iGEM 2022 — Team east-china

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Notebook

July

|  |  |
| --- | --- |
| 16th | 1. Inoculation of pIB184   2. Inoculation of Streptococcus Brevis ATCC367 |
| 17th | 1. Prepare LB solid medium  2. Prepare LB liquid medium   3. Prepare MRS medium |
| 18th | 1. Amplification of genes thl, hbd, crt and ter by PCR  2. Agarose gel electrophoresis detection  3. Gel Extraction  4. One-step cloning  5. Plasmid pLY15-opt were transformed into competent cells, and LB plates were screened to obtain transformants, identify and preserve bacteria. |
| 19th | 1. Select the seed liquid of monoclonal culture  2. Plasmid extraction  3. Configure MRS liquid medium  4. Prepare 1mM magnesium chloride solution  5. Configure SM buffer |
| 20th | Preparation and electroporation of natural competence |
| 21th | 1. Culture of Streptococcus Brevis ATCC367  2. Plasmid extraction and nucleic acid detection |
| 23rd-30th | Butanol fermentation and detection |

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{% block title %}Engineering Success{% endblock %}
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Engineering

Introduction

N-butanol, an important chemical raw material, is expected to become one of the new generations of biofuels. At
present, the domestic industrial synthesis of N-butanol mainly adopts the low-pressure carbonyl synthesis method,
from the production process, propylene, CO, H2, and carbonyl are the main production materials. However, this
production method relies on non-renewable petroleum products as essential raw materials, which is not friendly to
the environment. Therefore, it is necessary to design an environmentally friendly production method for N-butanol
to meet the demand of environmental protection.

It has been found that N-butanol can be synthesized naturally in Clostridium, but the tolerance of Clostridium
bacteria is not good enough for large-scale production. Recently, Lactobacillus Brevis with better N-butanol
tolerance has been isolated by researchers. Therefore, this project develops an engineering N-butanol-producing
bacteria to improve the yield of N-butanol, meet the needs of industrial production, and lay a foundation for
subsequent improvement.

How we design our plasmid

The N-butanol pathway we developed according to the pathway in Lactobacillus Brevis
ATCC824. To achieve this, we designed the DNA sequences of thlA, crt, hbd, and ter gene into the ApaI and BglII
sites of the pIB184 vector (Figure 1.), and transferred the recombinant plasmid into Streptococcus Brevis ATCC367
for fermentation.

![](https://static.igem.wiki/teams/4515/wiki/t-east-china-engineering01.png)
Figure 1. The map of recombinant plasmid

How we build our plasmid

To build the plasmid, firstly amplified the thlA, crt, hbd, and ter genes
fragments from the Lactobacillus Brevis ATCC824 genomic DNA (Figure 2). The second step was to obtain the linear
carrier. The third step was to ligate the genes and linearized vector and transfer the ligation product into E.
coli DH5α competent.

![](https://static.igem.wiki/teams/4515/wiki/t-east-china-engineering02.png)
Figure 2. The PCR gel electrophoresis picture of gene fragments.

Line M: DNA marker; Line 1: The gene fragment of gene crt ; Line 2: The gene fragment of gene hbd ; Line 3: The
gene fragment of gene ter; Line 4: The gene fragment of gene thlA.

We send the constructed recombinant plasmid to a sequencing company for sequencing, and the results proved that
the plasmid was successfully constructed (Figure 3). And the last step was extracting the recombinant plasmid from
E. coli DH5α and transferring it into Streptococcus Brevis ATCC367, so that can be used to produce N-butanol.

![](https://static.igem.wiki/teams/4515/wiki/t-east-china-engineering03.png)
Figure 3. The results of the sequencing data mapped to the plasmids

How we produce N-butanol

a) Identification of transformed Streptococcus Brevis ATCC367

The constructed plasmid (containing 4 codon-optimized genes) was transformed into the Streptococcus Brevis ATCC367
by electroporation method and incubated on MRS solid medium plate at 37℃ for 24-48 hours. Next, we used colony PCR
to identify transformants (Figure 4). At this point, we got the engineered strain we wanted.

![](https://static.igem.wiki/teams/4515/wiki/t-east-china-engineering04.png)
Figure 4. Verification of transformant Streptococcus Brevis ATCC367

Line M: DNA marker; Line 1: Plasmid positive control; Line 2-7: Different
transformants; Line 8: negative
control; Line9: blank control without any DNA template.

b) Measure the yield of N-butanol

The identified single colony was cultured in MRS overnight, and antibiotics were added and put into the anaerobic
chamber (this step can convert glucose into N-butanol) overnight. N-Butanol was detected by gas chromatography.
Finally, we analyzed all data using ultra-performance liquid chromatography, UPCL, and Q Exactive Orbitrap Mass
spectrometer to measure the yield of N-butanol (Figure 5). As shown in the result, we successfully detected a
higher yield of N-butanol. This result is really encouraging.

![](https://static.igem.wiki/teams/4515/wiki/t-east-china-change2.png)
Figure 5. After pLY15-opt was transformed into Streptococcus Brevis, N-butanol production of ply15-opt
strain was measured at different times (48h, 69h, 95h, and 159h)

How we learn from our projectHow we learn from our project

We have already collected the figures from our experiments. We developed an N-butanol-producing pathway in
Streptococcus Brevis ATCC367 and successfully increase the yield of N-butanol. With the development of society,
people pay more and more attention to the impact of environmental problems while improving their own lives.
Nowadays the greenhouse effect is serious, one of the important factors is the overuse of fossil energy.
Because N-butanol is environmentally friendly energy, we believe that if we can use N-butanol in the future and
reduce our dependence on oil, it will relieve the pressure of resource shortage and make our environment better.

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{% block title %}Experiments{% endblock %}
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Experiments

A. Plasmid construction of pLY15-opt

1. Take the ATCC824 genome as the template, then thl, hbd, crt, and ter genes were amplified
by PCR using thl, hbd, crt and ter primers.

▶ Amplification
All reagents were melted at room temperature, mixed by shaking, centrifuged at 6000 rpm for 10 sec, and prepared
according to the following system:

| Name | Amount added | Final concentration |
| --- | --- | --- |
| 2×TOROBlue Flash KOD DyeMix | 25 μL | 1× |
| 10 μM upstream primer | 1.5 μL | 0.3μM |
| 10 μM downstream primer | 1.5 μL | 0.3μM |
| DNA template | 5 μL | GenomeDNA10-200 ng; Plasmid1-50ng; cDNA ~750 ng; Living specimen, crude extract 5 μL |
| ddH2O | Add to 50 μL |  |

▶ The PCR program

| Step | Temperature | Time | Numbers of circulation |
| --- | --- | --- | --- |
| Pre-denaturation | 98℃ | 2 min |  |
| Denaturation | 98°C | 10 sec | 25-45 |
| Annealing | (Tm-5) °C | 5 sec |
| Extension | 68°C | 1-10 sec/kb |

2. Agarose gel electrophoresis detection

▶ Prepare agarose gel: add 100 ml 1×TAE buffer into the conical flask, weigh 1.0 g agarose into a conical flask,
heat it in the microwave oven until it is completely melted, take it out and shake it well, then it is 1% agarose
gel.
▶ Add Dye: when agarose gel is cooled to 70°C, add 10 uL nucleic acid dye, and mix well.
▶ Preparation of gel board: waterproof tape is pasted on both ends of the glue bed to form an 8 mm high retaining
wall, press the tape, and place it on the horizontal table. Insert the comb, and the lower end of the comb is
about 1mm away from the bottom of the plate. Pour the 60℃ gel into the glue bed continuously, 3-4 mm high, avoid
bubbles, and solidify at room temperature. After complete solidification, tear off the adhesive tape, place it in
the electrophoresis tank, add the electrophoresis buffer, which is 1-2 mm higher than the glue surface, gently
pull out the comb obliquely from one end to remove the bubbles generated.
▶ Sampling: add 5 μL DNA maker first, and then take 10 μL PCR reaction system for sample addition.
▶ Electrophoresis: cover the electrophoresis tank, the sampling hole is located at the negative pole of the
electric field, and connects the power supply, 120V. When the front edge of the dye moves to the bottom edge for
1-2 mm, about 25-30 min, the electrophoresis ends.
▶ Observation results: turn off the power, take out the gel, put it in the UV detector (324 nm), and verify if
there is a band of target DNA.

3. Gel extraction.

![](https://static.igem.wiki/teams/4515/wiki/t-east-china-experiment01.jpg)
▶ 1. After DNA electrophoresis, quickly cut the gel containing the target DNA fragment under UV light. It is
recommended to blot the surface liquid of the gel with a paper towel and chop it, and try to remove the excess
gel. The gel was weighed (removing the empty tube weight) and 100mg of gel was equivalent to 100 μL volume, which
was used as a gel volume.
▶ 2. Add an equal volume of Buffer GDP. 50 ~ 55 '℃ water bath for 7-10min, adjust the time appropriately according
to the gel size to ensure that the gel block is completely dissolved. Water bath period upside down mixing 2 times
to accelerate sol. (Adding 1-3 times the volume of A Buffer GDP does not affect the DNA recovery rate. If the DNA
fragment less than or equal to 100 bp is recovered, add 3 times the volume of Buffer GDP, add the same volume of
isopropanol after water bath sol, mix and then proceed to the third step.)
▶ 3. Droplets on the wall of the tube were collected by brief centrifugation. The FastPure DNA Mini columns-G
adsorption column was placed in the Collection Tubes2ml Collection tube, and ≤700 μL sol solution was transferred
to the adsorption column, and centrifuged at 12,000 rpm (13,400×g) for 30-60 sec. If the sol volume is greater
than 700 μL, put the adsorption column back in the collection tube, and the remaining sol solution is transferred
to the column, 12,000 rpm (13,400×g) centrifugation 30-60 sec.
▶ 4. Discard the filtrate and place the column in the collection tube. Add 300 μL Buffer GDP to the column.
Centrifugation at 12,000 rpm(13,400×g) for 30-60 sec.
▶ 5. Discard the filtrate and place the adsorption column in the collection tube. Add 700 μL buffer GW (absolute
ethanol has been added) to the column. Centrifugation at 12,000rpm (13,400×g) for 30-60 sec. (Please add Buffer GW
around the wall of the column, or mix the cover upside down for 2-3 times after adding Buffer GW to help
completely wash the salt attached to the tube wall.)
▶ 6. Repeat Step 5. (Two flushes with Buffer GW can ensure that the salt coating is completely removed,
eliminating the impact on subsequent experiments.)
▶ 7. Discard the filtrate and put the adsorption column back into the collection tube. Centrifuge at 12,000 rpm
(13,400×g) for 2 min.
▶ 8. Place the column in a 1.5 ml sterilized centrifuge tube, add 20-30 μL Elution Buffer to the center of the
adsorption column, and place for 2 min. Centrifuge at 12,000 rpm (13,400×g) for 1 min. The column was discarded
and the DNA was stored at -20℃. (If maximum yield is required, it is recommended that the resulting solution be
readded to the centrifugal adsorption column and step 8 is repeated for secondary elution. When recovering
fragments larger than 3kb, it is recommended to preheat the Elution Buffer to 55 ° C to improve the recovery
efficiency.)

4. One-step cloning.

▶ Recombinant plasmids were transformed into competent cells, and LB plates were screened by colony PCR.
▶ Preparation of selective medium plate: LB liquid medium is prepared with 15g agar powder /1000ml LB solid
medium. After sterilization, it is cooled to below 55 ℃ and erythromycin is added. The final concentration is 300
μg/ mL, and immediately falls plate in disposable Petri dishes.
▶ Incubation on Ice: add recombinant plasmids into 50 μl prepared competent cells, mix well, and keep on ice for
30 minutes.
▶ Heat Shock: Put competent cells into 42℃ water bath for 42 seconds.
▶ Cooling: take it out and quickly keep it on ice for 2-3min.
▶ Resuscitation: 1 ml non-resistant LB liquid medium was added to each tube, 37℃, 150 rpm, 45 min.
▶ 5000rpm, 2min, suck and discard 700ul supernatant, resuspend the transformed competent cells or control samples
in the medium and coat them to the Petri dishes containing ampicillin respectively.

5. The culture dish is turned upside down and incubated at 37℃ overnight.

6. Pick monoclonal culture seed solution.

7. Plasmid extraction

▶ Add absolute ethanol to rinsing solution PWT.
▶ Centrifugate the overnight culture of bacteria at 12,000 rpm (~ 13,400 × g) for 1 min and remove the
supernatant.
▶ Add 150 μL solution P1 to the centrifuge tube to resuspend bacterial precipitation.
▶ Add 150 μl solution P2 into the centrifuge tube, and the tubes were gently turned up and down for 6-8
times.
▶ Add 350 μL solution P3 into the centrifuge tube, immediately and quickly mix up and down for 12-20 times, 12,000
rpm (~13,400 × g) centrifuge for 2 min.
▶ The supernatant collected in the previous step is transferred to the adsorption column CP3 with a pipette.
12,000 rpm (~ 13,400 × g) was centrifuged for 30 sec, the waste liquid in the collecting pipe was poured out, and
the adsorption column CP3 was put into the collecting pipe.
▶ 300 mL of rinsing solution PWT was added into the column CP3, and 12,000 rpm (~ 13,400 × g) was centrifuged for
30 sec. Discard the filtrate and reuse the collection tube.
▶ The adsorption column CP3 was put into the collection tube, and centrifuged at 12,000 rpm (~ 13,400 × g) for 1
min. Discard the filtrate and reuse the collection tube.
▶ Place the adsorption column CP3 in a clean centrifuge tube and add 50-100μL elution buffer TB drops to the
middle of the adsorption membrane, The plasmid solution was collected into a centrifuge tube after centrifugation
at 12, 000 rpm (~13400 × g) for 30 seconds.

B. Preparation of natural competence

1. Cultivate pIB184: LB medium (resistance: Erythromycin), 37° C, 220 rpm overnight. The plasmid was extracted the
next day.
2. Culture of Streptococcus Brevis ATCC367: Glycerol-preserving bacteria were isolated at 37° C for 24 ~ 48 h, and
single colonies were cultured in the MRS medium overnight. Overnight culture of 2 ml was seeded into 50 ml GMRS
(Mixed with 1% glycine) and incubated at 37 ° C until OD600 reached ~ 0.6. The cells were collected by
centrifugation at 4 ° C, 4000 rpm, 10 min. The supernatant was removed and the cell particles were washed twice
with 1 mM MgCl2. Cells were then suspended in 0.5 ml SM buffer (952 mM sucrose and 3.5 mM MgCl2), and 100μL of
aliquots were stored at -80 ° C.

C. Electroporation and Inoculation of pIB184 and streptococcus Brevis ATCC367

1. Plasmids were transformed into Streptococcus Brevis ATCC367 by electroporation.
2. During electroporation, competent cells were mixed with DNA (about 1 µ g, 5 µ L). Incubated on ice for 5min,
and then transferred to a pre-cooled 2mm electric rotating cup for electroporation under the conditions of 2kV,
25UF,200 Ω.
3. After electric shock, add 1ml fresh broth (add 500 mM sucrose, 20 mM MgCl2 and 2 mM CaCl2 to MRS), and
resuscitate at 37 ° C and 220rpm for 2~3h.
4. After that, the cells were inoculated in medium containing antibiotics and incubated at 37 ° C for 24~48h.

D. Butanol fermentation and product detection

1. After electroporation, the isolated colonies were cultured in MRS overnight and added with antibiotics.
2. Transfer 0.7 ml of overnight cultured medium to 70 ml of deoxy-MRS and seeded into an anaerobic chamber for
overnight incubation.
3. After culture, the cells were removed from the anaerobic chamber and incubated at 25 ° C for about 24 hours.

4. The cell density was measured with a DU730 photometer. Butanol and ethanol were detected by gas chromatography.

E. Extraction and detection of intracellular metabolites

1. The cells were cultured at 25 ° C, and 70 ml of fermentation broth was filtered by 0.45 μm filter. The
metabolic reaction of the sample on a filter using methanol: Acetonitrile: WATER = 40:40:20 was cooled at -4℃ for
20 min.
2. The samples were then transferred to a tube containing a quarter volume of 0.1 mm SiO2 dioxide beads and ground
three times for 3 minutes at 2,000 rpm.
3. The supernatant was collected by centrifugation at 12,000 rpm for 30 min at 4℃. The above extraction steps are
repeated, and the supernatant is merged and filtered prior to metabolite detection.
4. Metabolites were analyzed using High-performance liquid chromatography, UPLC and Q Exactive Orbitrap mass
spectrometer. The chromatographic column is T3 column (2.1×150 mM, 3 μm, Waters) , mobile phase A was 10 mM
ammonium acetate, mobile phase B was 100% acetonitrile, flow rate was 0.2 ml/min, elution gradient of Phase B was
0 min, 0% ; 1 min, 0% ; 1.1 min, 5% ; 7 min, 25% ; 8 min, 40% ; 11 min, 40% ; 11. 1min,0%；15 min,0%.
5. Full scans were performed at M/z 700-1300 using electrospray ionization (ESI) in a positive ion mode, and all
acquired data were analyzed with XCalibur 4.0

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Results

I. Construction of double knockout plasmid experiment

1. PCR amplification of codon-optimized gene thlA, hbd, crt, and ter.

We designed the program by thlA, crt, hbd, and ter genes into ApaI and BglII sites of
the pIB184 vector. In order to build our plasmids, we firstly amplified the gene fragments from the Lactobacillus
Brevis ATCC824 genomic DNA (Figure 1), double-enzyme digestion, and ligase to pIB184 carrier.

![](https://static.igem.wiki/teams/4515/wiki/t-east-china-results01.png)
Figure 1. Fragments of crt, hbd, ter, and thlA amplicons. A. crt gene, B. hbd gene, C. ter gene, D. thlA
gene.

In Figure 1, a clear and single DNA band for each gene was shown, indicating that the genes were successfully
amplified by PCR.
Then, the PCR products were purified by gel extraction, four gene fragments were ligated to the pIB184 carrier by
the one-step cloning method. Finally, recombinant plasmid pLY15-opt was transformed into E. coli DH5α competent
cells and coat on LB plate containing Erythromycin and incubated.

2. Transform plasmid pLY15-opt into Streptococcus Brevis ATCC367.

Streptococcus Brevis was cultured on a solid MRS plate, then a single colony was taken
and cultured overnight
in the MRS medium (Figure 2A), and remained 100 μL was mixed with an equal volume of 50% glycerin and stored at
-80℃. Then the constructed plasmid (containing 4 codon-optimized genes) was transformed into the Streptococcus
Brevis ATCC367 by electroporation method and incubated at 37℃ for 24-48 hours. We selected 7 monoclonal colonies,
1 was positive control, 8 was negative control (without any gene fragment or vector), 9 was blank control without
any template (only enzyme, without 4 gene fragments), and 4 failed (Figure 2B).Streptococcus Brevis was cultured
on a solid MRS plate, then a single colony was taken and cultured overnight in the MRS medium (Figure 2A), and
remained 100 μL was mixed with an equal volume of 50% glycerin and stored at -80℃. Then the constructed plasmid
(containing 4 codon-optimized genes) was transformed into the Streptococcus Brevis ATCC367 by electroporation
method and incubated at 37℃ for 24-48 hours. We selected 7 monoclonal colonies, 1 was positive control, 8 was
negative control (without any gene fragment or vector), 9 was blank control without any template (only enzyme,
without 4 gene fragments), and 4 failed (Figure 2B).

![](https://static.igem.wiki/teams/4515/wiki/t-east-china-results02.png)
Figure 2. A. Streptococcus Brevis Monoclonal colony on MRS plate. B.1: colony PCR verification of correct
transformed colony. 1. Plasmid positive control, 2-7: Different transformants, 8: negative control, 9: blank
control without any DNA template.

3. Growth curve measurement and Functional test

The identified single colony was cultured in MRS overnight, and antibiotics were added
and put into the anaerobic chamber (this step can convert glucose into N-butanol) overnight, and we also measured
its growth curve (Figure 3). N-Butanol was detected by gas chromatography. Finally, we analyzed all data using
ultra-performance liquid chromatography, UPCL, and Q Exactive Orbitrap Mass spectrometer to measure the yield of
N-butanol (Figure 4).

![](https://static.igem.wiki/teams/4515/wiki/t-east-china-change.jpg)
Figure 3. After the butanol expression plasmid pLY15-opt was transferred into Streptococcus Brevis, the
growth curve of the strain was measured at different times (48h, 69h, 95h, and 159h)

![](https://static.igem.wiki/teams/4515/wiki/t-east-china-change2.png)
Figure 4. After pLY15-opt was transformed into Streptococcus Brevis, N-butanol production of ply15-opt
strain was measured at different times (48h, 69h, 95h, and 159h)

As shown in Figure3, after culturing for 48h, the growth of the strain reached the stable phase, and after being
cultured for 95h, it turned into the decay phase, which means we improved the strain’s tolerance of N-butanol.

In Figure4, with the growth of the Streptococcus Brevis, the yield of N-butanol is also
increasing. Although this yield of N-butanol is still needed to be improved, it can be inferred from the above
result that with the engineered strain, we can finally optimize the workflow and produce enough N-butanol.In
Figure4, with the growth of the Streptococcus Brevis, the yield of N-butanol is also increasing. Although this
yield of N-butanol is still needed to be improved, it can be inferred from the above result that with the
engineered strain, we can finally optimize the workflow and produce enough N-butanol.

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