JUNE-JULY

Was trying to produce more protein from the 1.6L induced bacterial cultures.

Purified with Affinity chromatography successfully. (Although lost an amount of protein due to my mistakes)

Concentrated it with a protein concentrator since it has been diluted.

Polishing with size exclusion chromatography.

The peak of elution is a bit broad although it is very high peak.

After SDS PAGE analysis, the results shown a thick fat band at around 80kDa, but problem is they are not alone. some contaminants found at size 30-60kDa.

Are they complex with PhaC? malfunction of the column? or any other possibilities?

Optimization of gel filtration

These are the factors to be considered in order to optimize gel filtration resolution:

1. Sample Volume

2. Ratio of sample volume to column volume

3. Column dimensions

4. Particle size

5. Particle size distributions

6. Packing density

7. Pore size of the particles

8. Flow rate

9. Viscosity of the sample and buffer

cloning, protein expression and purification

Cloning

Cloning of genes and bacteria

Escherichia coli Rosetta-gami2(DE3) selected as bacterial host for this cloning.

Expression vector pET32 Xa/Lic used as expression vector.

PHA synthase from Chromobacterium spp. USM2 was amplified by PCR and cloned into expression vector and transformed into bacterial host.

The culture conditions for E.coli RG2 (DE3) pET32-phaCcs are 37degree C, shake 180 rpm.

For selection indicator, This transformant resist Chloramphenicol, Streptomycin, tetracycline and Ampicillin.

Protein Expression

Induction

Transformed bacteria were cultured at 37degree C, 180 rpm for 4-4hours 30mins to reach an OD600nm reading of ~0.5.

Then, a concentration of 0.4mM IPTG added into the culture.

The bacteria suspension were incubate at 30degree C, 180rpm for 8hours.

Sonication

Cells were break by sonicate @ 90V power output for 1 minute and cold for 1minute (5cycles).

Then, centrifugation at 4degreeC kubota at 8000rpm, 20minutes to pellet the insoluble fraction protein.

The supernatant was treated as soluble fraction protein.

SDS P.A.G.E

The uninduced total protein, induced total protein, Soluble fraction, Insoluble Fraction and protein ladder were used to run SDS P.A.G.E for analysis.

Purification

Metal affinity chromatography

Only the soluble fraction of the protein were proceeded with purification.

SDS P.A.G.E

The elution, binding flow through, washing flow through are used as SDS sample to analyse the purity.

Size exclusion chromatography

To remove Imidazole, further polishing the purity of the phaC protein.

Welcome

Welcome to my researcher's challenging life!

Currently, I'm working on bioplastic synthase, an enzyme catalyzing the polymerization of the bioplastic. The project is supervised under Assoc. Prof. Dr. Razip, School of biological Sciences, Universiti Sains Malaysia.

This project can be categorized into 3 parts, namely Cloning, Expression and Crystallization.

Research Laboratory 414 and MBBS crystallization suite are the working location throughout the whole experiment.

Research Lab 414 is a lab for cloning of bacteria and genes, protein expression and bacterial cultures, and some minor works of protein purification and SDS P.A.G.E.

While MBBS is a lab for all protein works such as protein purification (gel filtration/ metal affinity chromatography/ Ion exchange) and protein crystallization (equip with all the cold room facilities and crystallization suite).

This is my working bench in lab414. Is quite messy

(proof of hardworking ma...lol)