Difference between revisions of "Part:BBa K4033005"
| Line 32: | Line 32: | ||
2. Expression of protein | 2. Expression of protein | ||
| − | 1） Add 10ml LB medium into 50ml centrifuge tube and 4ul | + | 1） Add 10ml LB medium into 50ml centrifuge tube and 4ul Kanr. Make two tubes for each plasmid, one of which is the control |
2) 500ul of bacteria cultured overnight were added to 10ml LB medium and expanded at 37 ℃ and 220rpm for 2.5h | 2) 500ul of bacteria cultured overnight were added to 10ml LB medium and expanded at 37 ℃ and 220rpm for 2.5h | ||
| Line 42: | Line 42: | ||
5） The mixed bacteria and loading buffer were heated at 100 ℃ for 10 min | 5） The mixed bacteria and loading buffer were heated at 100 ℃ for 10 min | ||
| − | 6） Put the heated protein into - 20 ℃ for standby | + | 6） Put the heated protein into - 20 ℃ for later use |
| + | |||
| + | 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 | ||
Revision as of 17:17, 20 October 2021
Ant B
Ant B is extracted from Photorhabdus luminescens, and is responsible for charging Ant F which functionalizes Ant F with a 4’ phosphopantetheine arm necessary for activity.[1]
Biology and Usage
Ant B and Coenzyme A (CoA) ligase (Ant G), were required as alternative auxiliary enzymes to carry out the posttranslation modification - functionalize ACP (Ant F) with a 4’ phosphopantetheine arm necessary for the activity (Ant F is charged by Ant B)– which resulted in detectable biosynthesis of molecules putatively identified as SEK4, SEK4b. E. coli endogenous 4’ phosphopantetheinyl transferases (PPTases)—AcpS and Ent D—could not efficiently realize this process. Expression of the mPKS (antDEF) alone did not produce any detectable masses corresponding to SEK4/SEK4b.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Experimental approach
1. Pre growth
1) Prepare 50mg / ml streptomycin 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. Expression of protein
1） Add 10ml LB medium into 50ml centrifuge tube and 4ul Kanr. Make two tubes for each plasmid, one of which is the control
2) 500ul of bacteria cultured overnight were added to 10ml LB medium and expanded at 37 ℃ and 220rpm for 2.5h
3） 0.1M IPTG 50ul (final concentration: 0.5mm) was added to the LB medium of the induction group of two plasmids, which was induced at 37 ℃ and 220rpm for 4H; The control group of the two plasmids did not add IPTG, and the parallel experiment was carried out
4） Suck 1ml bacterial solution from the four cultures, add it to 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
5） The mixed bacteria and loading buffer were heated at 100 ℃ for 10 min
6） Put the heated protein into - 20 ℃ for later use
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] Cummings M, Peters AD, Whitehead GFS, Menon BRK, Micklefield J, Webb SJ, et al. (2019) Assembling a plug-and-play production line for combinatorial biosynthesis of aromatic polyketides in Escherichia coli. PLoS Biol 17(7): e3000347. https://doi.org/10.1371/journal.pbio.3000347