Appearance
Week 1 (7.7-7.11)
W1-1: to amplify NdmA, B, and C+E gene fragments by PCR
- The mix (50 μL):
| 20 μL | ddH2O |
|---|---|
| 2 μL | forward primer (10 μM) |
| 2 μL | reverse primer (10 μM) |
| 1 μL | synthetic NdmA, B, or C+E gene (1 ng) as the template |
| 25 μL | 2× Rapid Taq Master Mix (Vazyme, Nanjing, Jiangsu, China) |
- The PCR program:
| 95°C | for 3 min | pre-denaturation | |
|---|---|---|---|
| 95°C | for 15 sec | 35 cycles | denaturation |
| 55°C | for 15 sec | annealing | |
| 72°C | for x sec | extension | |
| 72°C | for 5 min | thorough extension |
Notably,
Duration x: according to the size of gene fragments. Taq polymerase extension speed: 15 sec/kb. NdmA or NdmB: 15 sec; NdmC+E: 24 sec.
Run 1% agarose gel electrophoresis to verify the size of amplified DNA fragments;
Cut the gel around desired bands and extract DNA from it using the FastPure Gel DNA Extraction Mini Kit (Vazyme, Nanjing, Jiangsu, China), and measure the purity and concentration of the DNA samples with an ultra-micro spectrophotometer (TUOHE, Shanghai, China).
Notes & Results:
Succeeded. Despite non-interference in our procedures and conclusions, there is a non-specific band in NdmC+E PCR samples which we avoid when cutting the gel.
Lane 1-5: 10 μL NdmC+E PCR products;
Lane 6: 5 μL FY5000 DNA marker (Best Enzymes, Lianyungang, Jiangsu, China).
Lane 1: 50 μL NdmA PCR products;
Lane 3: 50 μL NdmB PCR products;
Lane 4: 10 μL FY5000 DNA marker.
W1-2: to digest the plasmid pET28a-NdmD (as the vector) and the gene fragments NdmA, B, and C+E with EcoRI and SalI
1 ) The mix (40 μL):
| 4 μL | 10× H Buffer (Takara, Tokyo, Japan) |
|---|---|
| 2 μg | DNA |
| 1 μL | EcoRI (Takara, Tokyo, Japan) |
| 1 μL | SalI (Takara, Tokyo, Japan) |
| up to 40 μL | ddH2O |
2 ) The digestion program:
Incubated in 37°C for 2 h.
3 ) Run 0.5% agarose gel electrophoresis to verify the success of the vector digestion;
4 ) Extract the digested vectors from the gel and the digested gene fragments from the reaction buffer using the FastPure Gel DNA Extraction Mini Kit, and measure the purity and concentration of the DNA samples with the ultra-micro spectrophotometer.
Notes & Results:
Succeeded.
Lane 1-5: 50 μL pET28a-NdmD plasmid digested with EcoRI and SalI
Lane 6: 10 μL FY5000 DNA marker.
W1-3: to ligate the digested vector and gene fragments and
- The mix (10 μL):
| 2 μL | 10× Ligation buffer (Takara, Tokyo, Japan) |
|---|---|
| 25 ng | Vector DNA |
| x ng | Fragment DNA |
| 0.5 μL | T4 DNA Ligase (Takara, Tokyo, Japan) |
| up to 10 μL | ddH2O |
Notably,
x : At a molar ratio of approximately 10 : 1 to the vector DNA. Thus, x = 10 × 25ng × fragment length(bp) ÷ Vector length(bp)
- The ligation program:
Incubated in 16°C for 5 h.
W1-4: to transform the ligation products into competent E.coli DH5α, to yield the plasmids pET28a-NdmD+A, -NdmD+B, and -NdmD+C+E.
1 ) Add 5 μL ligation products to 50 μL competent E.coli DH5α and gently mix them.
2 ) Ice bath for 10 min.
3 ) 42°C water bath for 50~60 sec, then return to ice bath for 5 min.
4 ) Add 1 mL antibiotic-free LB liquid media and incubate them at 37°C, 200 rpm for 1 h.
5 ) Centrifuge at 4000 rpm for 3 min, then remove 900 μL supernatant.
6 ) In a biosafety cabinet, resuspend and transfer the remaining bacterial samples to a Petri dish with solid LB medium containing Kanamycin, and evenly distribute them.
7 ) Incubate them with dishes upside-down in a 37°C incubator overnight (~16 h).
8 ) Observe the colonies and pick some of them for further PCR verification.
W1-5: to verify the transformation by PCR.
- The mix (10 μL):
| 3 μL | ddH2O |
|---|---|
| 0.5 μL | Forward primer (10 μM) |
| 0.5 μL | Reverse primer (10 μM) |
| 1 μL | Bacterial samples |
| 5 μL | 2× Rapid Taq Master Mix |
- The PCR program:
| 95°C | for 10 min | |
|---|---|---|
| 95°C | for 15 sec | 35 cycles |
| 55°C | for 15 sec | |
| 72°C | for x sec | |
| 72°C | for 5 min |
Notably,
Duration x: according to the size of target sequences. NdmD+A or NdmD+B: 45 sec; NdmD+C+E: 28 sec.
- Run 1% or 1.5% agarose gel electrophoresis to verify the size of amplified DNA fragments.
Notes & Results:
Succeeded.
Lane 1-10: 10 μL PCR products of NdmD+A bacterial samples;
Lane 11: 5 μL FY5000 DNA marker.
Bacteria from all lanes contain NdmD+A sequence, except Lane 5 and 10.
Lane 1-10: 10 μL PCR products of NdmD+B bacterial samples;
Lane 11: 5 μL FY5000 DNA marker.
Bacteria from Lanes 2, 5, 7, and 8 contain NdmD+B sequence.
Lane 1-10: 10 μL PCR products of NdmD+C+E bacterial samples;
Lane 11: 5 μL FY5000 DNA marker.
Bacteria from all lanes contain NdmD+A sequence, except Lane 1 and 10.
Week 2 (7.14-7.18)
W2-1: to obtain sufficient purified plasmids pET28a-NdmD+A, -NdmD+B, and -NdmD+C+E, using the FastPure Plasmid Mini Kit (Vazyme, Nanjing, Jiangsu, China).
Culture above-verified bacteria (NdmD+A-Lane 4, NdmD+B-Lane 2, and NdmD+C+E-Lane 2) with 5 mL LB media overnight. Centrifuge at 12,000 rpm for 1 min to collect cell pellet.
Add 150 μL Buffer P1 (containing RNase) to resuspend the samples.
Add 150 μL Buffer P2 (containing SDS and NaOH) and invert the tubes.
Add 350 μL Buffer NP3 (neutralizing buffer) and invert the tubes until the precipitate forms.
Centrifuge at 12,000 rpm for 2 min and transfer the supernatant to the column.
Centrifuge at 12,000 rpm for 30 sec, discard the flow-through.
Add 700 μL Buffer PW (with ethanol), centrifuge at 12,000 rpm for 30 sec, discard the flow-through.
Centrifuge at 12,000 rpm for 30 sec, to remove residual ethanol.
Elute the column with 30 μL Elution Buffer, to collect purified plasmid DNA.
Measure the purity and concentration of the plasmid samples with the spectrophotometer.
W2-2: to transform the plasmids pET28a-NdmD+A, -NdmD+B, and -NdmD+C+E into competent E.coli BL21(DE3).
- Add 0.1 μL purified plasmids to 10 μL competent E.coli BL21(DE3) and gently mix them.
2-8) Same as W1-4
Week 3 (7.21-7.25)
W3-1: to induce and detect the expression of Ndm in E.coli BL21(DE3) by SDS-PAGE.
Culture these engineered E.coli with LB media at 37℃;
Add the inducer IPTG (Isopropyl-beta-D-thiogalactopyranoside) (Cwbio, Shanghai, China) and incubate at 16℃ overnight;
Centrifuge to harvest the cells;
Resuspend them in protein loading buffer (Beyotime, Shanghai, China) containing SDS and β-Mercaptoethanol, with protease inhibitor cocktail (Beyotime, Shanghai, China).
Run SDS-PAGE with 12% Precast Gel (Beyotime, Shanghai, China).
Notes & Results:
Succeeded. All Ndm proteins in BL21(DE3) are induced and detected by SDS-PAGE. Due to the similar size (around 40 kDa), bands of NdmA and NdmB overlapped in samples containing both proteins. We also concluded that the presence of caffeine didn’t inhibit the expression of all Ndm proteins.
M: 5 μL Prestained protein molecular weight standard marker (Beyotime, Shanghai, China)
EV: BL21 carrying pET28a empty vector, as a negative control.
W3-2: to examine whether caffeine affects the E.coli BL21(DE3) growth.
BL21(DE3)/DH5α + IPTG + standard caffeine (400 μg/mL)
Use 1 mL LB medium to assign zero for all samples, and read absorbance value at 600 nm.
| Time | BL21(DE3) | BL21(DE3)+caf |
|---|---|---|
| 0 | 0.002 | 0.005 |
| 100 | 0.154 | 0.101 |
| 200 | 0.382 | 0.182 |
| 250 | 0.65 | 0.362 |
| 300 | 1.055 | 0.6 |
| 350 | 1.48 | 1.032 |
| 400 | 1.906 | 1.43 |
| 450 | 2.35 | 1.85 |
| 500 | 2.67 | 2.3 |
| 550 | 2.85 | 2.622 |
| 600 | 2.954 | 2.76 |
| 650 | 2.958 | 2.852 |
| 700 | 2.963 | 2.868 |
| 750 | 2.967 | 2.872 |
| 800 | 2.969 | 2.886 |
W3-3: to examine whether the engineered BL21(DE3) promotes caffeine degradation.
- Fermentation
a) After 2 hours, the inducer IPTG was added to the bacterial solution (OD ≈ 0.4-0.6).
b) After 3 hours of induction, add caffeine. Leave to ferment overnight.
In short, BL21(DE3) + IPTG + standard caffeine (400 μg/mL)
- Sample Preparation
a) Centrifuge at 12,000 rpm for 3 min, to retain 300 μL supernatants.
b) Seal the tubes and incubate them at 95°C for 10 min.
c) Centrifuge at 12,000 rpm for 10 min and collect the supernatant as "the tested sample".
- Sample purification
a) In a new tube, 100 μL the test samples + 40 μL Buffer1 + 10 μL Buffer2 + 850 μL ddH2O
b) Mix well, and centrifuge at 8,000 x g for 10 min, to retain the supernatants.
c) In a new tube, 750 μL the supernatants + 30 μL Buffer3 + 720 μL ddH2O
d) Mix well, and centrifuge at 8,000 x g for 10 min, to retain the supernatants.
- Caffeine quantification
Use 2 μL ddH2O to assign zero for all samples, and read absorbance value at 274 nm.
Notes & Results:
Basically succeeded. BL21(DE3) carrying the gene NdmD+A, NdmD+B, or NdmD+C+E slightly degrades caffeine in the fermentation broth respectively, while BL21(DE3) carrying all these genes can degrade caffeine more efficiently. However, the extent did not meet our expectations. We decided to explore the reasons and optimize the fermentation conditions.
| Sample | OD274 nm | Percent(%) |
|---|---|---|
| EV1 | 0.884 | 100.34% |
| EV2 | 0.923 | 104.77% |
| EV3 | 0.836 | 94.89% |
| Mean | 0.881 | |
| DA1 | 0.673 | 76.39% |
| DA2 | 0.754 | 85.58% |
| DA3 | 0.686 | 77.87% |
| DB1 | 0.724 | 82.18% |
| DB2 | 0.648 | 73.55% |
| DB3 | 0.698 | 79.23% |
| DCE1 | 0.763 | 86.61% |
| DCE2 | 0.736 | 83.54% |
| DCE3 | 0.635 | 72.08% |
| ABCDE1 | 0.449 | 50.96% |
| ABCDE2 | 0.533 | 60.50% |
| ABCDE3 | 0.516 | 58.57% |
Week 4 (7.28-8.1)
W4-1: to examine whether caffeine affects the E.coli DH5α growth.
DH5α + IPTG + standard caffeine (400 μg/mL)
Use 1 mL LB medium to assign zero for all samples, and read absorbance value at 600 nm.
| Time | DH5α | DH5α+caf |
|---|---|---|
| 0 | 0.001 | 0.002 |
| 100 | 0.253 | 0.201 |
| 200 | 0.582 | 0.382 |
| 250 | 0.854 | 0.56 |
| 300 | 1.25 | 0.812 |
| 350 | 1.68 | 1.218 |
| 400 | 2.102 | 1.643 |
| 450 | 2.55 | 2.05 |
| 500 | 2.87 | 2.521 |
| 550 | 3.058 | 2.825 |
| 600 | 3.151 | 2.986 |
| 650 | 3.159 | 3.053 |
| 700 | 3.162 | 3.068 |
| 750 | 3.167 | 3.081 |
| 800 | 3.172 | 3.079 |
W4-2: to determine whether DH5α degrades caffeine more efficiently than BL21.
- Fermentation
BL21(DE3) + IPTG + standard caffeine (400 μg/mL)
DH5α + IPTG + standard caffeine (400 μg/mL)
- Sample Preparation, purification, and caffeine quantification, same as W3-2
Notes & Results:
Succeeded. Compared with BL21(DE3), DH5α incorporating the Ndm genes degrades caffeine more efficiently. Given that the T7 promoter controls the expression of downstream Ndm genes in E.coli BL21(DE3) where T7 RNA polymerase is expressed, we assume that there was a leaky expression of Ndm genes in E.coli DH5α via an unrevealed mechanism.
| Sample | OD274 nm | Percent(%) | |
|---|---|---|---|
| BL21(DE3) | EV1 | 0.986 | 103.17% |
| EV2 | 0.924 | 96.69% | |
| EV3 | 0.957 | 100.14% | |
| Mean | 0.955666667 | ||
| ABCDE1 | 0.622 | 65.09% | |
| ABCDE2 | 0.611 | 63.93% | |
| ABCDE3 | 0.659 | 68.96% | |
| DH5α | EV1 | 0.98 | 100.89% |
| EV2 | 0.981 | 101.00% | |
| EV3 | 0.953 | 98.11% | |
| Mean | 0.971333 | ||
| ABCDE1 | 0.281 | 28.93% | |
| ABCDE2 | 0.388 | 39.95% | |
| ABCDE3 | 0.471 | 48.49% |
W4-3: to detect the expression of NdmA-E proteins in E.coli DH5α by SDS-PAGE.
Same as W3-1
Notes & Results:
Succeeded. Compared with the expressions of NdmA-E proteins in BL21(DE3), they were not significantly detected in DH5α. We supposed the “leaky expression” of NdmA-E in E.coli DH5α is so low and hard to be detected by SDS-PAGE.
Week 5 (8.4-8.8)
W5-1: to probe for the best fermentation temperature and endpoint for caffeine degradation.
- Fermentation
DH5α + IPTG + standard caffeine (400 μg/mL) at 16, 20, 24, 28, 32, and 36°C for 24 hours
DH5α + IPTG + standard caffeine (400 μg/mL) at 28°C for 12, 24, 36, 48, 60, and 72 hours
- Sample Preparation, purification, and caffeine quantification, same as W3-2
Notes & Results:
Succeeded. 28°C is the best fermentation temperature and 48h is the best fermentation endpoint for the engineered DH5α.
| Sample | OD274 nm | Percent(%) |
|---|---|---|
| EV1 | 1.157 | 99.00% |
| EV2 | 1.165 | 99.69% |
| EV3 | 1.184 | 101.31% |
| Mean | 1.168667 | |
| 16 °C-1 | 0.61 | 52.20% |
| 16 °C-2 | 0.624 | 53.39% |
| 16 °C-3 | 0.697 | 59.64% |
| 20 °C-1 | 0.584 | 49.97% |
| 20 °C-2 | 0.594 | 50.83% |
| 20 °C-3 | 0.64 | 54.76% |
| 24 °C-1 | 0.574 | 49.12% |
| 24 °C-2 | 0.572 | 48.94% |
| 24 °C-3 | 0.581 | 49.71% |
| 28 °C-1 | 0.467 | 39.96% |
| 28 °C-2 | 0.552 | 47.23% |
| 28 °C-3 | 0.481 | 41.16% |
| 32 °C-1 | 0.569 | 48.69% |
| 32 °C-2 | 0.564 | 48.26% |
| 32 °C-3 | 0.603 | 51.60% |
| 36 °C-1 | 0.823 | 70.42% |
| 36 °C-2 | 0.786 | 67.26% |
| 36 °C-3 | 0.82 | 70.17% |
| Sample | OD274 nm | Percent(%) |
|---|---|---|
| EV1 | 1.192 | 94.03% |
| EV2 | 1.353 | 106.73% |
| EV3 | 1.258 | 99.24% |
| Mean | 1.267667 | |
| 12h-1 | 0.721 | 56.88% |
| 12h-2 | 0.714 | 56.32% |
| 12h-3 | 0.698 | 55.06% |
| 24h-1 | 0.614 | 48.44% |
| 24h-2 | 0.594 | 46.86% |
| 24h-3 | 0.602 | 47.49% |
| 36h-1 | 0.484 | 38.18% |
| 36h-2 | 0.5 | 39.44% |
| 36h-3 | 0.512 | 40.39% |
| 48h-1 | 0.428 | 33.76% |
| 48h-2 | 0.406 | 32.03% |
| 48h-3 | 0.406 | 32.03% |
| 60h-1 | 0.424 | 33.45% |
| 60h-2 | 0.425 | 33.53% |
| 60h-3 | 0.411 | 32.42% |
| 72h-1 | 0.419 | 33.05% |
| 72h-2 | 0.421 | 33.21% |
| 72h-3 | 0.441 | 34.79% |
W5-2: to determine the best carbon and nitrogen source for bacterial growth and caffeine degradation.
- Fermentation
DH5α + IPTG + standard caffeine (400 μg/mL) + M9 medium + glucose, sucrose, maltose, and corn starch, at 28°C for 48h
DH5α + IPTG + standard caffeine (400 μg/mL) + M9 medium + tryptone, NaNO3, NH4Cl, and urea, at 28°C for 48h
- Sample Preparation, purification, and caffeine quantification, same as W3-2
Notes & Results:
Succeeded. Corn starch is the best carbon source and NaNO3 is the best nitrogen source for bacterial growth and caffeine degradation.
| EV | ABCDE | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| M9 | M9 | M9+Glu | M9+Sucro | M9+Mal | M9+Starch | |||||||
| OD274 | 0.851 | 100.00% | 0.48 | 56.40% | 0.194 | 22.80% | 0.268 | 31.49% | 0.14 | 16.45% | 0.052 | 6.11% |
| OD600 | 2.046 | 2.016 | 2.585 | 2.45 | 2.666 | 2.773 |
| ABCDE | ||||||||
|---|---|---|---|---|---|---|---|---|
| M9+Tryptone | M9+NaNO3 | M9+NH4Cl | M9+Urea | |||||
| OD274 | 0.153 | 17.98% | 0.07 | 8.23% | 0.123 | 14.45% | 0.232 | 27.26% |
| OD600 | 2.603 | 2.715 | 2.659 | 2.594 |
Week 6 (8.11-8.15)
W6-1: to further explore the best concentration of different nutrients for bacterial growth and caffeine degradation.
- Fermentation
DH5α + IPTG + standard caffeine (400 μg/mL) + M9 medium + different concentrations of corn starch, NaNO3, ZnSO4, FeSO4, MgSO4, at 28°C for 48h
- Sample Preparation, purification, and caffeine quantification, same as W3-2
Notes & Results:
Succeeded.
| EV | ABCDE+Starch | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| M9 | M9+0 | M9+4g/L | M9+2g/L | M9+1g/L | M9+0.67g/L | |||||||
| OD274 | 0.837 | 100.00% | 0.42 | 50.18% | 0.221 | 26.40% | 0.17 | 20.31% | 0.107 | 12.78% | 0.252 | 30.11% |
| OD600 | 2.054 | 1.969 | 2.127 | 2.285 | 2.3 | 2.126 |
| ABCDE+NaNO3 | ||||||||
|---|---|---|---|---|---|---|---|---|
| M9+4g/L | M9+2g/L | M9+1g/L | M9+0.67g/L | |||||
| OD274 | 0.21 | 25.09% | 0.152 | 18.16% | 0.181 | 21.62% | 0.292 | 34.89% |
| OD600 | 1.94 | 2.29 | 2.099 | 1.981 |
| EV | ABCDE+ZnSO4 | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| M9 | M9+0 | M9+0.05g/L | M9+0.1g/L | M9+0.2g/L | M9+0.3g/L | |||||||
| OD274 | 0.74 | 100.00% | 0.505 | 68.24% | 0.37 | 50.00% | 0.206 | 27.84% | 0.29 | 39.19% | 0.368 | 49.73% |
| OD600 | 1.325 | 1.247 | 1.237 | 1.433 | 1.41 | 1.256 |
| EV | ABCDE+FeSO4 | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| M9 | M9+0 | M9+0.05g/L | M9+0.1g/L | M9+0.2g/L | M9+0.3g/L | |||||||
| OD274 | 0.74 | 100.00% | 0.376 | 50.81% | 0.173 | 23.38% | 0.273 | 36.89% | 0.4 | 54.05% | 0.376 | 50.81% |
| OD600 | 1.325 | 1.331 | 1.52 | 1.404 | 1.352 | 1.331 |
| EV | ABCDE+MgSO4 | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| M9 | M9+0 | M9+0.1g/L | M9+0.25g/L | M9+0.5g/L | M9+1g/L | |||||||
| OD274 | 0.74 | 100.00% | 0.331 | 44.73% | 0.25 | 33.78% | 0.2 | 27.03% | 0.403 | 54.46% | 0.331 | 44.73% |
| OD600 | 1.325 | 1.344 | 1.389 | 1.424 | 1.359 | 1.344 |
W6-2: to quantify the caffeine degradation in spent coffee grounds by fermentation using the engineered DH5α.
- Fermentation
DH5α + IPTG + SCG (100 g) + corn starch, NaNO3, ZnSO4, FeSO4, MgSO4, at 28°C for 48h
- Sample Preparation, purification, and caffeine quantification, same as W3-2
Notes & Results:
Succeeded.
| Sample | OD274 nm | Percent(%) |
|---|---|---|
| EV1 | 54.652 | 101.34% |
| EV2 | 53.654 | 99.49% |
| EV3 | 53.482 | 99.17% |
| Mean | 53.92933 | |
| ABCDE1 | 3.961 | 7.34% |
| ABCDE2 | 3.975 | 7.37% |
| ABCDE3 | 3.977 | 7.37% |
