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
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
Week 1:
-Siderophore production in wild-type (W3110, Top10) and mutant E. coli has been tested using CAS plates- the initially tested protocol did not work
Week 6:
-CAS plates from week 1 were tested again; instead of adding E.coli cells, EDTA iron chelators have been added
-Presence of yellow hollows suggested that our plates could change colour in the presence of iron chelators, but in the case of E.coli cells the approach was not successful as cells probably require additional carbon sources
-Multiple other protocols have been tested
Week 10:
- CAS plates have been poured again; a new protocol has been followed
-inoculated overnight cultures of W3110, JC28, Top10 and TonB on the CAS plates
-following a couple of days, yellow have been observed, suggesting that this approach was successful
GROWTH CURVES
Week 3:
-Growth curves of JC28 and W3110 have been set up; cells have been grown in liquid broth
Week 6:
-Growth curves of JC28 and W3110 have been set up; cells have been grown in defined rich media as well as M9 media
