iGEM 2022 — Team goettingen

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## Notebook

Notebook

##### Introduction

Following are the abbreviations for the organisms that were used during our project:

*Clostridium saccharoperbutylacetonicum* ATCC 27021 = Cba
*Clostridium beijerinckii* ATCC 51743 = C.bei A21
*Clostridium beijerinckii* NRRL B-593 = C.bei 8052
*Acetobacterium woodii* ATCC 29683 = Awo
*Clostridium ljungdahlii* ATCC 5538 = XY

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

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## Engineering

Introduction

##### Engineering success

We succeeded to construct the clostridial specific ClosTron plasmid pMTL83151 with the central gene acsB of the Wood-Ljungdahl pathway (WLP). The WLP enables acetogenic bacteria to assimilate CO2. AcsB was previously amplified from the acetogenic bacteria Clostridium ljungdahlii and Acetobacterium woodii. As our initial promoter was too strong, we switched to a lactose induced promoter (PbgaL) and could construct our vector with this promoter. By changing our recipient cells to low copy cells, we were able to compensate for the toxicity of the constitutively active thiolase promoter (PthlA) which we also originally intended to use. The constructed plasmids were successfully introduced into the high yield butanol producer Clostridium saccharoperbutylacetonicum. Furthermore, a 5 kb fragment consisting of the WLP genes ascB1, cooC2 and acsA was isolated from A. woodii for subsequent plasmid construction.

---

Engineering success

##### Overview and Planning

During our project, we faced multiple obstacles which we had to overcome and therefore have gone through several engineering cycles. We aimed to enable a clostridial butanol producer to utilize CO2 as metabolic substrate for biofuel production. For this purpose, a carbon dioxide assimilation pathway had to be introduced into these sugar oxidizing bacteria. The ancient Wood-Ljungdahl pathway (WLP), a conserved CO2, CO and H2 assimilating pathway in acetogenic bacteria[1], [2] was chosen for cloning as it is the most efficient pathway. The WLP does not generate energy, hence the bacteria using the WLP for carbon assimilation requires an additional energy source, an Ech or RNF complex[3], [4]. Therefore, our selection of host organisms was limited to such bacteria which are already harboring such energy sources. Recent publications indicate that some solventogenic bacteria (butanol-producing bacteria) harbor fragments of the required genes, e.g *Clostridium beijerinckii* (*C. beijerinckii*)[5]. However, these results were based only on genomic data without indication for functionality or expression of these genes. As reliable data for the proteome and cloning approaches in these bacteria are scarce in literature, we decided to clone first a minimal fragment into our chosen butanol producer, *Clostridium saccharoperbutylacetonicum* (*C. saccharoperbutylacetonicum*). For choosing the minimal fragment, we selected the acetyl-CoA synthase (acsB1) of the acetogene *Acetobacterium woodii* (*A. woodii*) and the same enzyme (acsB2) of *Clostridium ljungdahlii* (*C. ljungdahlii*). This central enzyme combines the carbonyl and methyl branches of the WLP and has pyruvate as a direct product. It is therefore absolutely necessary for a functional pathway. After functional assessment, in particular for CO2 assimilation, of *C. saccharoperbutylacetonicum*, we aimed to increase the cloning fragment to 5 kb, consisting of the genes ascB1, cooC2 and acsA of our donor organism A. woodii. These are the genes immediately upstream of acsB1. Since CO2 assimilation was also not possible as a result, this suggests that only genetic remnants of WLP without functional enzymes remain in these bacteria and that the entire metabolic pathway of *A. woodii* or *C. ljungdahlii* would have to be cloned. This poses another hurdle for our project, as all enzymes are organized in large clusters that would need to be separated from each other. Therefore, two plasmids would be required and the functional transcription units would have to be separated.

---

##### Plasmid Construction

For our vector construction to transfer the required CO2 assimilation genes into *C. saccharoperbutylacetonicum*, we used the Clostron plasmid system as it is the only well-established vector system for clostridia [6], [7]. We aimed to construct a plasmid (backbone pMTL83151) with the constitutively expressed thiolase promoter (pthlA) and our required genes of the Wood-Ljungdahl-pathway as insert (downstream of promoter). After common amplification of our target from the genome of *A. woodii* and *C. ljungdahlii* via PCR and numerous attempts of cloning the minimal fragment acsB into our plasmid backbone with the thiolase promoter, we succeeded. However, we figured out by sequencing that the thiolase promoter was heavily mutated in all our attempts. Due to the high number of identical cloning outcomes without succeeding a plasmid with intact thiolase promoter and acsB insert, we decided to change the promoter in our construct. As the thiolase promoter is one of the strongest promoters utilized in literature so far , we switched to a lactose induced promoter (pBgal). Therefore, if not induced, this promoter leads to low basal expression of the following insert. And finally, we succeeded to construct a plasmid with pBgal promoter and acsB1.

---

##### Plasmid Construction

We could conclude that one of the toxic parts in our construct is the promoter as we sequenced our vector system right after we retransformed it into *Escherichia coli* (*E. coli*). For this retransformation, the vector system consists only of the backbone and the thiolase promoter (without acsB1 insert). Even in our retransformed *E. colis*, the thiolase promoter was already mutated and functionless. Therefore, we summarized that not the insert, but the promoter must be a toxic part in the construct.

![35](https://static.igem.wiki/teams/4404/wiki/mutatedthiolasepromotor.png)

**Figure 1: Mutated thiolase promoter.** Sequencing of the original plasmid pMTL83151\_PthlA\_gusA after retransformation in *E. coli* XL-1 blue MRF. Light blue bands indicate coverage of aligned sequences, black bands indicate no coverage of aligned sequence. Mutations are visible in the thiolase promoter (PthlA). Analyzed in *Geneious Prime*.

---

##### Plasmid Construction

Nevertheless, the thiolase promoter would be ideal for our construct as it is constitutively expressed and acts as a strong promoter which is required to ensure a sufficient metabolic flux for the final organism. We therefore decided to change the competent *E. coli* cells in which we transform. Based on the protocol of the ClosTron plasmid construction we have used *E. coli* XL-1 blue MRF. This line is specialized on high efficiency cloning. We switched to *E. coli* Copy Cutter EPI 400, cells specialized on potential toxic genes or unstable DNA fragments by lowering the copy number. As the thiolase promoter was toxic for *E. coli* XL-1 blue MRF it seemed reasonable to us that cells specialized for toxic products can compensate for the promoter's toxicity. Ultimately, we succeeded to construct a plasmid pMTL83151 with the thiolase promoter and the acsB1 insert by using *E. coli* Copy Cutter EPI 400.

We subsequently managed to introduce all constructed plasmids in *C. saccharoperbutylacetonicum* and performed growth analysis. We verified our constructs by Sanger Sequencing.

![35](https://static.igem.wiki/teams/4404/wiki/engineeringfertigeplasmide.png)

**Figure 2: Plasmid constructions.** The plasmids we have successfully constructed: **A**: pMTL83151\_PbgaL\_acsB2\_CLJU, consisting of the gene *acsB2* from *C. ljungdahlii* as insert and the lactose inducible promoter (PbgaL). **B**: pMTL83151\_PbgaL\_acsB1\_Awo, consisting of the gene *acsB1* from *A. woodii* as insert and the lactose inducible promoter (PbgaL). **C**: pMTL83151\_PthlA\_acsB2\_CLJU, consisting of the gene *acsB2* from *C. ljungdahlii* as insert and the thiolase promoter. The insert is marked in green, in orange the promoter. Genes marked in blue are part of the backbone.

---

![35](https://static.igem.wiki/teams/4404/wiki/pcrawo5kb.png)

**Figure 3: Amplification of 5 kb fragment of** ***A. woodii*****.** 0.8 % agarose gel stained with ethidium bromide. A fragment of the size of 5 kb, consisting of the genes *ascB1*, *cooC2* and *acsA*, of *A. woodii* was amplified. 1-6: Varying PCR protocol (see notebook). In all PCR attempts, the desired fragment of a size of 5 kb was amplified. Additionally, a fragment was amplified with a size smaller than 250 bp. M: GeneRuler 1 kb DNA Ladder with known fragment sizes in base pairs (bp).

##### Isolation of 5 kb fragment of *A. woodii*

The minimal fragment was not sufficient for CO2 assimilation in *C. saccharoperbutylacetonicum*. Therefore, we attempted to construct a ClosTron plasmid pMTL83151 with the genes ascB1, cooC2 and acsA (5 kb fragment) of *A. woodii* as insert to check whether these genes are adequate for CO2 assimilation. However, PCR amplification of the 5 kb fragment, taking *A. woodii* gDNA as template was not successful. A smaller insert of 200 bp was always additionally amplified (even though no complementary structures of our primers were known in the entire *A. woodii* genome). We could conclude that the smaller insert is not the primer cloud usually visible after gel electrophoresis because after purification this smaller insert was still clearly visible. We additionally assumed that the smaller insert will have the required restriction sites due to our designed primers harboring a restriction site as overhang. Hence, the smaller insert would be digested and ligated in the same way as our desired 5 kb insert. Because during transformation, the host prefers to integrate plasmids of smaller sizes (therefore smaller inserts), removing the insert was required. After designing more specific primers and performing nested PCRs we still could not eliminate the smaller insert in our sample. After all attempts we came back to gel extraction. Before extraction of the 5 kb fragment directly from gel could not reach a sufficient amount of the PCR product for subsequent digestion, ligation and transformation. We adapted our PCR protocol to reach a sufficient amount of PCR product from the beginning. Additionally, we modified the protocol of gel extraction to finally extend the amount of PCR product. We ultimately had a sufficient quantity of the 5 kb PCR product to perform the cloning procedures.

We were challenged by many obstacles during our project. By modifying previously tested methods and overthinking and reviewing ideas we succeeded in coping with numerous problems and learnt how to deal with such obstacles

---

Engineering

##### Sources

[1] Ragsdale, S. W. (2008): Enzymology of the wood-Ljungdahl pathway of acetogenesis. In: Annals of the New York Academy of Sciences 1125, P. 129-136.

[2] Ragsdale, S. W.; Pierce, E. (2008): Acetogenesis and the Wood-Ljungdahl pathway of CO(2) fixation. In: Biochimica et biophysica acta 1784 (12), P. 1873-1898.

[3] Müller, V. (2003): Energy conservation in acetogenic bacteria. Appl Environ Microbiol. 69: 6345–6353

[4] Schuchmann, K.; Müller, V. (2014): Autotrophy at the thermodynamic limit of life: a model for energy conservation inacetogenic bacteria. Nat Rev Microbiol. 12: 809–821.

[5] Sandoval-Espinola, W. J.; Chinn, M. S.; Thon, M. R.; Bruno-Bárcena, J. M. (2017): Evidence of mixotrophic carbon-capture by n-butanol-producer Clostridium beijerinckii. In: Scientific reports 7 (1), P. 12759.

[6] Heap, J. T.; Pennington, O. J.; Cartman, S. T.; Carter, Glen P.; Minton, N. P. (2007): The ClosTron: a universal gene knock-out system for the genus Clostridium. In: Journal of microbial methods 70(3), P. 452-464.

[7] Heap, J. T.; Kuehne, S. A.; Ehsaan, M.; Cartman, S. T.; Cooksley, C. M.; Scott, J. C.; Minton, N. P. (2010): The ClosTron: Mutagenesis in Clostridium refined and streamlined. In: Journal of microbiological methods 80 (1), P. 49-55.

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## Experiments

These are the media we used

#####

Measure all components and dissolve them with approx. half of the total amount of water in a cylinder using a stir-fish. Prepare a Glucose stock solution (4%) in a separate cylinder. For 500 ml medium, use 50 ml of a 40% Glucose solution (= 20 g Glucose in 50 ml H2O).
Fill the medium solution up with demin. H2O (to 450 ml in case of 500 ml total, due to addition of Glucose later on) and pour the medium (for 500 ml) in a 1 l and the Glucose solution in a 500 ml bottle.
Gass them with N2 (2 cannulas, 30 min for 500 ml medium; 1 cannula, 20 min for 100 ml glucose).
Measure and note down the pH value of the medium, then gas it for another 1 min with N2 before closing the bottle with a plug and lid.
Autoclave both bottles at 121°C for 20 min.
Using a cannula, add 50 ml of a 40% Glucose solution to 500 ml medium.
Using a 5 ml syringe, take out 5 ml of the medium into a 15 ml falcon to measure the pH value. A pH value of ~ 6.60 is desired.

Clostridial growth medium (CGM):
![35](https://static.igem.wiki/teams/4404/wiki/cgmmedium.png)

Measure all components and dissolve them with approx. half of the total amount of water in a cylinder using a stir-fish. Prepare a Glucose stock solution (4%) in a separate cylinder. For 500 ml medium, use 50 ml of a 40% Glucose solution (= 20 g Glucose in 50 ml H2O).
Fill the medium solution up with demin. H2O (to 450 ml in case of 500 ml total, due to addition of Glucose later on) and pour the medium (for 500 ml) in a 1 l and the Glucose solution in a 500 ml bottle.
Gass them with N2 (2 cannulas, 30 min for 500 ml medium; 1 cannula, 20 min for 100 ml glucose).
Measure and note down the pH value of the medium, then gas it for another 1 min with N2 before closing the bottle with a plug and lid.
Autoclave both bottles at 121°C for 20 min.
Using a cannula, add 50 ml of a 40% Glucose solution to 500 ml medium.
Using a 5 ml syringe, take out 5 ml of the medium into a 15 ml falcon to measure the pH value. A pH value of ~ 6.60 is desired.

TYA medium:
![35](https://static.igem.wiki/teams/4404/wiki/tyamedium.png)

Measure all components and dissolve them with approx. half of the total amount of water in a cylinder using a stir-fish. Prepare a Glucose stock solution (40%) in a separate cylinder. For 500 ml medium, use 50 ml of a 40% Glucose solution (= 20 g Glucose in 50 ml H2O).
Fill the medium solution up with demin. H2O (to 450 ml in case of 500 ml total, due to addition of Glucose later on) and pour the medium (for 500 ml) in a 1 l and the Glucose solution in a 500 ml flask.
Gass them with N2 (2 cannulas, 30 min for 500 ml medium; 1 cannula, 20 min for 100 ml glucose).
Measure and note down the pH value of the medium, then gas it for another 1 min with N2 before closing the flask with a plug and lid.
Autoclave both flasks at 121°C for 20 min.
Using a syyringe, add 50 ml of a 40% Glucose solution to 500 ml medium.
Using a 5 ml syringe, take out 5 ml of the medium into a 15 ml falcon to measure the pH value. A pH value of ~ 6.60 is desired.

54b Clostridium sp. medium
![35](https://static.igem.wiki/teams/4404/wiki/closspmedium1.png)

Dissolve ingredients (note that most of the carbonate remains suspended) and sparge medium with 100% N2 gas for 30 -45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials while stirring to keep carbonate suspended and autoclave.

135 Acetobacterium medium:
![35](https://static.igem.wiki/teams/4404/wiki/acetomedium.png)
Dissolve ingredients except bicarbonate, fructose, vitamins, cysteine and sulfide, bring to the boil and cool to room temperature under 80% N2 and 20% CO2 gas mixture. Add bicarbonate (solid) and equilibrate the medium with the gas until a pH of around 7.4 is reached. Then distribute under the same gas atmosphere in anoxic Hungate-type tubes or serum vials and autoclave. Before use adjust the pH to 8.0 - 8.2 by adding a sterile anoxic stock solution of sodium carbonate (5% w/v) prepared under 80% N2 and 20% CO2 gas mixture (c. 0.25 ml per 10 ml medium) and add fructose, vitamins (sterilized by filtration), cysteine and sulfide from anoxic sterile stock solutions prepared under 100% N2.
Note: For autotrophic growth D-fructose is omitted and a gas atmosphere of 80% H2 and 20% CO2 is used

YTF medium:
![35](https://static.igem.wiki/teams/4404/wiki/ytfmedium.png)

879 Clostridium ljungdahlii medium:
![35](https://static.igem.wiki/teams/4404/wiki/xymedium1.png)
Dissolve ingredients (except bicarbonate, fructose, vitamins and reducing agents), sparge medium with 80% N2 and 20% CO2 gas mixture for 30 – 45 min to make it anoxic and adjust pH to 5.5. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave at 121°C for 15 min. Add fructose, vitamins (sterilized by filtration), cysteine and sulfide from sterile stock solutions prepared under 100% N2 gas atmosphere and bicarbonate from a sterile stock solution prepared under 80% N2 and 20% CO2 gas atmosphere. Adjust pH of complete medium to 5.9!

LB medium:
![35](https://static.igem.wiki/teams/4404/wiki/lbmedium.png)

M9 medium:
![35](https://static.igem.wiki/teams/4404/wiki/m9medium.png)
Autoclave MgSO4 and CaCl2 seperately.

SOB medium:
![35](https://static.igem.wiki/teams/4404/wiki/sobmedium.png)
Add MgCl2 x 6 H2O and MgSO4 x 7 H2O directly before use in autoclaved medium.

SOC medium:
![35](https://static.igem.wiki/teams/4404/wiki/socmedium.png)
Add components, except MgCl2 x 6 H2O solution (1 M), MgSO4 x 7 H2O solution (1 M) and glucose
solution (1 M) to ddH2O and mix thoroughly. Adjust pH to 7.0. Bring volume to 960.0 ml.
Autoclave for 20 min at 121 °C. Aseptically add sterile MgCl2 · 6 H2O solution (1 M), sterile MgSO4 · 7 H2O solution (1 M) and sterile glucose solution (1 M). Mix thoroughly.

TB medium:
![35](https://static.igem.wiki/teams/4404/wiki/tbmedium.png)
Adjust pH of dissolved Pipes, CaCl2 and KCl solution to 6.7 with 2M NaOH. Autoclave dry MnCl2 separately.

These are the solutions/additives we used:
Wolin‘s vitamin Solution:
![35](https://static.igem.wiki/teams/4404/wiki/wolinssolution.png)

Modified Wolin‘s vitamin solution:
![35](https://static.igem.wiki/teams/4404/wiki/wolinssolutionmod.png)
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH.

Amino Acid-Mix I:
![35](https://static.igem.wiki/teams/4404/wiki/aminoacidmix1.png)

Amino Acid-Mix II:
![35](https://static.igem.wiki/teams/4404/wiki/aminoacidmix2.png)
To dissolve L-Tyrosine adjust pH to 11.0.

These are the buffers we used:
50x TAE:
![35](https://static.igem.wiki/teams/4404/wiki/50xtae.png)

EPB S:
![35](https://static.igem.wiki/teams/4404/wiki/epbs.png)

EPB NS:
![35](https://static.igem.wiki/teams/4404/wiki/epbns.png)

---

Primers

##### These is the list of primers that we used:

![35](https://static.igem.wiki/teams/4404/wiki/primerlist1.png)
![35](https://static.igem.wiki/teams/4404/wiki/primerlist2.png)

---

Plasmids

#####

- **All backbones we used**:

![35](https://static.igem.wiki/teams/4404/wiki/pmtlpthla.jpg)

pMTL83151\_pthlA\_gusA

![35](https://static.igem.wiki/teams/4404/wiki/pmtlpbgal.jpg)

pMTL83151\_PbgaL\_gusA

- **All plasmids we created with new inserts**:

![35](https://static.igem.wiki/teams/4404/wiki/pmtl83151-acsb-xy-pbgal.jpg)

pMTL83151\_acsB\_XY\_PbgaL

![35](https://static.igem.wiki/teams/4404/wiki/pmtl83151-acsb-awo-pbgal.jpg)

pMTL83151\_acsB\_AWO\_PbgaL

![35](https://static.igem.wiki/teams/4404/wiki/pmtl83151-acsb-xy-pthla.jpg)

pMTL83151\_acsB\_XY\_pthlA

Cultivation of our organisms

#####

*E. coli*
- DH5-alpha: 37 °C, LB medium
- XL-1 blue MRF: 37 °C, LB or M9 medium
- TOP10: 37 °C, LB medium
- CopyCutter EPI400: 37 °C in LB or SOC medium

*Clostridium saccharoperbutylacetonicum* ATCC 27021
30 °C in TYA, XY or CGM medium; N2-gassed anaerobic hungates/flasks or anaerobic agar plate

*Clostridium beijerinckii* ATCC 51743
37 °C in TYA, XY or 54b medium; N2-gassed anaerobic hungates/flasks or anaerobic agar plate

*Clostridium beijerinckii* NRRL B-593
30 °C in TYA, XY or 54b Clostridium sp. medium in N2-gassed anaerobic hungates/flasks or anaerobic agar plate

*Clostridium ljungdahlii* ATCC 55383
30 °C in XY or YTF medium; N2-gassed anaerobic hungates/flasks or anaerobic agar plate

*Acetobacterium woodii* ATCC 29683
30 °C in TYA, XY medium pH 8.0 or 135 Acetobacterium medium; N2-gassed anaerobic hungates/flasks or anaerobic agar plate

---

PCR

#####

Fusion PCR
Fusion PCR was used to amplify our wanted inserts out of *Clostridium ljungdhalii* and *Acetobacterium woodii*. It was performed according to Harrison et al.
Pipetting scheme:
![35](https://static.igem.wiki/teams/4404/wiki/fusionpcr1.png)

PCR program:
![35](https://static.igem.wiki/teams/4404/wiki/fusionpcr2.png)

16S-rRNA DreamTaq-PCR
We performed 16S-rRNA PCR to check if we have pure cultures which are not contaminated.
Pipetting scheme:

![35](https://static.igem.wiki/teams/4404/wiki/fusionpcr1.png)

PCR program:

![35](https://static.igem.wiki/teams/4404/wiki/fusionpcr2.png)

PCR clean-up
We cleaned- up our insert PCR samples to use for digestion with the Qiagen QIAquick PCR purification Kit according to the manufacturer's protocol.

gDNA isolation
We isolated gDNA from *Clostridium ljungdahlii* ATCC 55383 and *Acetobacterium woodii* ATCC 29683 to use their gDNA as template for amplification of Wood-ljungdahl pathway genes.

Cell lysis and Protein precipitation
- Pellet 2 ml of culture by centrifugation, discard the supernatant.
- Add 150 µl TE-buffer and resuspend the cell pellet.
- Add 5 µl of lysozyme (100 mg/ml in ddH2O) and mix by vortexing briefly.
- Incubate at 37 °C for at least 30 min.
- Dilute 1 µl of Proteinase K (50 µg/µl) in 300 µl 2 % SDS solution for each sample.
- Add 300 µl of the Proteinase K/SDS-solution master mix to the sample and vortex briefly.
- Incubate at 65 °C for 15 min, invert every 5 min.
- Cool the samples at 37 °C for 5 min.
- Place the samples on ice for 5 min.
- Add 175 µl of MPC Protein Precipitation Reagent to the sample and vortex vigorously for 10 s.
- Pellet the debris by centrifugation at 4 °C for 10 min at >10.000 g in a centrifuge.
- Avoiding the pelleted debris, transfer the supernatant to a clean microcentrifuge tube.

DNA Precipitation
- Add 1 µl of RNase A (5µg/µl) to the sample and vortex briefly.
- Incubate at 37 °C for 30 min.
- Add 500 µl of ice-cold isopropanol and invert the tubes 40 times.
- Pellet the DNA by centrifugation at 4 °C for 10 min at >1-000 g in a centrifuge.
- Carefully pour off the isopropanol without dislodging the DNA pellet. Centrifuge briefly if the pellet is dislodged.
- Rinse the DNA pellet with 100 µl ice cold ethanol (70 %).
- Remove the ethanol with a pipet tip and dry the DNA pellet at room temperature until the ethanol has evaporated.
- Add 25 µl of EB-buffer (Qiagen) to the dried DNA pellet.
- Place the samples in the fridge and measure next day.

---

Gel electrophoresis

#####

We used gel electrophoresis for verification of PCR amplification and digestion.

- Pour appropriate percentage of Agarose Gel and pour TAE buffer on top
- load 2-3 µl GeneRuler 1 kb DNA Ladder, ready-to-use
- load 5 µl PCR product or digested plasmid + 1 µl 6x Loading Dye
or 2.5 µl digested plasmid + 0.5 µL 6x Loading Dye
- run at 150 V 33 min for PCR verification or at 85 V 50 min for digest verification

![35](https://static.igem.wiki/teams/4404/wiki/electrophoresis.png)

---

Plasmid prep

#####

We prepped our plasmids out of E. coli and Clostridium saccharoperbutylacetonicum with the Qiagen QIAprep Spin miniPrep Kit according to the manufacturer’s protocol.

Sequencing

#####

To check if our plasmids after transformation have any unwanted mutations, we send them for Sanger Sequencing. The samples for sequencing were prepared as follows:
700 – 1000 ng of plasmid were diluted in to a total of 9 µl and mixed with 4 µl of sequencing primer
Or 400 ng of plasmid were diluted in to a total of 6 µl and mixed with 4 µl of sequencing primer

---

Preparation of electrocompetent *E. coli* cells

#####

2x 5 ml LB
250 ml LB in 1 L Erlenmeyer flask with chicane
500 ml sterile ddH2O at 4°C
50 ml sterile glycerol (10% (w/v)) at 4°C
30 sterile labeled Eppendorf cups at – 20°C or -80°C
1x sterile GS3 jar at 4°C
1x Aquatron at 30°C
1x Sorvall RC6 centrifuge at 4°C
1x Universal 320R centrifuge at 4°C
1 L liquid nitrogen

Day 1
- Inoculate 2x 5 ml LB (if required with antibiotics) with your strain of interest, either from cryo-stock culture or from agar plate.
- Incubate overnight at 37°C and 150 rpm.

Day 2
- Inoculate 250 ml LB (without antibiotics) with 2% (5 mL) preculture.
- Incubate in the Aquatron at 30°C and 160 rpm until OD600 of 0.5-0.8.
- Check the culture via microscope for contaminations.
- Let the cells (OD600 of 0.5-0.8) cool down in ice water for 15 min. All further steps are performed under cool conditions.
- Decant the cells in sterile GS3 jar and centrifuge at 4°C and 5000 rpm (4500 g) for 20 min at 4 °C.
- Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 200 ml sterile 4°C ddH2O.
- Centrifuge the GS3 jar at 4°C and 5000 rpm (4500 g) for 5-10 min at 4 °C.
- Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 200 ml sterile 4°C ddH2O.
- Centrifuge the GS3 jar at 4°C and 5000 rpm (4500 g) for 5-10 min at 4 °C.
- Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 30 ml sterile 4°C glycerol (10% (w/v)). Afterwards, transfer the culture into a 50 ml Falcon tube.
- Centrifuge the Falcon tube at 4°C and 6200 rpm for 5-10 min at 4 °C.
- Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 10 mL sterile 4°C glycerol (10% (w/v)).
- Centrifuge the Falcon tube at 4°C and 6200 rpm (4230 g) for 5-10 min at 4 °C.
- Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 500-700 µl sterile 4°C glycerol (10% (w/v)).
- Aliquot the cells into the Eppendorf cups (40 µL per Eppendorf cup). While aliquoting, filled Eppendorf cups must be frozen directly in liquid nitrogen.
- Store the filled Eppendorf cups in a cryobox at -80°C.

Preparation of chemocompetent *E. coli* cells

#####

2x 5 ml SOB
250 ml SOB in 1 L Erlenmeyer flask with chicane
Ca. 240 sterile labeled Eppendorf cups at – 20°C or -70°C
1x sterile GS3 jar
1x Aquatron at 19°C
1 L liquid nitrogen

Day 1
- Inoculate 2x 5 ml SOB (supplemented with 50 µl 1M MgCl2 and 50 µl MgSO4 and if required with antibiotics) with your strain of interest, either from cryo-stock culture or from agar plate.
- Incubate overnight at 37°C and 150 rpm.

Day 2
- Inoculate 250 ml SOB (supplemented with 2.5 ml 1M MgCl2 and 2.5 ml MgSO4 without antibiotics) with 2% (5 mL) preculture.
- Incubate in the Aquatron at 19°C and 200-250 rpm until OD600 of 0.6.

Day 3
- Let the cells (OD600 of 0.6) cool down on ice for 10 min. All further steps are performed under cool conditions.
- Decant the cells in sterile GS3 jar and centrifuge at 4°C and 5000 U (2500 g) for 10 min at 4 °C.
- Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 80 ml sterile ice-cold TB very carefully.
- Incubation of the suspension for 10 min on ice.
- Decant the cells in sterile GS3 jar and centrifuge at 4°C and 5000 U (2500 g) for 10 min at 4 °C.
- Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 20 ml sterile ice-cold TB very carefully.
- Add 1.4 ml sterile-filtered DMSO.
- Incubation of the suspension for 10 min on ice.
- Aliquot the cells into Eppendorf cups (100 µL per Eppendorf cup). While aliquoting, filled Eppendorf cups must be frozen directly in liquid nitrogen.
- Store the filled Eppendorf cups in a cryobox at -80°C

---

Transformation of electrocompetent *E. coli cells*

#####

- Dialysis
- Fill Petri dish ~50 % with sterile demi H2O
- Place Dialysis filter on top of water: shiny side upwards DO NOT TOUCH THE MEMBRANE! USE FORCEPS!
- Carefully place entire reaction mixture on membrane by pipetting. Do not apply to much pressure otherwise it will sink
- Incubate for 30 min at room temperature
- Remove reaction mixture from mebrane by carefully pipetting into new 1.5 mL eppi
- Measure by NanoDrop. Attention! Concentration can be expected to be very low!
- add 2 µL ligation samples to 40 µl defrosted electrocompetent cells
- incubate on ice for 10 min
- transfer cells into precooled electroporation cuvette
- electroporation (2500 V, 25 F, 200 ?)
- directly add 1 mL LB or M9 medium
- incubation for 1h @37 °C
- plating on agar plates supplemented with appropriate antibiotic (here chloramphenicol)

---

Transformation of chemocompetent *E. coli* cells

#####

- Thaw 100 µL cell aliquots on ice for 10-15 min
- add 1-5 µL DNA to the competent cells
- incubate cells for 30 min on ice
- heat shock cells @42 °C for 1 min
- place tubes on ice for 5 min
- add 500 µL desired medium to the cells for recovery (M9/LB/SOC)
- incubate cells for 1h @37°C and 210 rpm
- centrifuge cells for 1 min at 13 000 rpm
- discard supernatant and resuspend cells in backflow
- plate all rest on appropriate medium
- incubate plates @37 °C

---

Transformation of *Clostridium saccharoperbutylacetonicum*

#####

Preparation:
- Sterile 500 mL anaerobic flask for incubation.
- Channeling into anaerobic tent a few days before: Cuvettes, Eppis, 20 mL syringe, glass beads,
15ml Falcon, channel Falcon centrifuge into the tent.
- Channeling into anaerobic tent at day of transformation: CGM medium, EPB S and EPB NS buffer (with and without MgCl2) and ice-cold metal blocks.
- Epo-machine needs to be connected.

Day1
- Inoculate 1x 5 ml pre-culture with 50 µl cryo-stock culture and incubate at 30°C

Day2
- Main culture is inoculated in an anaerobic flask from pre-culture: 50 mL CGM medium is inoculated to an OD 0.05 at the late afternoon

Day 3
The 50 ml main culture had to be reached an OD 1 - 1.2 (min. OD 0.8, max. OD 1.4).
Competence:
- Culture divided into 4 x 15 mL Falcon (12.5 mL/Falcon).
- Centrifuge set to rcf.
- Centrifuge 2600x g for 12 min.
- Discard supernatant
- Resuspend cells in 5 mL/Falcon (Total 20 mL) in EPB S buffer (with MgCl2).
- Centrifuged again at 2600x g for ca. 12 min.
- Discard supernatant
- Resuspend cells in 250 µL/Falcon (Total 1 mL) EPB NS buffer (without MgCl2).
- Pool the cells in a falcon.

Transformation:
- Add 200 µL of cells into electroporation cuvettes.
- Add 500-1000 ng DNA in 2 mm electroporation cuvettes.
- Incubate cuvettes for 5 min between cold metal blocks before transformation.
- Pulsing: exponential decay wave, 1500 V, 200 Ohm, 25 µF.
- 1 mL CGM was added and gently mixed with the cell suspension.
- Recovery between 2 h or ON at 30°C.
- Cells were shortly centrifuge down, supernatant was discarded and all cells were plated on CGM agar with appropriate antibiotic (here: Thiamphenicol).

---

Digestion

#####

Digest of Insert
- Prepare reaction mixture in order indicated:
For one restriction enzyme

![35](https://static.igem.wiki/teams/4404/wiki/digest1.png)

For two restriction enzymes

![35](https://static.igem.wiki/teams/4404/wiki/digest2.png)

- Incubate time and temperature was chosen according to used enzyme
- Heat shock at 65 °C for 5 min for enzyme deactivation

Digestion of Plasmid
In order to prevent re-ligation of plasmid and Fragment ?? Plasmid is simultaneously cut and dephosphorylated

![35](https://static.igem.wiki/teams/4404/wiki/digest3.png)

- Mix thoroughly, spin briefly and incubate at 37 °C for 10 min
- Heat shock at 65 °C for 5 min

Purification of Digest
We purified or digested samples to use for ligation either with the Qiagen QIAquick PCR purification Kit or the Qiagen MinElute PCR
 Purification Kit according to their manufacturer’s protocols.
Test digest

![35](https://static.igem.wiki/teams/4404/wiki/digest4.png)
- Incubate time and temperature was chosen according to used enzyme
- Heat shock at 65 °C for 5 min for enzyme deactivation

---

Ligation

#####

We ligated our desired digested inserts with our different digested plasmid backbones to obtain a plasmid that we later could transform into our solventogenic clostridia.

![35](https://static.igem.wiki/teams/4404/wiki/ligation.png)

Ligation was carried out in a 1:5 molar ration of Plasmid to Insert
Formula: required mass insert (g) = desired insert/vector molar ratio x mass of vector (g) x ratio of insert to vector lengths
We used the NEBioCalculator to determine correct amounts.

- Incubate over-night at room-temperature (store in dark)

Preparation of Cryo-Stocks

#####

To store all the strains we obtained during our iGEM period, we prepared cryo-stocks for storage at -80°C. Bacteria strains were grown in appropriate liquid medium. 750 µl of the culture were mixed with 750 µL 99.5 % glycerol. The cryo-stocks were immediately stored on ice until they were transferred to -80 °C.

---

Gel-Extraction

#####

Gel extraction was performed to specifically purify our 5 kB Insert fragments and to remove the unwanted PCR site-products. We used the QIAquick Gel extraction kit and followed the manufacturer’s protocol.

---

Measurement of *Clostridium saccharoperbutylacetonicum* growth curves

#####

To test if the plasmids have effects on the growth of our transformed clostridia, growth curves were measured until the stationary phase was reached.
Inoculation of 5 ml CGM medium preculture from cryo-stock and incubation overnight at 30 °C. Next day shifting of cultures in fresh 10 ml CGM medium and setting of OD to 0.05. The OD600 for each culture was measured each hour.

{% endblock %}

{% extends "layout.html" %}
{% block page\_content %}

## Results

Results

![35](https://static.igem.wiki/teams/4404/wiki/fig1.png)

**Figure 1: Genes of the Wood-Ljungdahl pathway.** Clustered in A: *A. woodii* and B: *C. ljungdahlii* (for details see supplementary Table 1).

##### Target gene analysis

For target gene analysis, we analysed the genome of our aimed recipient strain to identify which genes are required to generate a functional Wood-Ljungdahl pathway (WLP) in a non-CO2-assimilating host organism. Sandoval-Espinola *et al.* were first to identify WLP genes in Clostridia which are incapable of utilising CO2 as a carbon source[1]. This proposes that only the acetyl-CoA synthase (*acsB*) is required to enable a functional WLP. However, these investigations were based on genome annotations and RNA sequencing data. No enzymatic studies on translation, functionality, or interaction were made to further investigate these transcripts. Additionally, these studies were only conducted in the butanol producer *Clostridium beijerinckii* (*C. beijerinckii*), but not in our target organism *Clostridium saccharoperbutylacetonicum* (*C. saccharoperbutylacetonicum*). In order to identify required genes for a functional WLP in our target organism, we first thought about the essential genes of the WLP. Woods *et al*. did an in-depth essay to identify the minimal set of WLP genes[2]. They were able to identify a set of 21 genes required for the assimilation of CO2 and H2, the methyl- and carbonyl branch and the eventual synthesis of acetyl-CoA. By in-silico analysis we were able to identify that only the acetyl-CoA synthase (ACS), the formate dehydrogenase and the corrinoid-iron nickel enzyme annotations in the genome of *C. saccharoperbutylacetonicum* were missing. The absence of the formate dehydrogenase is of secondary importance for this project as *C. saccharoperbutylacetonicum*, same as *C. beijerickii* possess the Pfor/Pfl system. It catalyzes first the transfer of CO2 onto acetyl-CoA (Pfor) and then, by removal of the CoA, formate is generated (Pfl). Therefore, *C. saccharoperbutylacetonicum* can synthesize formate, which is the first step of the WLP, without the utilisation of the formate dehydrogenase. Furthermore, all essential genes for the Rnf energy conservation system are annotated in *C. saccharoperbutylacetonicum*, implying that the same energy system as in *Clostridium ljungdhalii* (*C. ljungdahlii*) and *Acetobacterium woodii* (*A. woodii*) is present (Figure 1). However, due to the limited time frame of the project, we were not able to conduct enzyme essays of RNA-seq. experiments in *C. saccharoperbutylacetonicum*.

---

Results

##### Plasmid Construction

During the course of our project, we constructed the following three plasmids (Figure 2):

![35](https://static.igem.wiki/teams/4404/wiki/fig2.png)

**Figure 2: Plasmid constructions.** The plasmids that we have successfully constructed: **A**: pMTL83151\_PbgaL\_acsB2\_CLJU, consisting of the gene *acsB2* from *C. ljungdahlii* as insert and the lactose inducible promoter (PbgaL). **B**: pMTL83151\_PbgaL\_acsB1\_AWO, consisting of the gene *acsB1* from *A. woodii* as insert and the lactose inducible promoter (PbgaL). **C**: pMTL83151\_PthlA\_acsB2\_CLJU, consisting of the gene *acsB2* from *C. ljungdahlii* as insert and the thiolase promoter. Inserts are marked green, promoters are orange. The genes marked in blue are part of the backbone.

The backbones used belong to the ClosTron plasmid system and were: pMTL83151\_PbgaL\_gusA (7506 bp) and pMTL83151\_PthlA\_gusA (6241 bp)
pMTL83151\_PbgaL\_gusA contains the promoter PbgaL which leads to low basal expression of the following coded gene on the plasmid and additionally is lactose inducible for stronger gene expression. pMTL83151\_PthlA\_gusA instead contains the PthlA promoter which is known to be a strong promoter in Clostridia. The empty plasmid pMTL83151 contains the *lacZ*-gene at the multiple cloning site, which means that by introduction of a transgene insert, the *lacZ*-gene becomes disrupted. Colonies of positive clones with correct transgene insertion should therefore appear white on a medium containing X-gal. Negative clones with faulty transgene integration in the plasmid can still express the β-galactosidase and therefore X-gal is hydrolyzed and the blue 5,5’-dibromo-4,4’-dichloro-indigo dye is produced. Additionally, the plasmids code for the catP cassette, so *Escherichia coli* (*E. coli*) or *C. saccharoperbutylacetonicum* transformants can grow on selective media containing the antibiotics Chloramphenicol (*E. coli*) or Thiamphenicol (*C. saccharoperbutylacetonicum*).

Our desired transgene we wanted to transform into *E. coli* and subsequently into *C. saccharoperbutylacetonicum* was the acetyl-CoA synthase *acsB*. We obtained the gene by amplification via PCR from the genomic DNA of our donor organisms. The donor organisms we used were *C. ljungdahlii* and *A. woodii*. The correct amplification of the 2.1 kb large gene can be seen by the band on the gel in Figure 3.

![35](https://static.igem.wiki/teams/4404/wiki/fig-3.png)

**Figure 3:** **Agarose gel electrophoresis verified correct PCR amplification of*****acsB1*****from** ***A. woodii*** **and** ***acsB2*** **from** ***C. ljungdahlii*** **genome.** Columns 1 to 3 show a band that matches the size of 2175 bp of *acsB1*. Columns 5 and 6 show a band that matches the size of 2127 bp of *acsB2*. Both results indicate that the *acsB1* and *acsB2* gene were successfully amplified. However, amplification was not successful in column 4, where no distinct band has emerged. A possible explanation for that could be that the gDNA used for this specific amplification sample was sheared. Nevertheless, the successful amplified ACS was sufficient to continue with the next step.

Additionally, we amplified a 5 kb large fragment from *A. woodii* using PCR, in case our minimal ACS fragment is not sufficient for CO2 assimilation in *C. saccharoperbutylacetonicum.* This 5 kb fragment contains the genes encoding for AcsA, CooC2 and AcsB1. Here, *acsA* and *cooC2* code for the CO dehydrogenase (CODH) whereas *acsB1* codes for the acetyl-CoA synthase, the ACS subunit of the CODH/ACS complex. The CODH/ACS complex catalyzes the fusion of the methyl- and carbonyl branches by transferring the methyl- and carbonyl groups to Co-enzyme A to form acetyl-CoA. Initial amplification attempts of the 5 kb fragment failed. We therefore tried different PCR master mixes (as indicated in the notebook). To check whether the amplification with one of the new master mixes was successful, the size of the PCR product was examined via agarose gel electrophoresis. The correct amplification of the 5 kb large gene can be seen by the band on the gel in Figure 4 for all samples. However, an additional band smaller than 250 bp is visible. To isolate only the 5 kb fragment, direct purification from gel was carried out.

![35](https://static.igem.wiki/teams/4404/wiki/fig-4.png)

**Figure 4: Agarose gel electrophoresis verified successful amplification of** ***acsA*****,** ***cooC2*** **and** ***acsB1*** **from the genome of** ***A. woodii*****.** Examination of the agarose gel showed that all samples (1-6) show a band for a 5kb PCR product which meets the expectations and indicates correct amplification of the desired fragment. However, an additional band for a fragment smaller than 250 bp can also be seen.

Next, we digested our backbone plasmids and insert-genes *acsB1* (from *A. woodii*) and *acsB2* (from *C. ljungdahlii*) with Xho1 and Nhe1. The *gusA* gene (1.8 kb) was cut out by the enzymes and the *acsB* (2.1 kb) was newly ligated into the backbone plasmid. The ligated plasmids were then transformed into *E. coli*. Plasmids from white colonies on transformation plates containing X-gal, IPTG and Chloramphenicol were prepared and controlled by test-digestion with Xho1 and Nhe1. With the digested samples a gel electrophoresis was conducted. In Figures 5-7, bands for the digested linearized plasmids without insert can be seen. At 5.7 kb for pMTL83151\_PbgaL\_acsB2\_CLJU and pMTL83151\_PbgaL\_acsB1\_AWO, as well as at 4.4 kb for pMTL83151\_PthlA\_acsB2\_XY can be seen. Furthermore, at 2.1 kb the excised *acsB* insert is present, demonstrating correct cloning could be verified.

![35](https://static.igem.wiki/teams/4404/wiki/fig-5.png)

**Figure 5:** **Agarose gel electrophoresis verified correct cloning of pMTL83151\_PbgaL\_acsB1\_AWO.** After restriction digest (Xho1 and Nhe1) of pMTL83151\_PbgaL\_gusA to remove gusA and ligation with *acsB1* at the previous location of gusA, the resulting plasmid was called pMTL83151\_PbgaL\_acsB1\_AWO. This plasmid was amplified through transformation in *E. coli* cells, followed by cultivation of the transformed *E. coli* cells on selective media and plasmid preparation. The obtained plasmids were test-digested with restriction enzymes Xho1 and Nhe1 cutting at the insert borders. The agarose gel shows the undigested and digested plasmids of different positive (white) *E. coli* clones. Furthermore, *acsB1* from Fig. 3, as well as plasmid pMTL83151\_PbgaL\_gusA after digestion with Xho1 and Nhe1 were used as controls. Fig. 5 shows that all undigested plasmid samples exhibited a band that matches the expected size of 7860 bp of pMTL83151\_PbgaL\_acsB1\_AWO as well as two other bands corresponding to the different forms of plasmid DNA, e.g. coiled-coil which passes through the gel faster than the circular form. The corresponding digested samples led to two bands located at sizes that match the 2175 bp of *acsB1* insert and the 5685 bp plasmid backbone without *acsB1*.

![35](https://static.igem.wiki/teams/4404/wiki/fig-6.png)

**Figure 6:** **Agarose gel electrophoresis verified correct cloning of pMTL83151\_PbgaL\_acsB2\_CLJU.** After restriction digest (Xho1 and Nhe1) of pMTL83151\_PbgaL\_gusA to remove gusA and ligation with *acsB2* at the previous location of gusA, the resulting plasmid was called pMTL83151\_PbgaL\_acsB2\_CLJU. This plasmid was amplified through transformation in *E. coli* cells, followed by cultivation of the transformed *E. coli* cells on selective media and plasmid preparation. The obtained plasmids were test-digested with restriction enzymes Xho1 and Nhe1 cutting at the insert borders. The agarose gel shows the undigested and digested plasmids of different positive (white) *E. coli* clones. Furthermore, *acsB2* from Fig. 3, as well as plasmid pMTL83151\_PbgaL\_gusA after digestion with Xho1 and Nhe1 were used as controls. Fig. 6 shows that all undigested plasmid samples exhibited a band that matches the expected size of 7812 bp of pMTL83151\_PbgaL\_acsB2\_XY as well as two other bands corresponding to the different forms of plasmid DNA, e.g. coiled-coil which passes through the gel faster than the circular form. The corresponding digested samples led to two bands located at sizes that match the 2127 bp of *acsB2* and the 5685 bp plasmid backbone without *acsB2*.

![35](https://static.igem.wiki/teams/4404/wiki/fig-7.png)

**Figure 7:** **Agarose gel electrophoresis verified correct cloning of pMTL83151\_PthlA\_acsB2\_CLJU.** After restriction digest (Xho1 and Nhe1) of pMTL83151\_PthlA\_gusA to remove gusA and ligation with *acsB2* at the previous location of gusA, the resulting plasmid was called pMTL83151\_PthlA\_acsB2\_CLJU. This plasmid was amplified through transformation in *E. coli* cells, followed by cultivation of the transformed *E. coli* cells on selective media and plasmid preparation. The obtained plasmids were digested with restriction enzymes Xho1 and Nhe1 cutting at the insert borders. The agarose gel shows the undigested and digested plasmids of different positive (white) *E. coli* clones. The figure shows that all undigested plasmid samples exhibited a band that matches the expected size of 6547 bp of pMTL83151\_PthlA\_acsB2\_CLJU as well as two other bands corresponding to the different forms of plasmid DNA, e.g. coiled-coil which passes through the gel faster than the circular form. The corresponding digested samples led to two bands located at sizes that match the 2127 bp of *acsB2* and the 4420 bp plasmid backbone without *acsB2*.

For final verification of the transformed plasmids and to check for any possible point mutations, plasmids pMTL83151\_PbgaL\_acsB1\_AWO, pMTL83151\_PbgaL\_acsB2\_CLJU and pMTL83151\_PthlA\_acsB2\_CLJU were completely sequenced. Alignment of the sequencing results obtained with the expected plasmid sequences revealed that no mutations were present.

After we successfully constructed our plasmids, we transformed them into our recipient organism *C. saccharoperbutylacetonicum* . We selected positive transformants on media containing Thiamphenicol. To check if the correct plasmids were integrated, we prepared the plasmids out of clones and again performed a test-digest with Xho1 and Nhe1. After gel electrophoresis, bands for the digested linearized plasmids without an insert at 5.7 kb for pMTL83151\_PbgaL\_acsB1\_Awo, pMTL83151\_PbgaL\_acsB2\_CLJU, as well as at 4.4 kb for pMTL83151\_PthlA\_acsB2\_CLJU can be seen (see Figures 8-9) which match with the bands of the digested plasmids out of the *E. coli* transformants. Furthermore, at 2.1 kb the excised acsB insert is present, demonstrating correct transformation.

![35](https://static.igem.wiki/teams/4404/wiki/fig-8.png)

**Figure 8:** **Agarose gel electrophoresis verified the correct transformation of** ***C. saccharoperbutylacetonicum*** **with pMTL83151\_PbgaL\_acsB1\_AWO and pMTL83151\_PbgaL\_acsB2\_CLJU.** After verification of correct plasmid amplification in *E. coli*, *C. saccharoperbutylacetonicum* was transformed with pMTL83151\_PbgaL\_acsB1\_AWO and pMTL83151\_PbgaL\_acsB2\_CLJU and cultivated on selective media for following plasmid preparation. The obtained plasmids were digested with restriction enzymes Xho1 and Nhe1 cutting at the insert borders. The agarose gel shows the undigested and digested plasmids of different positive *C. saccharoperbutylacetonicum* clones. Fig. 8 shows that the undigested plasmid samples exhibited bands that match the size of pMTL83151\_PbgaL\_acsB1\_AWO and pMTL83151\_PbgaL\_acsB2\_CLJU in the coiled-coil form respectively (compare Fig. 3-4). The corresponding digested samples showed two bands located at sizes that match the 2175 bp/2127 bp of *acsB1*/*acsB2* and the 5685 bp/5685 bp plasmid backbone without *acsB1*/*acsB2*. These observations meet the expectations and therefore indicate that *C. saccharoperbutylacetonicum* match the bands of digested pMTL83151\_PbgaL\_acsB2\_CLJU from *E. coli*. These observations meet the expectations and therefore indicate that *C. saccharoperbutylacetonicum* was successfully transformed and that the plasmids were not modified. However, the bands of the digested samples were rather faint, especially the ones that match the size of *acsB1*/*acsB2*. This may be due to a low overall concentration of the plasmid samples

![35](https://static.igem.wiki/teams/4404/wiki/fig-9.png)

**Figure 9:** **Agarose gel electrophoresis verified the correct transformation of** ***C. saccharoperbutylacetonicum*** **with pMTL83151\_PthlA\_acsB2\_CLJU.** After verification of correct plasmid amplification in *E. coli*, *C. saccharoperbutylacetonicum* was transformed with pMTL83151\_PthlA\_acsB2\_CLJU and cultivated on selective media for following plasmid preparation. The obtained plasmids were digested with restriction enzymes Xho1 and Nhe1 cutting at the insert borders. The agarose gel shows the undigested and digested plasmids of different positive *C. saccharoperbutylacetonicum* clones. Bands were only observed for the plasmid DNA of Clone 6 and undigested plasmid DNA of Clone 7 which may be due to the low plasmid DNA concentration of the samples. Two faint bands that match the size of *acsB2* (2127 bp) and the plasmid backbone without *acsB2* (4420 bp) were observed for the digested plasmid DNA prepared from Clone 6 which meets the expectations. The corresponding undigested sample as well as C.7 - u was located at a size below the expected value of 6547 bp which may be due to the fact that the band corresponds to the coiled-coil DNA form.

At the very end, we sent the plasmids to another round of sequencing to make sure that also our *C. saccharoperbutylacetonicum* transformants do not possess plasmids with any undesired mutations. Figures 10 and 11 show the alignment of the sequenced fragments with the plasmid reference and verified that our inserts were correctly cloned into the vector system. This supports the assumption that we successfully transformed *C. saccharoperbutylacetonicum* with the *acsB1* gene from *A. woodii* or *acsB2* gene from *C. ljungdahlii* under control of the two different promoters PbgaL and PthlA on the pMTL83151 ClosTron plasmids.

![35](https://static.igem.wiki/teams/4404/wiki/fig-10.png)

**Figure 10:** **Sequencing of acsB2\_CLJU insert and PbgaL promotor region of pMTL83151\_PbgaL\_acsB2\_CLJU verified successful transformation of** ***C. saccharoperbutylacetonicum*****.** After plasmid preparation from *C. saccharoperbutylacetonicum* strains transformed with pMTL83151\_PbgaL\_acsB2\_CLJU, the isolated plasmid DNA was sequenced. Sequencing was carried out by an external sequencing laboratory and the fragment sequences thus obtained were aligned to the expected plasmid sequence of pMTL83151\_PbgaL\_acsB2\_CLJU. It is visible in the top line that *acsB2* and the promoter region of PbgaL is covered by the sequenced fragments. The display of the individual fragment sequences indicates that the plasmid is correct and does not contain any mutations.

![35](https://static.igem.wiki/teams/4404/wiki/fig-11.png)

**Figure 11.** **Sequencing of acsB2\_CLJU insert and PthlA promotor region of pMTL83151\_PthlA\_acsB2\_CLJU verified successful transformation of** ***C. saccharoperbutylacetonicum*****.** After plasmid preparation from *C. saccharoperbutylacetonicum* strains transformed with pMTL83151\_PthlAL\_acsB2\_CLJU, the isolated plasmid DNA was sequenced. Sequencing was carried out by an external sequencing laboratory and the fragment sequences thus obtained were aligned to the expected plasmid sequence of pMTL83151\_PthlA\_acsB2\_CLJU. It is visible in the top line that *acsB2* and the promoter region of PthlA is covered by the sequenced fragments. The display of the individual fragment sequences indicates that the plasmid is correct and does not contain any mutations.

---

Results

##### Growth analysis of transformed *C. saccharoperbutylacetonicum*

We have successfully integrated the constructed plasmids pMTL83151\_PbgaL\_acsB2\_CLJU, pMTL83151\_PbgaL\_acsB1\_Awo and pMTL83151\_PthlA\_acsB2\_CLJU into our chosen host organism *C. saccharoperbutylacetonicum*.

 For verification whether the inserted subunit of the acetyl-CoA synthase (*acsB*) is sufficient for autotrophic growth of *C. saccharoperbutylacetonicum*, growth analysis was performed. If CO2 could be assimilated and utilised for further metabolic processes by our transformed strains, the bacteria should be viable and grow on CO2 as only C-source. To test if this was the case, we inoculated *C. saccharoperbutylacetonicum* strains transformed with pMTL83151\_PbgaL\_acsB2\_CLJU, pMTL83151\_PbgaL\_acsB1\_Awo and pMTL83151\_PthlA\_acsB2\_CLJU and without plasmid (wildtype as negative control) on *C. ljungdahlii* medium without fructose. For inoculation, we picked a colony of our *C. saccharoperbutylacetonicum* strains from an *C. ljungdahlii* agar plate to avoid picking up any carbon source from the medium. Afterwards, we gassed all cultures with a mix of hydrogen (66%) and carbon dioxide (34%). No growth could be detected for all investigated strains. This indicates that inserting *acsB* alone is not sufficient for CO2 assimilation and autotrophic growth of *C. saccharoperbutylacetonicum*.

 To ensure that the inserted plasmid does not impair or reduce the growth of the *C. saccharoperbutylacetonicum* transformants, further growth analysis was conducted. Growth experiments were carried out with all our transformants: *C. saccharoperbutylacetonicum* containing pMTL83151\_PbgaL\_acsB2\_CLJU, pMTL83151\_PbgaL\_acsB1\_Awo and pMTL83151\_PthlA\_acsB2\_CLJU respectively. Growth experiments were performed in CGM medium supplemented with Thiamphenicol. Additionally, we induced *C. saccharoperbutylacetonicum* pMTL83151\_PbgaL\_acsB2\_CLJU and pMTL83151\_PbgaL\_acsB1\_Awo with 20 mM lactose to induce the PbgaL promoter and activate stronger gene expression of the *acsB* insert. The growth of the *C. saccharoperbutylacetonicum* wildtype strain in CGM medium without thiamphenicol was used as a reference. All cultures were inoculated to an OD600 of 0.05. As seen in Figure 12, all transgenic strains were viable on CGM medium supplemented with Thiamphenicol. *C. saccharoperbutylacetonicum* with pMTL83151\_PbgaL\_acsB1\_Awo (not induced) showed the most rapid growth. It switched from lag- to the exponential stage after two hours. All other investigated strains reached the exponential phase after six hours, whereas *C. saccharoperbutylacetonicum* with pMTL83151\_PthlA\_acsB2\_CLJU reached this phase after 8 hours only. The slope during the exponential growth phase was similar for the entire investigated strains and no significant difference was detected. All strains reached the stationary stage after 27 hours where no notable increase of the OD600 could be noticed anymore. The transformed strains (induced and not induced for the lactose inducible promoter) grew slightly better than the wildtype. This indicates that the inserted plasmid does not reduce or limit the growth of the transformed cultures. Hence, we assumed that the undetectable growth on CO2 as the sole carbon source was not cause by the integration of the plasmid. Instead, we concluded that the inserted *acsB* gene may not be sufficient for CO2 assimilation in *C. saccharoperbutylacetonicum*.

![35](https://static.igem.wiki/teams/4404/wiki/fig-13.png)

**Figure 12: Growth analysis of** ***C. saccharoperbutylacetonicum*** **and transformants.** Displayed is the growth behaviour of *C. saccharoperbutylacetonicum* wildtype, with integrated pMTL83151\_PbgaL\_acsB2\_CLJU (Cba\_pBgal\_acsB\_XY), pMTL83151\_PbgaL\_acsB1\_Awo (Cba\_pBgal\_acsB\_Awo) and pMTL83151\_PthlA\_acsB2\_CLJU (Cba\_pthla\_acsB\_XY) over a range of 30 hours. Additionally, *C. saccharoperbutylacetonicum* with pMTL83151\_PbgaL\_acsB2\_CLJU (Cba\_pBgal\_acsB\_XY\_induced) and pMTL83151\_PbgaL\_acsB1\_Awo (Cba\_pBgal\_acsB\_Awo\_induced) were induced with 20mM lactose. All cultures were inoculated in CGM medium supplemented with Thiamphenicol to an OD600 of 0.05. All cultures are showing a similar growth but individual differences occur. Cba\_pBgal\_acsB\_Awo shows the fastest and Cba\_pthla\_acsB\_XY the slowest growth.

---

Results

##### Transcription unit design

As the results above indicate that the expression of the acetyl-CoA synthase is not sufficient to enable an autotrophic growth of *C. saccharoperbutylacetonicum*, therefore more pathway genes are required to convey this capability. Since the PCR for the 5 kb unit of *A. woodii* (as described above) proved challenging, most likely due to the low GC content of Clostridia, we chose a commercial de-novo synthesis approach for the construction of the larger transcription units. As we are currently unable to identify which genes are required to allow a functional Wood-Ljungdahl pathway (WLP), we targeted the WLP cluster of *A. woodii* for transforming further genes. This is a particularly well-suited target as, in this organism, the WLP is organised into two clusters which are subdivided into smaller transcription units respectively. To ensure a successful synthesis we selected genes of higher priority for the pathway and designed two transcription units respectively so that they can be expressed together from one plasmid system. For the first plasmid construct we have chosen the pMTL83151 backbone and designed two subunits for expression. The first subunit contains the *acsD*-*acsC*-*acsE* fragment and the second unit contains *acsA*-*cooC2*-*acsB1*. To combine these units on the plasmid, they were connected via the *Sfa*A1 restriction site. By this, we created one frame in the order: *acsD-acsC-acsE-acsA-cooC2-acsB1*. For the insertion of this unit into the plasmid, we utilised the *Sal*1-*Nhe*1 combination. By this, all subunits of the ACS and CODH should be expressed. For the second unit, we choose the pMTL82151 backbone. This backbone is organised identically to the pMTL83151 backbone (Figure 13). However, it contains a pBP1 replication origin instead of a pCB102 replication origin. On this backbone, we inserted the unit of *cooC1-acsV*-Awo\_c10690-Awo\_c10700 and *metV-metF* by connecting them with a *Pac*1 restriction site. This fragment adds important units of the methylene-THF reductase and accessory proteins. To this point we did not successfully transform these plasmids into *C. saccharoperbutylacetonicum*.

![35](https://static.igem.wiki/teams/4404/wiki/fig-14.png)

**Figure 13:** **Transcription unit design.** The two transcriptional subunits from *A. woodii* are combined on the plasmid backbone (pMTL83151 or pMTL82151) by either the restriction site *Sfa*A1 or *Pac*1.

---

Outlook

##### Cloning aims

Based on the results described above, we concluded that *acsB* of *A. woodii* and *C. ljungdahlii* respectively is not sufficient to enable autotrophic growth of *C. saccharoperbutylacetonicum*. Hence, further Wood-Ljungdahl pathway (WLP) proteins are required for a functional WLP in *C. saccharoperbutylacetonicum*. In order to expand the gene set for cloning, we amplified 5 kb out of the genome of *A. woodii*. This fragment contains the following genes: *acsA*, *cooC2* and *acsB1*. We aim to insert the 5 kb fragment into the plasmids pMTL83151\_PbgaL\_gusA and pMTL83151\_PthlA\_gusA for future transformation of *C. saccharoperbutylacetonicum.* Subsequently, the ability of the new *C. saccharoperbutylacetonicum* transformant to assimilate CO2 and to grow autotrophically should be evaluated.

In case that the organism is still not able to grow autotrophically, further required genes must be integrated to complement the WLP in *C. saccharoperbutylacetonicum*. For cloning of further required WLP genes, we were able to obtain *A. woodii* transcription units by de-novo synthesis as described in the transcription unit design section. However, due to the limited time frame and an accumulation of technical problems related to the synthesis, we were not able to express these genes in *C. saccharoperbutylacetonicum* yet. Successful integration of these *A. woodii* genes would introduce entirely new genes into *C. saccharoperbutylacetonicum*, and genes that are already annotated but it is unclear whether they are functionally expressed. We would apply the same cloning strategy as described in the results section for *acsB*. With the combination of our integrated gene clusters and the annotated genes in *C. sacchroperbutylacetonicum*, all the required genes of the WLP are present and it is likely that our transformed organism will be capable of CO2 assimilation.

In order to increase the eventual biofuel yield of our engineered Clostridium, we aim to clone an even stronger promoter in front of the pathway genes. We decided to utilise the improved ferredoxin promoter as proposed by iGEM Nottingham 2019 ([part BBa\_K2715011](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2992016)) which is the strongest promoter they analysed. However, we did not succeed to construct a plasmid with a ferredoxin promoter yet as we experienced toxic effects in *E. coli*. This improved ferredoxin promoter could therefore be directly integrated via the RiboCas system in *C. saccharoperbutylacetonicum* and the transformation of the potentially toxic plasmid into *E. coli* could be bypassed.

As the entirety of the Wood-Ljungdahl genes are ~18 kb in size, problems can occur during plasmid construction and subsequent transformation of such large sized plasmids. Even though ClosTron plasmids with a size of ~12 kb were already constructed and successfully transformed (see Human Practices), we could come across obstacles even when splitting the gene clusters onto several plasmids. Therefore, other more suited methods for cloning larger sized fragments such as gene clusters should be used. During our [integrated human practice](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D) activities, Prof. Winzer explained alternative transformation systems. The RiboCas system is a CRISPR-based tool for genome editing specifically for the genus Clostridia with a transformation efficiency rate of 100 %[3]. Additionally, the allelic exchange system is a further possible approach for gene editing and introducing metabolic pathways into Clostridia[4].

##### Gene expression and enzymatic studies

To gain insights into whether the annotated genes are actually transcribed or whether they need to be cloned, and whether transformed genes are expressed in *C. saccharoperbutylacetonicum*, we aim to perform a Reverse Transcription quantitative PCR (RT-qPCR) screen or RNA-sequencing.

A crucial step of the WLP is the dimer formation of the CODH and the ACS in order to obtain acetyl-CoA. In previous studies it was indicated that this interaction seems to vary depending on the respective organisms[5]. We would therefore employ enzymatic studies to identify whether our cloned ACS can form a functional dimer with the CODH of *C. saccharoperbutylacetonicum*. Furthermore, a comparison of the amounts of acetyl-CoA synthesised by the native CODH-ACS complex to the cloned equivalent can be carried out.

##### Improvement of butanol yield

In an industrial strain, it must be taken into account that various aspects can reduce or limit the product yield.

For industrial scale-up the desired strain must fulfil certain requirements: it must have sufficient resistance to the end product, in our case butanol, as it normally is toxic in high concentrations. There are already first approaches to increase the butanol resistance and yield of *C. saccharoperbutylacetonicum* and thus make it more suitable and profitable for industrial use. Tanaka et al. performed a mutant screen and found a strain with a 1.6-fold increased butanol production[6]. The highest butanol yield so far was reached by using the genome editing tool CRISPR-Cas9. A yield of 19.0 g/liter was achieved by the deletion of the genes *pta* and *buk* in *C. saccharoperbutylacetonicum*[7]. Koepke et al. additionally indicated an adapted metabolism of *C. autoethanogenum* depending on the use of syngas or CO [8]. It would therefore be useful to assess the product yield under different gas compositions including CO, CO2, H2 gas mixtures together with varying temperatures.

Once a prototype strain is generated, the scale-up process would accelerate significantly due to the great progress made by LanzaTech and numerous other companies working with *C. saccharoperbutylacetonicum* on an industrial scale. So, we would be able to utilize the already existing infrastructure on industrial anaerobic fermentation.

---

Results

##### Sources

[1] Sandoval-Espinola, W. J.; Chinn, M. S.; Thon, M. R.; Bruno-Barcena, J. M. (2017): Evidence of mixotrophic carbon-capture by n-butanol-producer Clostridium beijerinckii. *Sci Rep* **7**, 12759.

[2] Woods, C.; Humphreys, C. M.; Tomi-Andrino, C.; Henstra, A. M.; Köpke, M.; Simpson, S. D.; Winzer, K.; Minton, N. P. (2022); Required Gene Set for Autotrophic Growth of *Clostridium autoethanogenum*. App Environ Microbiol. 12;88(7):e0247921. [DOI: 10.1128/aem.02479-21.](https://journals.asm.org/doi/10.1128/aem.02479-21)

[3] Canadas, I. C., Groothuis, D., Zygouropoulou, M., Rodrigues, R., Minton, N. P. (2019): RiboCas: A Universal CRISPR-Based Editing Tool for *Clostridium*. *ACS Synthetic Biology* 8 (6), 1379-1390. [DOI: 10.1021/acssynbio.9b00075.](https://pubs.acs.org/doi/full/10.1021/acssynbio.9b00075)

[4] Ehsaan, M.; Kuit, W.; Zhang, Y.; Cartman, S. T.; Heap, J. T.; Winzer, K.; Minton, N. P. (2016): Mutant generation by allelic exchange and genome resequencing of the biobutanol organism *Clostridium acetobutylicum* ATCC 824. *Biotechnol Biofuels* 9, 4. [DOI: 10.1186/s13068-015-0410-0.](https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-015-0410-0)

[5] Cohen, S. E; Can, M.; Wittenborn, E. C.; Hendrickson, R. A.; Ragsdale, S. W.; Drennan C. L. (2020): Crystallographic Characterization of the Carbonylated A-Cluster in Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase. In: *ACS Catal.*, vol. 10, no. 17, 9741–9746. [DOI: 10.1021/acscatal.0c03033.](https://pubs.acs.org/doi/10.1021/acscatal.0c03033)

[6] Tanaka, Y.; Kasahara, K.; Hirose, Y.; Morimoto, Y.; Izawa, M.; Ochi, K. (2017): Enhancement of butanol production by sequential introduction of mutations conferring butanol tolerance and streptomycin resistance. In: *Journal of Bioscience and Bioengineering*, vol. 124, no. 4, 400-407. [DOI: 10.1016/j.jbiosc.2017.05.003.](https://www.sciencedirect.com/science/article/abs/pii/S138917231730213X)

[7] Wang, S.; Dong, S.; Wang, P.; Tao, Y.; Wang, Y. (2017): Genome Editing in Clostridium saccharoperbutylacetonicum N1-4 with the CRISPR-Cas9 System *Appl Environ Microbiol*, vol. 83, no. 10, pp. e00233-17. [DOI: 10.1128/AEM.00233-17.](https://journals.asm.org/doi/10.1128/AEM.00233-17)

[8] Koepke, M.; Mihalcea, C.; Liew, F.; Tizard, J. H.; Ali, M. S.; Conolly, J. J.; Al-Sinawi, B.;, Simpson, S. D. (2011): 2,3-Butanediol Production by Acetogenic Bacteria, an Alternative Route to Chemical Synthesis, Using Industrial Waste Gas’, *Applied and Environmental Microbiology*, vol. 77, no. 15, 5467-5475. [DOI: 10.1128/AEM.00355-11.](https://journals.asm.org/doi/10.1128/AEM.00355-11)

Results

##### Supplementary

**Table 1: Description of *A. woodii* and *C. ljungdahlii* genes required for the WLP.**

![](https://static.igem.wiki/teams/4404/wiki/supresult.png)

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