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

Constitutive hemerythrin expression

Week 4:
-Synthetic hemerythrin genes as well as primers required for amplifying these genes have arrived
-Synthetic genes have been amplified (PCR) and the success of the PCR reactions has been tested (PCR and Nano Drop)
-Synthetic genes as well as plasmid backbones (pSB1C3 and pBSKII) have been digested using appropriate restriction enzymes
-Ligation reactions have been set up
-Ligated plasmids have been transformed in TOP10 competent cells

Week 5:
-Plasmid mini-preps have been made from the TOP10 cells transformed with constitutively expressed hemerythrin genes (both pSB1C3+hemerythrin and pBSKII+hemerythrin)
-Small fractions of harvested pSB1C3+hemerythrin plasmids have been digested with appropriate restriction enzymes; restriction products have been run through an agarose gel and screened for the presence of desired inserts
-Plasmids containing desired inserts have been transformed in W3110 (wild-type) and JC28 (siderophore-deficient) E.coli strains
-Plasmid mini-preps have been made again from transformed W3110 and JC28 strains

Week 6:
-Plasmid mini-preps (from W3110 and JC28 strains transformed with pSB1C3+hemerythrin) have been digested with appropriate restriction enzymes
- Small fractions of harvested pSB1C3+hemerythrin plasmids have been digested with appropriate restriction enzymes; restriction products have been run through an agarose gel and screened for the presence of desired inserts
-Plasmids containing the appropriate inserts have been sent for sequencing
-Sequences have been analysed

Week 7:
- Measured absorbance at 500nm in overnight cultures of Dcr and Mc in JC28 and W3110 strains and Td in W3110 strain in chloramphenicol LB
-No change in colour has been detected between hemerythrin encoding mutant, wild-type strains and negative controls (no hemerythrin expression) suggesting that protin was not expressed
-Tried to figure out what was going on– realised that there were several methylation sites at adjacent positions to the promoters used
-Decided that we should replace the promoters and remove the methylation sites
-New promoters have been ordered

Week 10:
-Promoters have been cut out from the linear hemerythrin constructs
-Promoterless hemerythrin genes have been cloned into pSB1C3
-pSB1C3+promoterless hemerythrin plasmids have been cloned into TOP10
-Very few colonies of transformant TOP10 have been observed on every plate

Week 11:
-Cloning of promoterless hemerythrin genes into pSB1C3 has been carried out again, allocating longer incubation times for digestion and ligation reactions to take place
-Ligated plasmids have been transformed in TOP10
-Plasmids have been re-harvested from TOP10 and screened for the presence of the insert (samples were digested with appropriate restriction enzymes and run through an agarose gel)
-The new promoters have been cloned into the pSB1c3+promoterless hemerythrin genes containing desired inserts; these plasmids have been transformed into DH5α cells

Week 12:
-pSB1C3+new promoter+promoterless hemerythrin genes plasmids have been harvested from DH5α cells and screened for the presence of desired inserts
-Whole cell lysates of the transformed cells have been made and the soluble and insoluble protein fractions have been separated
-Soluble and insoluble protein fractions have been run through an SDS-PAGE gel
-Gel was stained with Cromassie Blue
-SDS-PAGE gels shown that hemerythrin genes have not been successfully expressed

Weeks 13-17:
-Cloning and submitting iGEM BioBricks

Overexpression of hemerythrin

Week 4:
-Synthetic hemerythrin genes as well as primers required for amplifying these genes have arrived
-Synthetic genes have been amplified (PCR) and the success of the PCR reactions has been tested (PCR and Nano Drop)

Week 6:
-PCR amplifications of the hemerythrin genes – experiment failed due to issues with the primers
-in silico design of new primers

Week 7:
-All 3 hemerythrin genes (Dcr, Td and Mc) have been amplified and inserted into each of the 2 overexpression plasmids- pET15b and pET28a
-Ligated plasmids have been transformed in TOP10 competent cells, but unexpected cell morphologies have been observed, therefore the cloning procedure has been repeated again (week 8)

Week 8:
-Hemerythrin genes have been cloned again in pET15b and pET28a
-Ligated plasmids have been transformed in TOP10
-Overexpression plasmids have been re-harvested from transformant TOP10 cells

Week 9:
-Harvested plasmids have been checked for the presence of the desired inserts (samples have been digested with appropriate restriction enzymes and run through an agarose gel)
-Plasmids containing the desired insert have been transformed in BL21 cells

Week 11:
-Confirming that the transformed BL21 cells contain plasmids with the appropriate inserts (samples were digested with appropriate restriction enzymes and run through an agarose gel)
-IPTG induction of hemerythrin genes
-Characterisation of protein expression profile (SDS-PAGE)