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</p> | </p> | ||
<h3> Constitutive hemerythrin expression </h3> | <h3> Constitutive hemerythrin expression </h3> | ||
+ | |||
+ | <p> <b> <u>Week 4:</u> </b> <br> | ||
+ | -Synthetic hemerythrin genes as well as primers required for amplifying these genes have arrived <br> | ||
+ | -Synthetic genes have been amplified (PCR) and the success of the PCR reactions has been tested (PCR and Nano Drop)<br> | ||
+ | -Synthetic genes as well as plasmid backbones (pSB1C3 and pBSKII) have been digested using appropriate restriction enzymes <br> | ||
+ | -Ligation reactions have been set up <br> | ||
+ | -Ligated plasmids have been transformed in Top10 competent cells </p> | ||
+ | |||
+ | |||
+ | <p> <b> <u>Week 5:</u> </b> <br> | ||
+ | -Plasmid mini-preps have been made from the Top10 cells transformed with constitutively expressed hemerythrin genes (both pSB1C3+hemerythrin and pBSKII+hemerythrin) <br> | ||
+ | -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 <br> | ||
+ | -Plasmids containing desired inserts have been transformed in W3110 (wild-type) and JC28 (siderophore-deficient) <i> E.coli </i> strains <br> | ||
+ | -Plasmid mini-preps have been made again from transformed W3110 and JC28 strains </p> | ||
+ | |||
+ | |||
+ | <p> <b> <u>Week 6:</u> </b> <br> | ||
+ | -Plasmid mini-preps (from W3110 and JC28 strains transformed with pSB1C3+hemerythrin) have been digested with appropriate restriction enzymes <br> | ||
+ | - 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 <br> | ||
+ | -Plasmids containing the appropriate inserts have been sent for sequencing <br> | ||
+ | -Sequences have been analysed </p> | ||
+ | |||
+ | |||
+ | <p> <b> <u>Week 7:</u> </b> <br> | ||
+ | - Measured absorbance at 500nm in overnight cultures of Dcr and Mc in JC28 and W3110 strains and Td in W3110 strain in chloramphenicol LB <br> | ||
+ | -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 <br> | ||
+ | -Tried to figure out what was going on– realised that there were several methylation sites at adjacent positions to the promoters used <br> | ||
+ | -Decided that we should replace the promoters and remove the methylation sites <br> | ||
+ | -New promoters have been ordered</p> | ||
+ | |||
+ | |||
+ | <p> <b> <u>Week 10:</u> </b> <br> | ||
+ | -Promoters have been cut out from the linear hemerythrin constructs <br> | ||
+ | -Promoterless hemerythrin genes have been cloned into pSB1C3 <br> | ||
+ | -pSB1C3+promoterless hemerythrin plasmids have been cloned into Top10 <br> | ||
+ | -Very few colonies of transformant Top10 have been observed on every plate</p> | ||
+ | |||
+ | |||
+ | <p> <b> <u>Week 11:</u> </b> <br> | ||
+ | -Cloning of promoterless hemerythrin genes into pSB1C3 has been carried out again, allocating longer incubation times for digestion and ligation reactions to take place <br> | ||
+ | -Ligated plasmids have been transformed in Top10 <br> | ||
+ | -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) <br> | ||
+ | -The new promoters have been cloned into the pSB1c3+promoterless hemerythrin genes containing desired inserts; these plasmids have been transformed into DH5α cells </p> | ||
+ | |||
+ | |||
+ | <p> <b> <u>Week 12:</u> </b> <br> | ||
+ | -pSB1C3+new promoter+promoterless hemerythrin genes plasmids have been harvested from DH5α cells and screened for the presence of desired inserts <br> | ||
+ | -Whole cell lysates of the transformed cells have been made and the soluble and insoluble protein fractions have been separated <br> | ||
+ | -Soluble and insoluble protein fractions have been run through an SDS-PAGE gel <br> | ||
+ | -Gel was stained with Cromassie Blue <br> | ||
+ | -SDS-PAGE gels shown that hemerythrin genes have not been successfully overexpressed</p> | ||
+ | |||
+ | |||
+ | <p> <b> <u>Weeks 13-17:</u> </b> <br> | ||
+ | -Cloning and submitting iGEM BioBricks </p> | ||
+ | |||
<h3> Overexpression of hemerythrin </h3> | <h3> Overexpression of hemerythrin </h3> | ||
</div> | </div> |
Revision as of 21:12, 18 October 2016
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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 overexpressed
Weeks 13-17:
-Cloning and submitting iGEM BioBricks