Difference between revisions of "Part:BBa K5492403"

Latest revision as of 11:42, 2 October 2024

Optimised HNMT - pETDUET-1

Optimised coding sequence of human Histamine-N-Methyltransferase appropiated for insertion into pETDUET-1. The part was assembled from notable features of pETDUET-1, and the optimised HNMT gene.

This part also serves as a contribution to part BBa_K364777.

Usage and Biology

Histamine-N-Methlytransferase (HNMT) takes part in the inactivation of histamine. It transfers a methyl group to histamine, and the resulting N-methyl histamine isn’t able to bind to histamine receptors, thus reducing the biological effect of the compound. Limiting the level of histamine effect can be an appropriate approach in cases of inflammation, where you can anticipate the histamine level increase. On our skin this includes some chronic diseases such as psoriasis, but more often would be useful in handling the acute increase of histamine level, such as a wasp bite. The decrease in oral histamine administration may also help to prevent some acute GI inflammation and smoothing the symptoms of some chronic bowel diseases.

Experiments

PCR – optimized HNMT

We have found the following PCR protocol the most optimal for the part:

HNMT According to the previous PCR reaction we decided to use S1 and N1 (from our previous reactions.) We added the following substrates in order: (each optimized and wild type)

We added 6X DNA Loading dye to each PCR tubes. We loaded the wells of the gels with 12μL of DNA solution in the following order: 

1. S1O (optimized) 2. S1W (wild type) 3. N1O (optimized) 4. N1W (wild type) 5. Ladder 

Restriction – optimised HNMT

We performed digestion of our sequences using EcoRI and BglII. Our protocol: We added the following substrates in order: The used green buffer contained the loading dye too.

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 flick the 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.

ELISA

ELISA assay performed on HNMT proteins.

Table of measured absorbance at 450nm

A1-A8: Standard stock, in 2X serial dilution.
B1-B8 ; C1-C8; D1-D8: Capsuled enzyme with liposome, samples taken at different times.
E1-E8; F1-F8; G1-G8: Referential enzyme without liposome, samples taken at different times.
H1-H2: Optimised HNMT
H3-H4: Wild type HNMT
H5-H6: 10X diluted opt. HNMT
H7-H8: 10X diluted wild type HNMT