Difference between revisions of "Part:BBa K4033001"
 
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<b><font size="3">Experimental approach</font></b>
 
<b><font size="3">Experimental approach</font></b>
  
 +
1) Absorb 500ul of overnight cultured bacterial solution and transfer it to 50ml (1:100) liquid LB medium containing corresponding antibiotics KanR. Shake at 37 ℃ and 220rpm for about 2.5h until E. coli is in logarithmic phase (a550 is about 0.6-0.8)
  
 +
2) IPTG was added to the final concentration of 0.5mmol/l and induced at 37 ℃ for 4hours
 +
 +
3) Centrifuge at 4 ℃ 6000rpm for 10min and discard the supernatant
 +
 +
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)
 +
 +
5) 12000rpm, centrifugation at 4 ℃ for 20min, collect supernatant (large floor centrifuge, distilled water balancing)
 +
 +
6) Freezing at - 80 ℃，or later soon used in expression of protein.
 +
 +
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
 +
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
  
  

Latest revision as of 17:07, 20 October 2021

ZhuI

ZhuI is a monodomain ARO/CYC that promotes the regiospecific C7–C12 first-ring cyclization for unreduced polyketide chains of variable lengths (C16 and C18–C20). And C7–C12 specificity for first-ring cyclization is unique among nonreducing type II PKS systems.


Biology and Usage

ZhuI encodes the cyclases that catalyzes formation of the C7-C12 first ring closure, which is unique among nonreducing type II PKS systems. ZhuI is proposed to catalyze aldol condensation between C7 and C12 (followed by aromatization) to yield intermediate SEK4b, which is then transferred to ZhuJ for second-ring cyclization between C5 and C14 (followed by aromatization) to give intermediate SEK4. After third-ring cyclization between C2 and C15, presumed to occur spontaneously, flavokermesic acid anthrone forms（Fig.1）[2].

ZhuI and ZhuJ are usually used together for typeⅡPKS reconstitution.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 229
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 203
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Experimental approach

1) Absorb 500ul of overnight cultured bacterial solution and transfer it to 50ml (1:100) liquid LB medium containing corresponding antibiotics KanR. Shake at 37 ℃ and 220rpm for about 2.5h until E. coli is in logarithmic phase (a550 is about 0.6-0.8)

2) IPTG was added to the final concentration of 0.5mmol/l and induced at 37 ℃ for 4hours

3) Centrifuge at 4 ℃ 6000rpm for 10min and discard the supernatant

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)

5) 12000rpm, centrifugation at 4 ℃ for 20min, collect supernatant (large floor centrifuge, distilled water balancing)

6) Freezing at - 80 ℃，or later soon used in expression of protein.

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 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