Difference between revisions of "Part:BBa K4033002"
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<b><font size="3">Experimental approach</font></b>
 
<b><font size="3">Experimental approach</font></b>
  
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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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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 ℃
 +
 +
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
  
  

Revision as of 17:15, 19 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) 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)

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 ℃

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


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