Difference between revisions of "Part:BBa K4033002"
 
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1. Pre-growth
 
1. Pre-growth
  
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1) Absorb 500ul of overnight cultured bacterial solution and transfer it to 50ml (1:100) liquid LB medium containing corresponding antibioticsKanR. Shake at 37 ℃ and 220rpm for about 2.5h until E. coli is in logarithmic phase (a550 is about 0.6-0.8)
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1) Prepare 50mg / ml KanR mother liquor, filter and sterilize with filter membrane
  
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2) IPTG was added to the final concentration of 0.5mmol/l and induced at 37 ℃ for 4hours
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2) Add 5ml LB liquid medium into 10ml centrifuge tube and 2ul streptomycin
  
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3) Centrifuge at 4 ℃ 6000rpm for 10min and discard the supernatant
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3) Scrape some agar (including bacteria) along the puncture line with the inoculation ring, and extend the inoculation ring into the LB medium in the corresponding centrifuge tube to complete the inoculation
  
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4) Add at least three times the volume of PBS buffer into the bacteria, and conduct ultrasonic crushing under ice bath conditions until the E. coli suspension becomes a non viscous liquid (output power 1200W, ultrasonic time 2s, interval 10s, ultrasonic times 40 times)
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4) 37 ℃ 220 RPM overnight grow
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5) 12000rpm, centrifugation at 4 ℃ for 20min, collect supernatant (large floor centrifuge, distilled water balancing)
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6) Freezing at - 80 ℃, or later used in expression.
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2. Protein Expression
 
2. Protein Expression

Latest revision as of 17:21, 20 October 2021


ZhuJ

ZhuJ is a CYC2 that promotes the regiospecific C5-C14 second-ring cyclization for non-reduced polyketide chains of the R1128 family.[1]


Biology and Usage

ZhuJ and OKS encodes the aromatase that catalyzes the C5-C14 second-ring closure, which spontaneously leads C2-C15 to form the third-ring, and produce a 2.7-fold increase in flavokermesic acid levels(Fig.a). ZhuJ is proposed to catalyze the condensation between C5 and C14 to yield intermediate SEK4, which is the byproduct of the reaction. After the cyclization of the third-ring (C2-C15) forms spontaneously, flavokermesic acid anthrone forms.[2]


ZhuI and ZhuJ are usually used together for typeⅡPKS reconstitution.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 312
    Illegal NgoMIV site found at 594
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 160


Experimental approach

1. Pre-growth

1) Prepare 50mg / ml KanR mother liquor, filter and sterilize with filter membrane

2) Add 5ml LB liquid medium into 10ml centrifuge tube and 2ul streptomycin

3) Scrape some agar (including bacteria) along the puncture line with the inoculation ring, and extend the inoculation ring into the LB medium in the corresponding centrifuge tube to complete the inoculation

4) 37 ℃ 220 RPM overnight grow

2. Protein Expression

7) Take 500ul of bacteria cultured overnight, add them to 10ml LB medium containing corresponding antibiotics KanR, and expand the culture at 37 ℃ and 220rpm for 2.5h

8) 0.1M IPTG 50ul (final concentration: 0.5mm) was added to the LB medium of the induction group of the three bacteria, which was induced at 37 ℃ and 220 rpm for 4 hours; The control group of the three bacteria did not add IPTG, and the parallel experiment was carried out

9) Draw 1ml bacterial solution from the cultures of the experimental group and the control group of each strain, add it to the 1.5ml EP tube, centrifuge at 12000rpm for 1min, discard the supernatant, add 50ul ddH2O to resuspend the bacteria, and then add 50ul SDS loading buffer

The mixed bacteria and loading buffer were heated at 100 ℃ for 10 min

10) Put the heated protein into - 20 ℃ for standby

3. protein extraction

1) Draw 1ml bacterial solution from the cultures of the experimental group and the control group of each strain, add it to the 1.5ml EP tube, centrifuge at 12000rpm for 1min, discard the supernatant, add 50ul ddH2O to resuspend the bacteria, and then add 50ul SDS loading buffer

2) The mixed bacteria and loading buffer were heated at 100 ℃ for 10 min

3) Put the heated protein into - 20 ℃ for standby

4. SDS polyacrylamide gel Preparation for protein detection

1) Put the dried glass plate on the support of electrophoresis tank and fix the glass plate

2) Equipped with 12% separating glue

3) After the separation glue is configured, immediately pour it into the electrophoresis tank between the two glass plates, stop about 3cm away from the upper mouth, and then add a thin layer of water for water sealing for about 40 minutes, waiting for the glue to condense naturally. After condensation, pour out distilled water to absorb the water.

4) Equipped with 4% condensation glue

5. Electrophoresis

1) Connect the electrophoresis tank to the power supply of the electrophoresis instrument and start electrophoresis. First, the constant voltage is 80V. After the sample enters the separation gel, the constant voltage is 120V

2) When bromophenol blue moves to the leading edge, cut off the power supply and stop electrophoresis

6. Dyeing

1)Preparation of dyeing solution:

0.03g Coomassie Brilliant Blue + 7.5ml isopropanol + 3ml acetic acid

2)dyeing

Remove the gel from the electrophoresis tank, carefully remove the gel, immerse the gel in dyeing solution for half an hour.

7. Decolorization

1) Preparation of decolorizing solution:

15ml isopropanol + 6ml glacial acetic acid + 39ml water

2) Decolorization: decolorize until the strip is clear


References

[1] Ames, B. D., Lee, M. Y., Moody, C., Zhang, W., Tang, Y., & Tsai, S. C. (2011). Structural and biochemical characterization of ZhuI aromatase/cyclase from the R1128 polyketide pathway. Biochemistry, 50(39), 8392–8406. https://doi.org/10.1021/bi200593m

[2] Frandsen, R.J.N., Khorsand-Jamal, P., Kongstad, K.T. et al. Heterologous production of the widely used natural food colorant carminic acid in Aspergillus nidulans. Sci Rep 8, 12853 (2018). https://doi.org/10.1038/s41598-018-30816-9