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)

CLONING OF Ryb-GFP

Week 6:
-in silico design of the GFP-Ryb construct; synthetic genes as well as primers required for cloning have been ordered

Week 8:
-Primers and synthetic GFP-Ryb constructs have arievd
-PCR amplification of the constructs
-pSB1C3 and amplified GFP-Ryb constructs have been digested with appropriate restriction enzymes
-Ligated plasmids have been transformed in Top1
-Following antibiotic selection no bacterial growth was observed so all experiments had to be repeated (week 9)

Week 9:
-Attempts to clone Ryb-GFP constructs into pSB1C3, but cloning experiments did not work due to issues with the forward primers
-Designed new primers for amplifying these constructs

Week 10:
-New genes and primers ordered have arrived
-PCR of the new Ryb-GFP constructs

Week 11:
-PCR amplified GFP-Ryb constructs have been cloned into both pSB1C3 and pBSKII
-Ligated plasmids have been transformed into Top10
-Transformed Top10 cells have been screened for the expression of GFP
-Plasmids from transformed cells have been harvested, checked for the presence of desired inserts and transformed into W3110 and JC28

Week 12:
-Fluorescence –based assays have been carried out in order to analyse the intensity of GFP when W3110 and JC28 are incubated with low and high iron concentrations

Weeks 13-17:
-Further characterisation of the Ryb-GFP constructs
-Cloning and submitting iGEM BioBricks

IRON MEASUREMENTS

Week 1:
-Assessing sensitivity range of ferene for measurement of iron concentrations

Week 2:
-Assessing cell lysis and iron reduction/dissolution techniques for intracellular iron concentration measurement
-Ascorbic acid selected as reducing agent, effect of pH on ferene absorbance investigated
-Different cell lysis methods were performed on E.coli cells and absorbance compared. Sonication found to be more effective than lysis with SDS

Week 3:
-Assessing cell lysis and iron reduction/dissolution techniques for intracellular iron concentration measurement
-Attempted to optimise pH to produce higher absorbances
-Using ferene assay on cells grown in different media, produced low absorbances

Week 4:
-Assessing cell lysis and iron reduction/dissolution techniques for intracellular iron concentration measurement
-Attempted to optimise pH to produce higher absorbances
-Produced accurate calibration curve
-Acetate buffer investigated and found to be ineffective

Week 6:
-Literature survey of alternative intracellular iron measurement techniques

Week 7:
-Developed ICP sample preparation protocol

Week 8:
-Attempted ICP sample preparation, issues with overnight cultures and CFU count

Week 9:
-Attempted ICP sample preparation, issues with overnight cultures and CFU counts

Week 11:
-Investigation of using concentration step in ferene assay. Some issues with precipitates forming
-Investigated adding tonB supernatant to LB cultures to observe any effect of siderophores that may be present

Week 12:
-ICP samples prepared, measurement performed by university analytical chemistry service service

CAS PLATES

GROWTH CURVES

MODELLING

DESIGNING THE DEVICE

POLICES AND PRACTICES

WIKI AND MEDIA DESIGN