Part:BBa_K3797006
Fre-L-SttH
Fre-linker-SttH is used to produce the regiospecific 6-brominated precursors.This year, we are trying to find a novel approach for bio-production of Tyrian purple. Fre and SttH are both used in the first step of our bio-production. SttH can help adding Br to tryptophan to produce the regiospecific 6-brominated precursors while Fre transfer FAD to FADH2. Combining SttH to Fre can also help solve the problem of the low solubility of SttH, making the protein function better in the cell.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1479
Illegal PstI site found at 942 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1479
Illegal NheI site found at 7
Illegal PstI site found at 942 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1479
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1479
Illegal PstI site found at 942 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1479
Illegal PstI site found at 942 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1029
Illegal SapI.rc site found at 318
Illegal SapI.rc site found at 438
Design and Properties
We connected it to the inducible lac promoter and induced its expression to complete the final oxidation reaction in the production of indigo violet.
Experimental approach
1. Use the EasyPure® HiPure Plasmid MiniPrep Kit from Beijing transgene biology to extract The plasmid, and the operation was carried out according to its instructions.
2. Prepare a double enzyme digestion system for plasmid digestion. The enzyme digestion system consists of 1 μL of two restriction enzymes and 5 μ l of enzyme digestion buffer to ensure that the plasmid is about 2 μg and supplemented with double distilled water to a total volume of 50 μL. The enzyme digestion time shall be determined according to the restriction enzyme instructions.
3. The agarose gel was prepared with gel concentration of 0.8%. GelGreen nucleic acid dye was used for gel dyeing, and the dye was added in the proportion of one thousandth. When loading, the volume ratio of loading buffer to sample solution is 10:1, and the volume of sample solution is 1 μL. 1Kb standard marker was used for electrophoresis.
4. Induced expression. Absorb 500ul of overnight cultured bacterial solution and transfer it to 50ml (1:100) liquid LB medium containing corresponding antibiotics. Shake at 37 ℃ and 220rpm for about 2.5h until E. coli is in logarithmic phase (a550 is about 0.6-0.8). IPTG was added to the final concentration of 0.5 mmol/L and induced at 37 ℃ for 4h.
5. Protein extraction. Centrifuge at 4 ℃ 6000rpm for 10min and discard the supernatant. Add at least three times the volume of PBS buffer into the bacteria, and conduct ultrasonic crushing under the condition of ice bath until the E. coli suspension becomes a non viscous liquid (output power 1200W, ultrasonic time 2s, interval 10s, ultrasonic times 40 times). 12000rpm, centrifugation at 4 ℃ for 20min, and collect the supernatant.
6. SDS-PAGE。Protein samples and 4 × Mix the loading buffer and keep it at 100 ℃ for 3-5 min. 12000r / min, centrifugation for 10min, and take the supernatant for use. Place the purchased preformed glue and apply the sample. Add the sample and broad-spectrum protein marker into various product holes one by one, and the amount of sample application is 10ml. Connect the electrophoresis tank to the power supply of the electrophoresis instrument and start electrophoresis with a constant voltage of 200V. When bromophenol blue moves to the leading edge, cut off the power supply and stop electrophoresis. Remove the gel from the electrophoresis tank, carefully remove the gel, soak the gel in R250 dyeing solution, dye 2h, decolorizing liquid to decolorization until the zone is clear.
Reference
[1] Lee J, Kim J, Song JE, Song WS, Kim EJ, Kim YG, Jeong HJ, Kim HR, Choi KY, Kim BG. Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli. Nat Chem Biol. 2021 Jan;17(1):104-112. doi: 10.1038/s41589-020-00684-4. Epub 2020 Nov 2. PMID: 33139950.
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