer: XY 1+2
Awo 4+5
Template: Awo & XY gDNA 20ng/µL
Elongation time: 1min 4s
Annealing temperature: 62.9 °C
- Gel electrophoresis for acsB PCR verification

Week 8 (18.04.24.04.)
20.04.
- Preparation of XY medium without fructose
- Preparation of Na2SxH2O
- Preparation of sterile Chloramphenicol Stock (300 mg + 10 mL 96% Ethanol)
Preparation of LB agar plates with Chloramphenicol, IPTG, X-Gal

Week 9 (25.04.-01-05.)
25.04.
- PCR for amplification of acsB gene of XY&Awo according to Fusion protocol (50µL) with new Primers
Primer: XY 13+14
Awo 11+12
Template: Awo & XY gDNA 20ng/µL
Elongation time: 1min 10s
Annealing temperature: 52.4 °C
- Gel electrophoresis for acsB PCR verification
- PCR product purification with Qiagen QIAquick PCR purification Kit according to protocol
- Inoculation of E.coli+pMTL8351 gusA in 5 mL LB with Chloramphenicol

26.04.
- Plasmid Prep pMTL8351 gusA with Qiagen QIAprep Spin mini Prep Kit according to protocol
- Inoculation of XY in XY medium

27.04.
- Digest of Insert (acsB XY/Awo) + digest of pMTL83151_pthlA-gusA with Xho1 and Nhe1 for 10 min @37 °C + 5 min @ 65 °C
- Purification of digest with Qiagen PCR Purification kit
- Ligation of acsB XY/Awo with pMTL83151_pthlA over night @RT in dark
2 µL Plasmid (50ng)
15 µL Insert (90 ng)
2 µL 10xT4 DNA ligase buffer
1 µL T4 DNA ligase
- Inoculation of Cba in XY medium

28.04.
- Inactivation of ligation samples (10 min @ 65°C)
- Preparation of X-gal stock solution 48 mg/µl
- Preparation of IPTG stock solution 40 mg/µl
- Dialysis of ligation samples (acsB XY/Awo with pMTL83151_pthlA) for 30 min
- Electroporation of E.coli XL1-blue MRF’Kan with pMTL83151_pthlA acsB XY/Awo ligation sample. Plating on LB+IPTG+X-gal+Chloramphenicol plates
- Test-digest of pMTL83151_pthlA acsB XY/Awo ligation with Nhe1 and Xho1 for
10 min @37 °C + 5 min @ 65 °C

Week 10 (02.05.-08.05.)
02.05.
- Verification of transformation from 28.04.: unfortunately all colonies were blue 🡪 still inoculation of 3 colonies acsB XY/Awo with pMTL83151_pthlA per transformation in LB+Chloramphenicol
- Check of growth: Cba from 27.04. did not grow
- Gel electrophoresis for verification of test-digest of pMTL83151_pthlA acsB XY/Awo ligation

03.05.
- Preparation of XY medium without fructose
- Plasmid Prep of pMTL83151_pthlA acsB XY and pMTL83151_pthlA acsB Awo (inoculated 02.05.)
- Test-digest of pMTL83151_pthlA acsB XY + pMTL83151_pthlA acsB Awo and pMTL83151_pthlA-gusA as control with Nhe1 and Xho1 for 10 min @37 °C + 5 min @ 65 °C
- Gel electrophoresis for verification of Test-digest of pMTL83151_pthlA acsB XY + pMTL83151_pthlA acsB Awo and pMTL83151_pthlA-gusA as control
- Re-inoculation of 3 colonies acsB XY/Awo with pMTL83151_pthlA per transformation in 5 mL LB

04.05.
- Inoculation of XY and Cba in XY medium from cryo
- Inoculation of C.bei A21 and Awo in XY medium
- Plasmid Prep of of pMTL83151_pthlA acsB XY and pMTL83151_pthlA acsB Awo (inoculated 03.05.)
- Test-digest of pMTL83151_pthlA acsB XY + pMTL83151_pthlA acsB Awo and pMTL83151_pthlA-gusA as control with Nhe1 and Xho1 for 10 min @37 °C + 5 min @ 65 °C
- Gel electrophoresis for verification of Test-digest of pMTL83151_pthlA acsB XY + pMTL83151_pthlA acsB Awo and pMTL83151_pthlA-gusA as control

05.05.
- Re-run: Gel electrophoresis for verification of Test-digest of pMTL83151_pthlA acsB XY + pMTL83151_pthlA acsB Awo and pMTL83151_pthlA-gusA as control
- Sequencing of pMTL83151_pthlA acsB XY C1,C2,C3 + pMTL83151_pthlA acsB Awo C1,C3 with pMTL83151_Pol_fw and pMTL83151_pol_rev primer

06.05.
- Verification of sequencing results (send 05.05.): ne clear results, potentially several sequences sequenced

Week 11 (09.05.-15.05.)
09.05.
- Preparation of sterile and anaerobic L-Cysteine-HClxH2O solution
- Check of growth: C.bei A21, Cba, Awo and XY in XY medium grew
- Shifting of C.bei A21, Cba, Awo and XY in fresh XY medium
- Inoculation of pMTL83151_pthlA acsB XY C1,C2,C3 + pMTL83151_pthlA acsB C1,C3 in 5mL LB with Chloramphenicol, respectively 2x for each clone

10.05.
- Plasmid prep of pMTL83151_pthlA acsB XY C1,C2,C3 + pMTL83151_pthlA acsB Awo C1,C3
- Colony PCR preparation:
Pipet Tip with E.coli colony of pMTL83151_pthlA acsB XY C1 and pMTL83151_pthlA acsB Awo C1 transferred in 50 µL H20 and boiling at 95 °C for 3 min 🡪 stored @-20 °C
- Inoculation of pMTL83151_pthlA acsB XY C1,C2,C3 + pMTL83151_pthlA acsB Awo C1,C3 in 5mL LB with Chloramphenicol (for preparation of cryo)
- Sequencing of pMTL83151_pthlA acsB Awo C3 with Awo_acsB_F and pMTL83151_pthlA acsB XY C1 with XY_acsB_F

11.05.
- Verification of sequencing results: Sequencing of XY/Awo acsB was succesful 🡪 Insert is in the plasmid
- Colony PCR for verification if acsB insert is in pMTL83151_pthlA1 plasmid
As template either the boiled-frozen E.colis or directly the fresh E.colis pMTL83151_pthlA acsB Awo C1 and pMTL83151_pthlA acsB XY C1 were used
Primer: 11+14 and 15+16
Annealing temperature: 52.4 °C (11+14), 59.6 °C (15+16)
Elongation: 1min 10s (11+14), 1min 30s (15+16)
- Gel electrophoresis for verification of colony PCR
- Test-digest pMTL83151_pthlA acsB XY C1,C2,C3 + pMTL83151_pthlA acsB Awo C1,C3 with Xho1 and Nhe1 15 min @37 °C + 5 min @ 65 °C
- Gel electrophoresis for verification of test-digest pMTL83151_pthlA acsB XY C1,C2,C3 + pMTL83151_pthlA acsB Awo C1,C3
- Sequencing of pMTL83151_pthlA acsB Awo C1 with pMTL83151_pol_fw (15) and Awo_S_acsB1_3_fw (17) primers
- Inoculation of pMTL83151_pthlA acsB XY C2 + pMTL83151_pthlA acsB Awo C3 for cryo stock preparation

12.05.
- Verification of sequencing (11.05.): With pMTL83151_pol_fw (15) did not work
With Awo_S_acsB1_3_fw (17) worked
- Sequencing of pMTL83151_pthlA_GusA with pMTL83151_pthlA_pol_fw and pMTL83151_pthlA_pol_rv Primers

13.05.
- Inoculation Cba and Awo in XY medium
- Verification of sequencing (12.05.): did not work
- Sequencing of pMTL83151_pthlA_GusA with pMTL83151_pol_fw and pMTL83151_pol_rv Primers re-done
+ Sequencing of pMTL83151_pthlA acsB Awo C1 with Awo_acsB1_r Primer

Week 12 (16.05.-22.05.)
17.05.
- Verification of sequencing (13.05.): did not work
- Dialysis pMTL83161_pthlA-gusA for 30 min
- Sequencing pMTL83161_pthlA-gusA with pMTL83151_pol_fw primer

18.05.
- Verification of sequencing of pMTL83161_pthlA-gusA: plasmid seems to be correct
- Re-transformation of pMTL83161_pthlA-gusA in electrocompetent E.coli XL1-blue MRF’Kan (20ng plasmid used). Plating on LB+IPTG+X-gal-Chloramphenicol plate.

19.05.
- Received 5 E.coli pellets that should contain pMTL83151_pthla-gusA
- Plating of the remaining transformation solution from 18.05. on LB+IPTG+X-gal+Chloramphenicol plates
- Test-digest of Clones 5a-e pMTL83151_pthla-gusA with Xho1 and Nhe1

20.05.
- Gel electrophoresis for verification of Test-digest of Clones 5a-e pMTL83151_pthla-gusA

Week 13 (23.05.-29.05.) - Test-digest pMTL831515_pthlA-gusA clones 5a-e with either Xho and Nhe1 or BamH1 single digest 20 min @37 °C + 5 min @ 65 °C
- Gel electrophoresis for verification of test-digest pMTL831515_pthlA-gusA clones 5a-e

24.05.
- Sequencing of pMTL831515_pthlA-gusA clone 5a with pMTL_83151-seq_f_1-9

25.05.
- Verification of Sequencing pMTL831515_pthlA-gusA clone 5a: Seq1&2 did not work, Seq3-9 worked
- Preparation of liquid M9 medium and M9+IPTG+X-gal+Chloramphenicol agarplates

Week 14 (30.05.-05.06.)
30.05.
- Re-transformation of pMTL83161_pthlA-gusA in electrocompetent E.coli XL1-blue MRF’Kan (20ng plasmid used). Recovery in M9 medium and plating on M9+IPTG
+X-gal-Chloramphenicol plate.

31.05.
- Re-transformation M9 plate from 30.05. did not grow 🡪 further incubation + additional plating of the rest of the re-trafo solution on M9+IPTG+X-gal-Chloramphenicol plate
- Inoculation of Cba in XY medium from cryo

02.06.
- Sequencing of pMTL831515_pthlA-gusA clone 5e&5a with pMTL83151-pol-fw Primer

03.06.
- Verification of sequencing of pMTL831515_pthlA-gusA clone 5e&5a: Moderate or ambiguous results
Week 15 (06.06.-12.06.)

07.06.
- Preparation of HPG medium
- Check of growth: Re-transformation plates (30.05.) did not grow
- Transformation of pMTL831515_pthlA-gusA in chemocompetent E.coli DH10B. Plating on LB +IPTG+X-gal-Chloramphenicol plate.
- Inoculation of Cba from 31.05. in fresh XY medium

08.06.
- Digest of Insert (acsB XY/Awo) + digest of pMTL83151_pBgal-gusA with Xho1 and Nhe1
for 10 min (plasmid) / 15 min (insert) @37 °C + 5 min @ 65 °C
- Purification of digest with Qiagen PCR Purification kit
- Ligation of acsB XY/Awo with pMTL83151_pBgal over night @RT in dark
- Preparation of LB +IPTG+X-gal-Chloramphenicol plates
- Inoculation of pMTL83151_pBgal-gusA in 5 mL LB with Chloramphenicol

09.06.
- Plasmid Prep of pMTL83151_pBgal-gusA
- Inactivation of ligation samples (10 min @ 65°C)
- Dialysis of ligation samples (acsB XY/Awo with pMTL83151_pBgal) for 30 min
- Electroporation of E.coli XL1-blue MRF’Kan with pMTL83151_pBgal acsB XY/Awo ligation sample. Plating on LB+IPTG+X-gal+Chloramphenicol plates
- Test-digest of pMTL83151_pBgal acsB XY/Awo ligation with Nhe1 and Xho1 for
25 min @37 °C + 5 min @ 65 °C,
- Gel electrophoresis for verification of test-digest of pMTL83151_pBgal acsB XY/Awo ligation

Week 16 (13.06.-19.06.)
14.06.
- PCR for amplification of acsB gene of XY&Awo and 6/5 kB unit according to Fusion protocol (50µL)
acsB: Primer: XY 13+14
Awo 11+12v
Template: Awo & XY gDNA 20ng/µL
Elongation time: 1min 10s
Annealing temperature: 52.4 °C
6/5 kB unit: Primer: XY 14+19
Awo 12+20
Template: Awo & XY gDNA 20ng/µL
Elongation time: 3min 30s
Annealing temperature: 53.1 °C

15.06.
- Gel electrophoresis for PCR verification of 14.06.
- PCR product purification of XY-acsB and Awo-acsB with Qiagen QIAquick PCR purification Kit according to protocol
- Check of pMTL83151_pBgal acsB XY/Awo transformation
pBgal83151 Awo-acsB transformation: blue colonie, but also 3 non-blue colonies grew 😊
pBgal83151 XY-acsB transformation: only blue colonies grew ☹
Re-ligation: only blue colonies grew 😊
- 2x Inoculation of the 3 non-blue pBgal83151 Awo-acsB clones in 5 mL LB with Chloramphenicol + streaking out of the clones on LB+IPTG+X-Gal+Chloramphenicol plate
- Digest of Insert (acsB XY/Awo) + digest of pMTL83151_pBgal-gusA with Xho1 and Nhe1
for respectively 15 min / 45 min @37 °C and over night @ RT + 5 min @ 65 °C

16.06.
- Purification of digest with Qiagen PCR Purification kit
- Ligation of acsB XY/Awo with pMTL83151_pBgal over night @RT in dark. Respectively the 15 min, 45 min and overnight digest samples were ligated.
For calculation of the NEBioCalculator online tool was used and a 5:1 vector:insert ratio was chosen
- Plasmid Prep of the 3 non-blue pBgal83151 Awo-acsB clones 🡪 Problem: Clones also became blue after they had been streaked out on new plate

17.06.
- Inactivation of pMTL83151_pBgal acsB XY/Awo ligation samples (10 min @ 65°C)

Week 17 (20.06.-26.06.)

20.06.
- Preparation of LB+IPTG+X-Gal+Chloramphenicol plates
- Plasmid prep of pMTL83151_pthlA-gusA C5i (Mechthild)
- Dialysis of ligation samples (16.06.) acsB XY/Awo with pMTL83151_pBgal
- Electroporation of E.coli XL1-blue MRF’Kan with pMTL83151_pBgal acsB XY/Awo ligation sample 15min/45min/ON. ON ligation sample transformation was recovered in M9 Medium.
15min/45min ligation sample transformation was recovered in LB. Plating on M9/LB+IPTG+X-gal+Chloramphenicol plates
- Sequencing of pMTL83151_pthlA-gusA C5i with Seq1, Seq8 and Seq9 primers

21.06.
- Verification of sequencing results: Sequencing of pMTL83151_pthlA-gusA C5i worked and clone seems to be correct!
- Preparation of 10% Na2SxH20
- Preparation of XY medium without fructose
- Preparation of M9 medium

22.06.
- Finalisation of XY and M9 medium
- Check of growth: Transformation plates from 20.06. show no growth except for neg. control
- Plating of pMTL83151_pthlA-gusA C5i on M9+IPTG+X-gal+Chloramphenicol and
LB+IPTG+X-gal+Chloramphenicol plates
- We obtained chemocompetent E.coli XL1-blue MRF-tet with a transformation efficiency of 8.25x107

23.06.
- Transformation of chemocompetent E.coli XL1-blue MRF-tet with pMTL83151_pBgal acsB XY ligation sample 45min and pMTL83151_pBgal acsB Awo ligation sample ON
Plating on M9+IPTG+X-gal+Chloramphenicol plates.
- Inoculation of Cba in XY medium
- Further incubation of transformation plates from 20.06. @RT

24.06.
- Check of growth:
pMTL83151_pthlA-gusA C5i (22.06.) shows strong growth on LB plate and light growth on M9 medium plate 🡪 further incubation on RT
pMTL83151_pBgal acsB XY/Awo transformation plates (23.06.) so far show no growth 🡪 further incubated @RT

Week 18 (27.06.-03.07.)
27.06.
- Preparation of M9 medium
- 2x Inoculation of pMTL83151_pthlA-gusA C5i from plate (22.06.) in 50 mL M9 and 50 mL LB medium with Chloramphenicol
- Check of growth: small white colonies on pMTL83151_pBgal acsB XY/Awo transformation plates (23.06.)
🡪 all colonies were picked, streaked out on plate and inoculated in 5 mL M9 with Chloramphenicol

28.06.
- Inoculation of Cba in XY medium
- Finalisation of M9 medium
- Plasmid Prep of pMTL83151_pthlA-gusA C5i (27.06.). 5 mL of LB culture and 10 mL of M9 culture was used 🡪 the rest of the cultures were pelleted and stored @-20 °C
- Test-digest of pMTL83151_pthlA-gusA C5i LB vs. M9 with Nhe1 and Xho1 for 45 min @37 °C + 5 min @ 65 °C

29.06.
- All pMTL83151_pBgal acsB XY/Awo clones that grew (27.06.) were pelleted and 5 clones (XY: 8,23,26,38,39 – Awo: 3,4,8,10,11) were also spotted on new M9+IPTG+X-gal+Chloramphenicol plates
- Plasmid Prep of pMTL83151_pBgal acsB clones XY: 8,23,26,38,39 – Awo: 3,4,8,10,11 clones
- Purification of tets-digest of pMTL83151_pthlA-gusA C5i LB vs. M9 and acsB XY/Awo fragment with Qiagen Min Elute PCR Purification Kit
- Ligation of acsB XY/Awo with pMTL83151_pthlA C5i for 2h @RT in dark.
- Transformation of chemocompetent E.coli XL1-blue MRF-tet with pMTL83151_pthlA acsB C5i XY/Awo ligation samples. Plating respectively on M9+IPTG+X-gal+Chloramphenicol and LB+IPTG+X-gal+Chloramphenicol plates.
- Gel electrophoresis for verification of test-digest of pMTL83151_pthlA-gusA C5i LB vs. M9 (28.06.)

30.06.
- Check of growth: No colonies so far on transformation (29.06.) E.coli XL1-blue MRF-tet with pMTL83151_pthlA acsB C5i XY/Awo 🡪 further incubated @RT
- Test-digest of pMTL83151_pBgal acsB clones XY: 8,23,26,38,39 – Awo: 3,4,8,10,11 clones with Nhe1 and Xho1 for 45 min @37 °C + 5 min @ 65 °C
Controls: pMTL83151_pBgal-gusA, pMTL83151_pthla clone2 (+ control), - control, acsB XY/Awo PCR products
- Gel electrophoresis for verification of test-digest of pMTL83151_pBgal acsB clones XY: 8,23,26,38,39 – Awo: 3,4,8,10,11 clones
- Sequencing of pMTL83151_pBgal acsB XY C38 and Awo C10 with pMTL83151_Seqf8 and pMTL83151_Pol_rev primers

Week 19 (04.07.-10.07.)
04.07.
- Check of growth: All colonies on LB transformation plate (29.06.) with chemocompetent E.coli XL1-blue MRF-tet with pMTL83151_pthlA acsB C5i XY/Awo are blue, on M9 colonies seem to be white but were still small 🡪 further incubation of M9 plate
- Gel electrophoresis for verification of correct insert integration in pMTL83151_pBgal acsB clones XY: 8, 38 – Awo: 10
- Inoculation of pMTL83151_pBgal acsB Awo C10 and pMTL83151_pBgal acsB XY C38 in 5 ml M9 with Chloramphenicol

05.07.
- Preparation of M9 medium
- Plasmid Prep of pMTL83151_pBgal acsB Awo C10 and pMTL83151_pBgal acsB XY C38
- Inoculation of pMTL83151_pBgal acsB Awo C10 and pMTL83151_pBgal acsB XY C38 in 5 mL M9 with Chloramphenicol + plating on M9+IPTG+X-gal+Chloramphenicol plate
- Sequencing of pMTL83151_pBgal acsB Awo C10 and pMTL83151_pBgal acsB XY C38 with pMTL_83151_Seqf1,2,3,4,5,6,7,9 Primers
- 10 clones of pMTL83151_pthlA acsB C5i XY/Awo respectively from transformation 29.06. grown on M9 medium were picked, streaked out on M9+IPTG+X-gal+Chloramphenicol plate ans inoculated in 5 mL M9 with Chloramphenicol

06.07.
- Check of growth: pMTL83151_pthlA acsB C5i XY/Awo clones inoculated 05.07. did not grow well so far 🡪 further incubation
- Plasmid Prep of pMTL83151_pBgal acsB Awo C10 and pMTL83151_pBgal acsB XY C38
- Sequencing of pMTL83151_pBgal acsB XY C38 with pMTL_83151_pol_fwd and NheI_acsB2_rev_XY Primers and of pMTL83151_pBgal acsB Awo C10 with pMTL_83151_pol_fwd, NheI_acsB2_rev_XY and seq_AcsB1_3_f Primers
- Inoculation of pMTL83151_pBgal acsB Awo C10 and pMTL83151_pBgal acsB XY C38 in 5 mL M9 with Chloramphenicol

07.07.
- Check of growth: 8 clones respectively of pMTL83151_pthlA acsB C5i XY/Awo inoculated 05.07. did grow
- Plasmid Prep of pMTL83151_pthlA acsB C5i XY/Awo
- Sequencing of pMTL83151_pBgal acsB XY C38 with NheI_acsB2_rev_XY Primer
- Test-digest of pMTL83151_pthlA acsB C5i XY/Awo for 45 min @37 °C + 5 min @ 65 °C with NheI&XhoI
- Gel electrophoresis for verification of test-digest of pMTL83151_pthlA acsB C5i XY/Awo

Week 20 (11.07.-17.07.)
11.07.
- PCR for amplification of acsB gene of XY&Awo according to Fusion protocol (50µL)
Primer: XY 13+14
Awo 11+12
Template: pMTL83151_pBgal acsB Awo C10 / XY C38 20ng/µL
Elongation time: 1min 10s
Annealing temperature: 52.4 °C
- Gel electrophoresis for PCR verification of acsB gene
- Test-digest of pMTL83151_pthlA acsB C5i XY/Awo for 45 min @37 °C + 5 min @ 65 °C with NheI&XhoI
- Gel electrophoresis for verification of test-digest of pMTL83151_pthlA acsB C5i XY/Awo

12.07.
- Inoculation of Cby in XY-Medium from cryo
13.07.
- Inoculation of pMTL83151_pBgal acsB Awo C10 / XY C38 in M9 liquid medium with Chloramphenicol, respectively 2x
- Sequencing of pMTL83151_pBgal acsB Awo C10 with Primer 13 + pMTL83151_pBgal acsB XY C38 with Primer 11 + pMTL83151_pthlA acsB C5i Awo C7 with pMTL83151_pol_fwd Primer
- Test-digest of pMTL83151_pthlA acsB C5i Awo C1 for 45 min @37 °C + 5 min @ 65 °C with NheI&XhoI
- Gel electrophoresis for verification of test-digest of of pMTL83151_pthlA acsB C5i Awo C1
- Digest of all pMTL83151_pthlA acsB C5i Awo/XY clones over night @RT + 5 min @ 65 °C with NheI&XhoI
- Digest of pMTL83151_pthlA acsB C5i Awo C7 over night @RT + 5 min @ 65 °C with BamHI

14.07.
- Preparation of cryo stock of pMTL83151_pBgal acsB Awo C10 / XY C38
- Plate pMTL83151_pBgal acsB Awo C10 / XY C38 over night culture on M9+IPTG+X-gal+Chloramphenicol agarplates
- Inoculation of XY in XY medium
- Gel electrophoresis for verification of over night digest
- Inoculation of pMTL83151_pthlA acsB C5i Awo C7 in M9 medium with Chloramphenicol
- Inoculation of pMTL83151_pBgal acsB Awo C10 / XY C38 in M9 liquid medium with Chloramphenicol

Week 21 (18.07.-24.07.)
18.07.
- Gradient PCR for amplification of 6/5 kB unit of XY&Awo according to Fusion protocol (50µL)
Primer: XY 14+20
Awo 12+19
Template: Awo & XY gDNA 20ng/µL
Elongation time: 3min 30s
Annealing temperatures: 11 samples between 51.3 – 70.7 °C
🡪 PCR stopped after first 98 °C step!
- Re-transformation of pMTL83161_ptA-Ack-gusA in chemocompetent E.coli XL1-blue MRF’Kan (100ng plasmid used). Plating on M9+IPTG+X-gal-Chloramphenicol plate.
- Inoculation of pMTL83151_pthlA acsB C5i from cryo in M9 liquid medium with Chloramphenicol

19.07.
- Preparation of XY medium without fructose
- Gel electrophoresis for verification of 6/5kb unit amplification 🡪 no PCR products
- Gradient PCR for amplification of 6/5 kB unit of XY&Awo according to Fusion protocol (50µL)
Primer: XY 14+20
Awo 12+19
Template: Awo & XY gDNA 20ng/µL
Elongation time: 3min 30s
Annealing temperatures: 11 samples between 51.3 – 70.7 °C
- Plasmid Prep pMTL83151_pBgal acsB Awo C10 / XY C38 (14.07.) + pMTL83151_pthlA acsB C5i (18.07.) with Qiagen QIAprep Spin mini Prep Kit according to protocol

20.07.
- Check of growth: small colonies for re-transformation of pMTL83161_ptA-Ack-gusA 🡪 further incubated
- Gel electrophoresis for verification of 6/5kb unit amplification
- Digest of pMTL83151_pthlA acsB C5i with Sma1 and Xho1 for 45 min @30 °C + 5 min @ 65 °C

21.07.
- Gel electrophoresis for verification of digest of pMTL83151_pthlA acsB C5i
- Inoculation of pMTL83151_pthlA acsB C5i, pMTL83151_pBgal acsB Awo C10 / XY C38 in M9 liquid medium with Chloramphenicol

Week 22 (25.07.-31.07.)
25.07.
- Digest of pMTL83151_pBgal acsB Awo C10 / XY C38 with Sma1 and Xho1 for 1x 1.5h + 1x 2h @30 °C + 5 min @ 65 °C
- Plasmid Prep of pMTL83151_pBgal acsB Awo C10 / XY C38
- Check of growth: colonies for re-transformation of pMTL83161_ptA-Ack-gusA were grown enough
- Inoculation of 20 colonies pMTL83161_ptA-Ack-gusA in M9 liquid medium with Chloramphenicol

26.07.
- Preparation of M9 medium
- Gel electrophoresis for verification of digest of pMTL83151_pBgal acsB Awo C10 / XY C38

27.07.
- gDNA isolation of received Awo_t_45 cell pellet
- Inoculation of Cba from Cryo in XY medium
- Preparation of Cryo-stock from of pMTL83151_pthlA acsB C5i
- 5 µl of M9 liquid culture of 14 pMTL83161_ptA-Ack-gusA clones were dropped onto M9+IPTG+X-gal-Chloramphenicol plate
- Plasmid Prep of all 14 pMTL83161_ptA-Ack-gusA clones that grew (the 6 other clones were further incubated)
- Test-digest of 14 pMTL83161_ptA-Ack-gusA clones with Sma1 and Xho1 for 30 min @37 °C + 5 min @ 65 °C
- Gel electrophoresis for verification of test-digest of 14 pMTL83161_ptA-Ack-gusA clones

28.07.
- Check of growth: The remaining 6 further pMTL83161_ptA-Ack-gusA clones grew and were stored in fridge
Cba in XY medium (27.07.) only show slight growth 🡪 further incubated
- Touch-up PCR for amplification of 6/5 kB unit of XY&Awo according to Fusion protocol (50µL)
Primer: XY 14+20
Awo 12+19
Template: Awo & XY gDNA 20ng/µL
Elongation time: 3min 30s
Annealing temperatures: 51 – 72 °C, 0.7 °C increase per cycle (30 cycles total)
- Gel electrophoresis for verification of 6/5kb unit amplification by touch-up PCR

Week 23 (01.08.-07.08.)
01.08.
- Plasmid Prep of the remaining 6 pMTL83161_ptA-Ack-gusA clones
- Repetition: Gel electrophoresis for verification of test-digest of 14 pMTL83161_ptA-Ack-gusA clones (27.07.)
- PCR for amplification of 6/5 kB unit of XY&Awo according to Fusion protocol (50µL)
Primer: Awo 12+19
Template: Awo gDNA 20ng/µL
Elongation time: 3min 30s
Annealing temperature: 57.4 °C
- Touch-down PCR for amplification of 6/5 kB unit of XY&Awo according to Fusion protocol (50µL)
Primer: XY 14+20
Awo 12+19
Template: Awo & XY gDNA 20ng/µL
Elongation time: 3min 30s
Annealing temperatures: 51 – 72 °C, 0.7 °C decrease per cycle (30 cycles total
) - Gel electrophoresis for verification of 6/5 kb unit of XY&Awo touch-down PCR for amplification
- Test-digest of 3 pMTL83161_ptA-Ack-gusA clones (9,11,16) with Nhe1 and Xho1 for 30 min @37 °C + 5 min @ 65 °C
- Digest of pMTL83151_pBgal acsB Awo C10 / XY C38 with Sma1 and Xho1 for 30 min @37 °C + 5 min @ 65 °C (for change of Promoter!)
- Gel electrophoresis for verification of test-digest of 3 pMTL83161_ptA-Ack-gusA clones
- Gel electrophoresis for verification of Digest of pMTL83151_pBgal acsB Awo C10 / XY C38

02.08.
- Inoculation of XY and Awo in XY medium
- Inoculation of 2x pMTL83151_pBgal acsB Awo C10 / XY C38 in M9 liquid medium with Chloramphenicol
- Touch-down PCR for amplification of 5 kB unit of Awo according to Fusion protocol (50µL)
Primer: Awo 12+19
Template: Awo gDNA 20ng/µL
Elongation time: 3min 30s
Annealing temperatures: 51 – 72 °C, 0.7 °C decrease per cycle (35 cycles total)
- Gel electrophoresis for verification of 5 kb unit of Awo touch-down PCR for amplification
- PCR clean-up of 5 kb unit of Awo touch-down PCR
- Digest of pMTL83151_pBgal acsB Awo C10 / XY C38 with Sma1 and Xho1 for 30 min @37 °C + 5 min @ 65 °C (for change of Promoter!)

03.08.
- Preparation of anaerobic Na2S
- Preparation of anaerobic vitamin solution

04.08.
- Plasmid Prep of pMTL83151_pBgal acsB Awo C10 / XY C38
- Preparation of CGM medium

05.08.
- PCR for amplification of pfdx and pthlA promoter according to Fusion protocol (50µL)
Primer: pfdx 55+56
pthlA 67+58
Template: 1 µl (10 ng/µl) of the corresponding received pure promoter
Elongation time: 15 s
Annealing temperature: pfdx 58.4 °C
pthlA 54.1 °C
- Gel electrophoresis for verification of amplification of pfdx and pthlA promoter
- Inoculation of Cba in XY and CGM medium

Week 24 (08.08.-14.08.)
08.08.
- PCR clean-up of pfdx and pthlA promoter PCR
- Digest of pfdx and pthlA promoter with Sma1 and Xho1 for 45 min @37 °C + 5 min @ 65 °C

09.08.
- Inactivation of ligation samples (10 min @ 65°C)
- Transformation of chemocompetent E. coli XL1-blue MRF-tet with pMTL83151_pfdx/pthlA acsB Awo C10 / XY C38 ligation samples. Plating on M9+IPTG+X-gal+Chloramphenicol
- Inoculation from Cryo of Cba, XY and Awo in XY medium + Cba in CGM

10.08.
- Preparation of TYA and CGM medium
- Shifting of Cba (from 09.08.) in fresh CGM medium
- Touch-down PCR for amplification of 5 kB unit of Awo according to Fusion protocol (50µL); 8 tubes à 50 µL prepared
Primer: Awo 12+19
Template: Awo gDNA 20ng/µL
Elongation time: 3min 30s
Annealing temperatures: 51 – 72 °C, 0.7 °C decrease per cycle (35 cycles total)
- Inoculation of Copy Cutter E. coli in LB medium without antibiotic

11.08.
- Preparation of EPB S- and EPB NS- Buffer
- Preparation of Cryo-stock of Copy Cutter E. coli
- Sequencing of pMTL83151_PbgaL acsB Awo C10 / XY C38 with PbgaL_S1_f and PbgaL_S2_f primers

12.08.
- Shifting of Cba (from 10.08.) in fresh CGM medium
- Check of growth: pMTL83151_pfdx/pthlA acsB Awo C10 / XY C38 Transformation so far no growth 🡪 further incubated
Week 25 (15.08.-21.08.)

15.08.
- Gel electrophoresis and Gel-Ex of 5 kB unit of Awo from touch-down PCR
- Inoculation of: Cba in CGM
pMTL83151_PbgaL acsB Awo C10 in M9 with chloramphenicol
Copy Cutter E. coli in 5, 10 and 100 ml LB medium

16.08.
- Chemocompetent Copy Cutter E. coli were prepared
- Shifting of Cba in fresh CGM medium
- Gel-electrophoresis for determination of concentration of 5 kB unit of Awo after Gel-Ex

17.08.
- Transformation-Efficiency Test of chemocompetent Copy Cutter E. coli with Puc19
- Transformation of chemocompetent Copy Cutter E. coli with pMTL83151_pfdx/pthlA acsB Awo C10 / XY C38 ligation samples. Plating on LB+IPTG+X-gal+Chloramphenicol
- Plasmid Prep of pMTL83151_PbgaL acsB Awo C10
- Inoculation of Cba from Cryo in CGM medium
- Preparation of Thiamphenicol Stock
- Preparation of CGM agar plates supplemented with Thiamphenicol

18.08.
- Check of growth: No colonies on the transformation plates from 17.08. grew
- Repetition of: Transformation of chemocompetent Copy Cutter E. coli with pMTL83151_pfdx/pthlA acsB Awo C10 / XY C38 ligation samples. Plating on LB+IPTG+X-gal+Chloramphenicol
- Shifting of Cba in fresh CGM and TYA medium
- Touch-down PCR for amplification of 5 kB unit of Awo according to Fusion protocol (50µL); 8 tubes à 50 µL prepared
Primer: Awo 47+50
Template: Awo gDNA 20ng/µL
Elongation time: 3min 30s
Annealing temperatures: 51 – 72 °C, 0.7 °C decrease per cycle (35 cycles total)
- Sequencing of pMTL83151_PbgaL acsB Awo C10 / XY C38
Primer: Awo: 37, 38, 17 61 60 26
XY: 30, 39, 31, 61

19.08.
- Shifting of Cba in fresh TYA and CGM medium
- Gel electrophoresis for verification of amplification of 5 kB unit of Awo
- Transformation-Efficiency Test of chemocompetent Copy Cutter E. coli with Puc19 + with and without added induction solution

21.08.
- Inoculation of Copy Cutter E. coli in SOB medium

Week 26 (22.08.-28.08.)
22.08.
- Inoculation of Awo and XY from Cryo in CGM and TYA medium
- Plating of Cba on CGM plates
- Preparation of Ampicillin Stock
- Preparation of LB plates with Ampicillin
- Touch-down PCR for amplification of 5 kB unit of Awo according to Fusion protocol (50µL); 6 tubes à 50 µL prepared
Primer: Awo 12+19
Template: Awo gDNA 20ng/µL
Elongation time: 3min 30s
Annealing temperatures: 51 – 72 °C, 0.7 °C decrease per cycle (35 cycles total)
- Gel electrophoresis and Gel-Ex of 5 kB unit of Awo from touch-down PCR
- Inoculation of Copy Cutter E. coli in SOB medium
- Preparation of TE buffer

23.08.
- Chemocompetent Copy Cutter E. coli were prepared
- Transformation-Efficiency Test of chemocompetent Copy Cutter E. coli with Puc19
- Transformation of chemocompetent Copy Cutter E. coli with pMTL83151_PbgaL acsB Awo C10 / XY C38 and pMTL83151_pthlA-gusA C5i. Plating on LB+IPTG+X-gal+Chloramphenicol
- Touch-down PCR for amplification of 5 kB unit of Awo according to Fusion protocol (50µL); 6 tubes à 50 µL prepared
Primer: Awo 12+19
Template: Awo gDNA 20ng/µL
Elongation time: 3min 30s
Annealing temperatures: 51 – 72 °C, 0.7 °C decrease per cycle (35 cycles total)
- Gel electrophoresis and Gel-Ex of 5 kB unit of Awo from touch-down PCR
- Preparation of XY medium
- Shifting of Cba in fresh TYA and CGM medium

24.08.
- Check of growth: Cba on CGM plate grew
- Preparation of Na2S
- Nested Touch-down PCR for amplification of 5 kB unit of Awo according to Fusion protocol (50µL)
Primer: Awo 12+19
Template: 1.1 ng, 2.2 ng, 11 ng, 22 ng of 5 kB unit of Awo from Gel-Ex (23.08.)
Elongation time: 3min 30s
Annealing temperatures: 51 – 72 °C, 0.7 °C decrease per cycle (35 cycles total)
- Gel electrophoresis for verification of amplification of 5 kB unit of Awo by nested Touch-down PCR
- Shifting of Cba in fresh TYA and CGM medium

25.08.
- Plasmid Prep of Puc19 by obtained E. coli culture
- Transformation-Efficiency Test of chemocompetent Copy Cutter E. coli with Puc19 + with and without induction solution
- Inoculation of XY and Awo from Cryo in XY medium
- Nested Touch-down PCR for amplification of 5 kB unit of Awo according to Fusion protocol (50µL)
Primer: Awo 12+19
Template: 1:1, 1:100, 1:1.000, 1:10.000 Dilutions of 5 kB unit of Awo from Gel-Ex (23.08.)
Elongation time: 3min 30s
Annealing temperatures: 51 – 72 °C, 0.7 °C decrease per cycle (35 cycles total)
- Digest of Insert (5 kB unit of Awo from Gel-Ex 23.08.) with Xho1 and Nhe1 for 45 min @37 °C + 5 min @ 65 °C
- Purification of digest of Insert 5 kB unit of Awo with Qiagen Min Elute PCR Purification Kit
- Shifting of Cba in fresh TYA and CGM medium
Week 27 (29.08.-04.09.)

29.08.
- Transformation of chemocompetent Copy Cutter E. coli with pMTL83151_pfdx/pthlA acsB Awo C10 / XY C38 (6 samples total). Plating on LB+IPTG+X-gal+Chloramphenicol
- Shifting of Cba in fresh TYA and CGM medium
- Touch-down PCR for amplification of 5 kB unit of Awo according to Fusion protocol (50µL)
Primer: Awo 12+19
Template: Awo gDNA 20ng/µL
Elongation time: 3min 30s
Annealing temperatures: 51 – 72 °C, 0.7 °C decrease per cycle (35 cycles total)
- Gel electrophoresis verification of amplification of 5 kB unit of Awo from touch-down PCR
- Inoculation of Cba from Cryo in CGM

30.08.
- Digest of pMTL83151_PbgaL acsB Awo C10 with Xho1 and Nhe1 for 45 min @37 °C + 5 min @ 65 °C
- Purification of digest of pMTL83151_PbgaL acsB Awo C10 with Qiagen Min Elute PCR Purification Kit
- Ligation of 5 kB unit of Awo insert with pMTL83151_PbgaL acsB Awo C10 backbone over night @RT in dark
- Gel electrophoresis of Awo_t_45 gDNA to check for possible shearing/degradation
- Shifting of Cba in fresh TYA and CGM medium

31.08.
- Transformation of chemocompetent E. coli XL1-blue MRF-tet with pMTL83151_PbgaL 5kB Awo. Plating on M9+IPTG+X-gal+Chloramphenicol
- Repeat: Ligation of pfdx/pthlA promoter with pMTL83151_PbgaL acsB Awo C10 / XY C38 backbone over night @RT in dark
- Touch-down PCR for amplification of 5 kB unit of Awo according to Fusion protocol (50µL)
Primer: Awo 12+19
Template: Awo gDNA 20ng/µL
Elongation time: 3min 30s
Annealing temperatures: 51 – 72 °C, 0.7 °C decrease per cycle (35 cycles total)
- Gel electrophoresis verification of amplification of 5 kB unit of Awo from touch-down PCR
- Shifting of Cba in fresh TYA and CGM medium

01.09.
- Shifting of Cba in fresh TYA and CGM medium
- Transformation of chemocompetent Copy Cutter E. coli with pMTL83151_pfdx/pthlA acsB Awo C10 / XY C38 (6 samples total). Plating on LB+IPTG+X-gal+Chloramphenicol
- Nested Touch-down PCR for amplification of 5 kB unit of Awo according to Fusion protocol (50µL)
Primer: Awo 12+19
Template: received 5 kB unit (Mechthild)
Elongation time: 3min 30s
Annealing temperatures: 51 – 72 °C, 0.7 °C decrease per cycle (35 cycles total)
- Gel electrophoresis and Gel-Ex of 5 kB unit of Awo from Mechthild’s PCR
- Digest of pMTL83151_PbgaL acsB Awo C10 and 5 kB unit with Nhe1 and Xho1 for 45 min @37 °C + 5 min @ 65 °C

02.09.
- Purification of digest of pMTL83151_PbgaL acsB Awo C10 and 5 kB unit with Qiagen Min Elute PCR Purification Kit
- Ligation of 5 kB unit of Awo insert with pMTL83151_PbgaL acsB Awo C10 backbone for 4h @RT in dark
- Transformation of chemocompetent Copy Cutter E. coli with pMTL83151_PbgaL 5kB Awo. Plating on LB+IPTG+X-gal+Chloramphenicol
- Gel electrophoresis for verification of correct Gel-Ex of 5 kB unit of Awo
- Preparation of 2x Cryo-stock of Cba
- Shifting of Cba in Fresh TYA and CGM medium
Week 28 (05.09.-11.09.)

05.09.
- Inoculation of colonies from pMTL83151_PbgaL 5kB Awo transformation in E. coli XL1-blue MRF-tet (31.08.) in LB with Chloramphenicol
- Inoculation of Copy Cutter E. coli in LB
- Inoculation from Cryo of pMTL83151_PbgaL acsB Awo C10 / XY C38
- Preparation of Glycerol and LB medium and LB plates
- Digest of Insert (acsB XY/Awo) + digest of pMTL83151_pthlA-gusA C5i with Xho1 and Nhe1 for 45 min @37 °C + 5 min @ 65 °C
- Purification of digest with Qiagen Min Elute PCR Purification Kit
- Ligation of acsB XY/Awo with pMTL83151_pthlA-gusA C5i backbone over night @RT in dark
- Shifting of Cba in fresh TYA and CGM medium

06.09.
- Preparation of Cryo-stock of Copy Cutter E. coli
- Shifting of Cba in fresh TYA and CGM medium
-Transformation of chemocompetent Copy Cutter E. coli with pMTL83151_pthlA acsB XY/Awo. Plating on LB+IPTG+X-gal+Chloramphenicol
- Plasmid Prep of pMTL83151_PbgaL acsB Awo C10 / XY C38 and pMTL83151_PbgaL 5kB Awo clones
- Test-digest of pMTL83151_PbgaL 5kB Awo clones with Nhe1 and Xho1 for 45 min @37 °C + 5 min @ 65 °C
- Digest of pMTL83151_pthlA-gusA C5i with Xho1 and Nhe1 for 45 min @37 °C + 5 min @ 65 °C
- Purification of digest with Qiagen Min Elute PCR Purification Kit
- Ligation of acsB XY/Awo with pMTL83151_pthlA-gusA C5i backbone over night @RT in dark
- Gel electrophoresis for verification of test-digest of pMTL83151_pthlA acsB XY/Awo
- Inoculation of Cba from Cryo in TYA and CGM medium

07.09.
- Shifting of Cba in fresh TYA and CGM medium
- Transformation of chemocompetent Copy Cutter E. coli with pMTL83151_pthlA acsB XY/Awo. Plating on LB+IPTG+X-gal+Chloramphenicol
- Plasmid Prep of pMTL83151_PbgaL 5kB Awo clones from transformation in E. coli XL1-blue MRF-tet
- Gel electrophoresis to check for plasmid sizes of pMTL83151_PbgaL 5kB Awo clones
- Test-digest of pMTL83151_PbgaL 5kB Awo clones O&T with Nhe1 and Xho1 for 45 min @37 °C + 5 min @ 65 °C
- Gel electrophoresis for verification of test-digest of pMTL83151_PbgaL 5kB Awo clones O&T
- Inoculation of pMTL83151_PbgaL 5kB Awo O, U and V
- Inoculation of pMTL83151 empty vector from cryo in LB with Ampicillin

08.09
- Transformation of Cba with pMTL83151_PbgaL acsB Awo C10 / XY C38. Plating on CGM+Thiamphenicol
- Plasmid Prep of pMTL83151_PbgaL 5kB Awo O, u and V
- Inoculation of Cba in CGM
- Sequencing of pMTL83151_PbgaL 5kB Awo O with 23-29, 40-44, 37,38,17,59-61 primers

09.09.
- Transformation of Cba with pMTL83151_PbgaL acsB Awo C10 / XY C38. Plating on CGM+Thiamphenicol
- Test-digest of pMTL83151_PbgaL 5kB Awo clones U&V with Nhe1 and Xho1 for 45 min @37 °C + 5 min @ 65 °C
- Gel electrophoresis for verification of test-digest of pMTL83151_PbgaL 5kB Awo clones U&V
- Inoculation of pMTL83151 empty vector and pMTL83151_pthlA acsB Awo in LB

Week 29 (12.09.-18.09.)
12.09.
- Plasmid Prep of pMTL83151 empty vector and pMTL83151_pthlA acsB Awo
- Digest of pMTL83151 empty vector with Xho1 and Sma1 for 45 min @37 °C + 5 min @ 65 °C
- Test-digest of pMTL83151_pthlA acsB XY clone A,B,C with Nhe1 and Xho1 for 45 min @37 °C + 5 min @ 65 °C
- Test-digest of pMTL83151_PbgaL 5kB Awo with Sal1 for 45 min @37 °C + 5 min @ 65 °C
- Gel electrophoresis for verification of test-digest of pMTL83151_pthlA acsB XY clone A,B,C and pMTL83151_PbgaL 5kB Awo
- Ligation of pfdx/pthlA promoter with pMTL83151 empty vector over night @RT in dark
- Digest of 5 kB unit of Awo and pMTL83151 empty vector with Nhe1 and Sal1 for 45 min @37 °C + 5 min @ 65 °C
- Inoculation of pMTL83151 and pMTL84151 in LB
- Inoculation of Cba pMTL83151_PbgaL acsB Awo C10 / XY C38 clones in CGM with Thiamphenicol
- Preparation of XY Cryo-stock
- Sequencing of pMTL83151_pthlA acsB Awo C with 15 and primers
- Shifting of Cba in fresh TYA and CGM medium

13.09.
- Preparation of Cryo-stock of pMTL83151_pthlA acsB XY A,B,C and Cba
- Preparation of CGM and YTF medium
- Inoculation of Cba on CGM plate
- Spotting of pMTL83151_pthlA acsB XY A,B,C on LB with Chloramphenicol
- Plasmid Prep of Cba pMTL83151_PbgaL acsB Awo Ca-i and Cba pMTL83151_PbgaL acsB XY 1-10
-Transformation of chemocompetent Copy Cutter E. coli with pMTL83151_pfdx/pthlA. Plating on LB+IPTG+X-gal+Chloramphenicol
- Test-digest of Cba pMTL83151_PbgaL acsB Awo Ca-i and Cba pMTL83151_PbgaL acsB XY 1-10
with Xho1 and Nhe1 for 45 min @37 °C + 5 min @ 65 °C
- Gel electrophoresis for verification of test-digest of Cba pMTL83151_PbgaL acsB Awo Ca-i and Cba pMTL83151_PbgaL acsB XY 1-10
- Shifting of Cba in fresh TYA and CGM medium

14.09.
- Test-digest of Cba pMTL83151_PbgaL acsB Awo Ca-b and Cba pMTL83151_PbgaL acsB XY 1
with Xho1 and Nhe1 for 50 min @37 °C + 5 min @ 65 °C
- Gel electrophoresis for verification of test-digest of Cba pMTL83151_PbgaL acsB Awo Ca-b and Cba pMTL83151_PbgaL acsB XY 1
- Purification of digest (12.09.) with Qiagen Min Elute PCR Purification Kit
- Ligation of 5 kB unit of Awo with pMTL83151_PbgaL acsB Awo C10 backbone over night @RT in dark
- Shifting of Cba in fresh TYA and CGM medium
- Inoculation of XY in YTF

15.09.
- Shifting of Cba in fresh TYA and CGM medium
- Transformation of chemocompetent Copy Cutter E. coli with pMTL83151_PbgaL 5kB Awo. Plating on LB+IPTG+X-gal+Chloramphenicol
- Plasmid Prep of pMTL83151_pthlA acsB XY A,B,C
- Inoculation of pMTL83151_pthlA acsB XY A,B,C in Lb with chloramphenicol
- Inoculation of Awo from Cryo in YTF
- Inoculatio of Cba from Cryo in CGM

16.09.
- Transformation of Cba with pMTL83151_pthlA acsB XY A,B,C. Plating on CGM+Thiamphenicol
- Shifting of Cba in fresh TYA and CGM medium
- Inoculation of pMTL83151_pthlA acsB XY A,B,C in LB with induction solution and Chloramphenicol
Week 30 (19.09.-25.09.)

19.09.
- Test-digest of induced pMTL83151_pthlA acsB XY A,B,C with Xho1 and Nhe1 for 50 min @37 °C + 5 min @ 65 °C
- Gel electrophoresis for verification of test-digest of induced pMTL83151_pthlA acsB XY A,B,C
- Digest of pfdx, pthlA, pMTL83151_pthlA acsB XY C, pMTL83151_PbgaL acsB Awo C10 with Xho1 and Sma1 for 45 min @37 °C + 5 min @ 65 °C
- Ligation of pfdx with pMTL82151, pMTL84151, pMTL83151_PbgaL acsB XY C38 backbone over night @RT in dark
- Plasmid Prep of pMTL83151_pthlA acsB XY A,B,C

20.09.
- Plasmid Prep of 7 Cba pMTL83151_pthlA acsB XY clones
- Test-digest of 7 Cba pMTL83151_pthlA acsB XY clones with Xho1 and Nhe1 for 45 min @37 °C + 5 min @ 65 °C
- Gel electrophoresis for verification of test-digest of 7 Cba pMTL83151_pthlA acsB XY clones

22.09.
- Test- digest of pMTL82151 empty vector with Xho1 and Sal1 for 45 min @37 °C + 5 min @ 65 °C
- Gel electrophoresis for verification of test- digest of pMTL82151 empty vector
- Preparation of YTF plates
- Inoculation of Cba pMTL83151_pthlA acsB XY C1,C6,CA,CB,B1.1,C2.2,C2.3,C1.4-7 in CGM medium
- Inoculation of Cba pMTL83151_PbgaL acsB Awo Ca-b and Cba pMTL83151_PbgaL acsB XY C1&C6 in CGM and XY medium

23.09.
- Ligation of pfdx promoter with pMTL83151_PbgaL acsB Awo XY C38, pMTL83151_pthlA acsB XY, pMTL82151, pMTL84151 backbones for 4h @RT in dark
- Transformation of chemocompetent Copy Cutter E. coli with pMTL83151_pfdx acsB Awo XY C38, pMTL83151_pfdx acsB XY , pMTL82151_pfdx, pMTL84151_pfdx. Plating on LB+IPTG+X-gal+Chloramphenicol
- Digest of 5 kB unit of Awo, pMTL83151_pta-ack _gusA and pMTL83151_PbgaL acsB Awo XY C38 with Sal1 and Nhe1 for 45 min @37 °C + 5 min @ 65 °C
- Plasmid Prep of 7 Cba pMTL83151_pthlA acsB XY clones
- Gel electrophoresis for visualizing 7 Cba pMTL83151_pthlA acsB XY clones plasmids
- Preparation of Cryo-stocks of Cba pMTL83151_PbgaL acsB XY C1&6
- Ligation of 5 kB unit of Awo with pMTL83151_PbgaL acsB XY C38 backbone over night @RT in dark

Week 31 (26.09.-02.10.)
26.09.
- Transformation of chemocompetent Copy Cutter E. coli with pMTL83151_PbgaL 5 kB Awo ligation. Plating on LB+IPTG+X-gal+Chloramphenicol
- Ligation of 5 kB unit of Awo with pMTL83151_PbgaL acsB XY C38 backbone over night @RT in dark
- Preparation of XY medium
- PCR for amplification of acsB gene of XY&Awo according to Fusion protocol (50µL) with new Primers
Primer: XY 13+14
Awo 11+12
Template: Awo & XY gDNA 20ng/µL
Elongation time: 1min 10s
Annealing temperature: 52.4 °C
- Sequencing of Cba pMTL83151_PbgaL acsB XY C1, Cba pMTL83151_PbgaL acsB Awo Cb and Cba pMTL83151_pthlA acsB XY C1.6 with 15 and 61 primers
- Inoculation of Cba from Cryo in CGM
- Plating of XY on YTF plate
- Preparing 0.5 M lactose

27.09.
- Gel electrophoresis for acsB PCR verification
- PCR for amplification of acsB gene of XY&Awo according to Fusion protocol (50µL) with new Primers
Primer: XY 13+14
Awo 11+12
Template: Awo & XY gDNA 20ng/µL
Elongation time: 1min 10s
Annealing temperature: 52.4 °C
- PCR clean-up of acsB amplification
- Sequencing of Cba pMTL83151_PbgaL acsB Awo Cb and Cba pMTL83151_pthlA acsB XY C1.6 with 60 and 61 primers
- Dialysis of ligation samples (pMTL83151_PbgaL 5 kB Awo, pMTL83151_pfdx acsB XY C38, pMTL83151_pthlA acsB XY, pMTL82151_pfdx; pMTL84151_pfdx) for 30 min
- Inoculation of Cba from Cryo in CGM
- Inoculation from Cryo of Cba pMTL83151_PbgaL acsB Awo Ca, Cba pMTL83151_PbgaL acsB XY C6 and Cba pMTL83151_pthlA acsB XY C1.6 in CGM and XY medium with Thiamphenicol
- Inoculation from Cryo of Cba pMTL83151_PbgaL acsB Awo Ca and Cba pMTL83151_PbgaL acsB XY C6 in CGM medium with 20mM lactose and Thiamphenicol

28.09.
- Transformation of Cba with pMTL83151_PbgaL 5 kB Awo, pMTL83151_pfdx acsB XY C38, pMTL83151_pthlA acsB XY, pMTL82151_pfdx; pMTL84151_pfdx direct dialysed ligation samples. Plating on CGM+Thiamphenicol
- Plating of XY on YTF medium
- Preparation of L-Apsaragin

29.09.
- Shifting of Cba pMTL83151_PbgaL acsB Awo Ca, Cba pMTL83151_PbgaL acsB XY C6 and Cba pMTL83151_pthlA acsB XY C1.6 in CGM and XY medium with Thiamphenicol + Cba pMTL83151_PbgaL acsB Awo Ca and Cba pMTL83151_PbgaL acsB XY C6 in CGM medium with 20mM lactose and Thiamphenicol cultures in fresh medium

30.09.
- Measuring of Growth curves of Cba pMTL83151_PbgaL acsB Awo Ca, Cba pMTL83151_PbgaL acsB XY C6 and Cba pMTL83151_pthlA acsB XY C1.6 in CGM and XY medium with Thiamphenicol + Cba pMTL83151_PbgaL acsB Awo Ca and Cba pMTL83151_PbgaL acsB XY C6 in CGM medium with 20mM lactose and Thiamphenicol cultures
- Inoculation of Cba pMTL83151_PbgaL acsB Awo Ca, Cba pMTL83151_PbgaL acsB XY C6 and Cba pMTL83151_pthlA acsB XY C1.6 in XY medium without fructose for growth on CO2 and H2
- Preparation of CGM medium
Week 32 (03.10.-09.10.)

04.10.
- Preparation of EPB S and EPB NS buffer
- 2x Digest of Insert (5 kB unit of Awo from Gel-Ex 01.09.) with Salö1 and Nhe1 for 45 min @37 °C + 5 min @ 65 °C
- Inoculation from Cryo of Cba pMTL83151_PbgaL acsB Awo Ca, Cba pMTL83151_PbgaL acsB XY C6 and Cba pMTL83151_pthlA acsB XY C1.6 in CGM with Thiamphenicol
- Inoculation from Cryo of Cba pMTL83151_PbgaL acsB Awo Ca and Cba pMTL83151_PbgaL acsB XY C6 in CGM medium with 20mM lactose and Thiamphenicol
- Ligation of 5 kB unit of Awo with pMTL82151 empty vector backbone over night @RT in dark

05.10.
- Preparation of CGM and XY medium
- Preparation of 40 % glucose
- Transformation of chemocompetent Copy Cutter E. coli with pMTL82151_5 kB Awo ligation. Plating on LB/M9+IPTG+X-gal+Chloramphenicol
- Measuring of Growth curves of Cba pMTL83151_PbgaL acsB Awo Ca, Cba pMTL83151_PbgaL acsB XY C6 and Cba pMTL83151_pthlA acsB XY C1.6 in CGM and XY medium with Thiamphenicol + Cba pMTL83151_PbgaL acsB Awo Ca and Cba pMTL83151_PbgaL acsB XY C6 in CGM medium with 20mM lactose and Thiamphenicol cultures

06.10.
- Measuring of Growth curves of Cba pMTL83151_PbgaL acsB Awo Ca, Cba pMTL83151_PbgaL acsB XY C6 and Cba pMTL83151_pthlA acsB XY C1.6 in CGM and XY medium with Thiamphenicol + Cba pMTL83151_PbgaL acsB Awo Ca and Cba pMTL83151_PbgaL acsB XY C6 in CGM medium with 20mM lactose and Thiamphenicol cultures
- Preparation of CGM medium

07.10.
- Measuring of Growth curves of Cba pMTL83151_PbgaL acsB Awo Ca, Cba pMTL83151_PbgaL acsB XY C6 and Cba pMTL83151_pthlA acsB XY C1.6 in CGM and XY medium with Thiamphenicol + Cba pMTL83151_PbgaL acsB Awo Ca and Cba pMTL83151_PbgaL acsB XY C6 in CGM medium with 20mM lactose and Thiamphenicol cultures
- Transformation of chemocompetent Copy Cutter E. coli and E. coli XL1-blue MRF-tet with pUCIDT-AMP_acsD-acsC-acsE. Plating on LB/M9+IPTG+X-gal+Chloramphenicol
- Plasmid Prep of Cba pMTL83151_PbgaL acsB Awo Ca and Cba pMTL83151_PbgaL acsB XY C6
- Sequencing of Cba pMTL83151_PbgaL acsB Awo Ca with 17, 23-29, 37, 38, 44, 59 primers
- Sequencing of Cba pMTL83151_PbgaL acsB XY C6 with 23-31, 39, 59 primers
- Sequencing of Cba pMTL83151_pthlA acsB XY C1.6 23-28, 30, 31, 39, 61 primers
- Inoculation of Cba pMTL83151_PbgaL acsB Awo Ca, Cba pMTL83151_PbgaL acsB XY C6 and Cba pMTL83151_pthlA acsB XY C1.6 in XY medium without fructose for growth on CO2 and H2

Notebook

Introduction

Overview