Team:UNSW Australia/Notebook

NOTEBOOK

WEEK 1

31st August - 6th September

Cloning

• Prepared LB broth and LB agar plates.
• Assembled HSP22E and HSP22F inserts into pET-19b backbone with Gibson Assembly.
• Transformed assembled plasmid into competent E. coli BL21 (DE3).
• Colony PCR of transformed colonies. PCR product run on agarose gel electrophoresis.
• Prepared starter cultures and performed QIAGEN Miniprep to extract and purify plasmids.
• Purified plasmid sample sent for Sanger Sequencing.

WEEK 2

7th September - 13th September

Protein Expression and Purification

• Prepared starter cultures of transformed colonies for induction.
• Took patch plates and made glycerol stocks
• Inoculated larger cultures and induced with IPTG.
• Harvested and froze cells.
• Conducted BugBuster protein expression check and SDS-PAGE to visualise results.
• Prepared binding buffer and elution buffer stocks.

WEEK 3

14th September - 20th September

Protein Expression and Purification

• Cell lysis of harvested cells by sonication
• Protein purification attempt #1 using gravity column.
• SDS-PAGE of purification attempt #1.
• Protein purification attempt #2 using centrifugation alongside gravity column.
• SDS-PAGE of purification attempt #2.

WEEK 4

21st September - 27th September

Protein Expression and Purification

• Research and design process to refine purification protocol.
• Protein purification attempt #3 using centrifugation alongside the gravity column.
• SDS-PAGE of purification attempt #3.

WEEK 5

28th September - 4th October

Characterisation and Assay

• Prepared Dialysis Buffer.
• Dialysis attempt #1.
• Prepared new Dialysis Buffer.
• Dialysis attempt #2.

WEEK 6

5th October - 11th October

Characterisation and Assay

• Research and design process to refine Bradford assay protocol.
• Protein concentration assay (Bradford Assay).
• Research and design process to create citrate synthase assay protocol.

WEEK 7

12th October - 18th October

Characterisation and Assay

• Citrate synthase assay attempt #1.
• Research and design process to refine citrate synthase assay protocol.
• Citrate synthase attempt #2.

WEEK 8

19th October - 23rd October

Characterisation and Assay

• Citrate synthase assay attempt #3.
• SDS-PAGEof purified HSP22E and HSP22F with and without DTT.
• Native PAGE of purified HSP22E and HSP22F.

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