Difference between revisions of "Part:BBa K5492101"

Latest revision as of 10:19, 2 October 2024

pETDUET-1

pETDUET-1 plasmid used for expression of Histamine-N-Methyltransferase BBa_K5492400, BBa_K5492401 and Diamine Oxidase BBa_K5492200 in multiple e.coli chassis.

Sequence and Features

As seen upon the map below, pETDUET-1 contains two lac operons, an AmpR gene, and contains multiple restriction sites. Our team has utilised the EcoRI site starting in position 112, and the BglII site starting in position 305. Additionally, we have utilised an RBS in position 58, and a start codon is located before the multiple cloning site, in position 71. A 6xHis tag is also found at position 83, which we have utilised in our gene designs in order to ease the protein purification process.

Restriction – pETDUET-1 plasmid

We performed the digestion of the plasmid five times with the same amount of plasmid. We added the following substrates in order:

The dilution of the plasmid: We took out 12.5µL (400 µg/µL) plasmid and we added 87.5µL water. Then we took out 10µL of this solution and added 990µL water. Thus the final concentration of the plasmid was 0.5 µg/µL.  

Ligation

We added the following substrates in order:

Transformation

Protocol for the transformation of DH10B E. coli strain

1. For C2987H: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for
10 minutes.
2. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flickthe tube 4-5 times to mix cells and DNA. 
Do not Vortex.
3. Place the mixture on ice for 30 minutes. Do not mix.
4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
5. Place on ice for 5 minutes. Do not mix.
6. Pipette 950 µl of room temperature SOC into the mixture.
7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8. Warm selection plates to 37°C.
9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
10. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C.
Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours. 

After transforming the DH10B E. coli strain using the heat shock method, bacterial colonies successfully grew on the antibiotic-containing culture medium.