The definition of biodegradability

Biodegradability definition

According to

Towards Common Ground –

Meeting Summary of the International Workshop on Biodegradability,

Annapolis, MD, USA, 1992.

· For all practical purposes of applying a definition, material manufactured to be biodegradable must relate to a specific disposal pathway such as composting, sewage treatment, denitrification, or anaerobic sludge treatment.

· The rate of degradation of a material manufactured to be biodegradable has to be consistent with the disposal method and other components of the pathway into which it is introduced, such that accumulation is controlled.

· The ultimate end products of aerobic biodegradation of a material manufactured to be biodegradable are carbon dioxide, water and minerals and that the intermediate products include biomass and humic materials. (Anaerobic biodegradation was discussed in less detail by the participants).

· Materials must biodegrade safely and not negatively impact on the disposal process or the use of the end product of the disposal.

Aerobic biodegradation

CPOLYMER + O2 -------> CO2 + H2O + CRESIDUE + CBIOMASS

Anaerobic biodegradation

CPOLYMER ---------> CO2 + CH4 + H2O + CRESIDUE + CBIOMASS

Stuck @ dec 2010

E. coli Rosettagami2(DE3) with pET32 harbouring phaCcs gene

problem 1: Expression level of PhaC is very low compare to previous batches

(less than 10% , will be very difficult to be purified)

problem 2: Almost 100% of the expressed PhaC is insoluble

(not a native functional protein we want)

Troubleshoot:

1. IPTG is not functioning?

- trial: induce with IPTG from colleage which is functioning for their expression.

Move forward!

This week will be a busy week starting from tomorrow.

Maybe should say it is a busy month too.

I do not want to waste my time anymore. So let's get to work.

An amount of bacterial hosts and expression vectors appearing in my minds now.

The classic - BL21(DE3) and BL21(DE3) pLysS.

The hybrid of origami and rossetta - Rosetta-gami2(DE3)

The NEB express - ER2523

+

pET32 Xa/Lic

pCold1 Xa

pMAL -c5x

pMAL -p5x

Good luck and all the best to me!

As well as the revival of glycerol stock of my precious Rosetta-gami2(DE3)pET32-PhaCcs which used to produce soluble PhaCcs.

Insoluble protein?!

According to SDS P.A.G.E for solubility which I did on 18 AUG, the result doesn't looks good.

No PhaC protein on the soluble fraction; but a estimated ~15% expressed PhaC in insoluble fraction. [Troubleshooting: I still got other stock, will sonicate other bacterial cells and check again]

Maybe it is better to subclone the stored plasmid to Rosetta-gami2 (DE3) and BL21 (DE3) , BL21 (DE3)/pLySs?

At the same time, design primer and clone into pCold and pMAL.

Will email Dr after I have troubleshoot for insolubility and plan the cloning experiments in this weekend.

August

Last 2 weeks screening for the culture of RG2 pET32 phaCcs between 2 culture from glycerol stock.

As a result, one of the bacteria was contamination, didn't express protein phaC and didnt contain the correct plasmids.

Then I continue to culture and induce the correct bacteria with a total amount of 2 litre media. (5 X 400ml culture)

After last weekend rest, I'm ready to continue to the next step, here is the plan.

17 AUG 2010 (Done)

1. SDS PAGE (for total protein expression)

18 AUG 2010

1. Sonication for 400ml culture (2 X 250ml centrifuge tube culture, others were kept for further uses at -20C) (DONE)

2. SDS PAGE (to check the solubility) (DONE)

3. PhaC temperature stability test. (just choose 2 different temperature, 4C and RT ~25C, I'm wondering the stability of my PhaC protein, so just wanna get more information about its degradation rate.) (Reschedule to 19 AUG, need to use pure protein)

19 AUG 2010

1. Purification by TALON

2. Purification by denatured conditions (TALON as well, doing this to check whether the suspicious 30kDa protein is the complex of PhaC protein or not)

3. SDS PAGE (to check the purity)

20 AUG 2010

1. Gel filtration (optimized condition, slower flow rate, smaller volume sample)

2. SDS PAGE

21/22 AUG 2010

1. PhaC enzyme activity assay (Joanne)

*Progress of the plan flow will be indicated and post will be edited accordingly.

Also as a guideline to push my laziness away.

*At the same time, 20AUG2010 will be our small group meeting for the structure of PhaC review which is 1 week later than our predicted schedule.

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)