Team:Sheffield/Notebook
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NOTEBOOK |
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CLONING OF HEMERYTHRIN
Week 1:
-Literature research
-Decided an experimental outline
-In silico design of hemerythrin constructs
-Synthetic genes and primers required for cloning have been ordered
Week 2:
-Make competent cells for the E.coli strains that we used during our project- Top10, JC28 and W3110
-Tested the efficiency of transformation in these strains
-Prepared buffers, growth media and pouring agar plates for the experiments we were planning to carry out during the following weeks
-Several experiments have been performed in order to monitor the growth of the wild type (W3110) and siderophore-deficient mutant (JC28)
Week 3:
-Ensured we had sufficient amounts of plasmid for further cloning experiments:
- transformed the pSB1C3, pUC18 and pBSKII plasmids into Top10 competent cells
- made plasmid mini-preps from these transformed cells
-Characterised the plasmid stocks obtained (Nano Drop and agarose gel)
-Characterised the genotype of wild-type (W3110) and mutant (JC28) strains:
- -genomic DNA extractions have been performed
- - entC gene has been amplified- PCR
- -PCR products have been analysed- agarose gel
