"
Team:Cambridge/Experiments
From 2011.igem.org
| Line 25: | Line 25: | ||
Assembly and amplification of constructs for reflectin A1 with and without a his tag, each on two different plasmids, and for reflectins A2 and 1B on low-copy plasmids [placeholder]. | Assembly and amplification of constructs for reflectin A1 with and without a his tag, each on two different plasmids, and for reflectins A2 and 1B on low-copy plasmids [placeholder]. | ||
| - | ===[[Team:Cambridge/Experiments/Protein_Purification | Protein Purification]]=== | + | ===[[Team:Cambridge/Experiments/Protein_Purification | Protein Purification - First attempt]]=== |
| - | Bacteria expressing his-tagged reflectin were lysed, and the protein was purified using a his-trap column and a denaturing protocol in order to solubilise reflectin. | + | Bacteria expressing his-tagged reflectin were lysed, and the protein was purified using a his-trap column and a denaturing protocol in order to solubilise reflectin. The isolated protein was then precipitated by dialysis and vacuum centrifugation. |
| - | =[[Team:Cambridge/Experiments/Plasmid_Constructs | Constructs]]= | + | ==[[Team:Cambridge/Experiments/Plasmid_Constructs | Constructs]]== |
A list of plasmid constructs made during the competition. | A list of plasmid constructs made during the competition. | ||
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}} | {{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}} | ||
Revision as of 18:42, 25 August 2011
Contents |
Experiments
Details of the experiments carried out throughout the project are linked from this page. These experiments should also be linked to from the appropriate blog entry.
Training Exercise
Initial exercise during our 2 weeks crash course in synthetic biology with the aim of familiarising us with common laboratory methods of preparing and assembling DNA.
Main Project - 'Bactiridescence'
Amplification of Reflectin Genes from the Squid Genomic DNA - Part 1
Reflectin genes were amplified directly from [http://en.wikipedia.org/wiki/Loligo Loligo] tissue. Tissue from the Loligo genus was commercially available from fishing bait suppliers and culinary wholesalers. Primers were designed from the nucleotide sequences of three reflectin proteins identified in L. pealei, and used in a PCR reaction upon L. vulgaris genomic DNA.
Amplification of Reflectin Genes from the Squid Genomic DNA - Part 2
Two new protocols for genomic DNA extraction were used in order to improve yield and purity of DNA. In addition to three sets of primers allowing for amplification of reflectin, an extra 'positive control' pair of primers was used in the PCR reaction.
Amplification of Synthesised Reflectin Genes
After failing to isolate reflectin genes from squid genomic DNA, we contacted several researchers who had previously worked on reflectin for advice. Dr. Wendy Crookes-Goodson very kindly offered to donate a sample of synthesised reflectin genes that she used in her research. These arrived on cloning (non-expressing) plasmids that had been spotted onto filter paper. Our first step was to elute the plasmids from the paper, and then to transform them into E. coli for storage and amplification. A standard miniprep then allowed us to recover the DNA from the bacteria.
Assembly of Reflectin Constructs
Assembly and amplification of constructs for reflectin A1 with and without a his tag, each on two different plasmids, and for reflectins A2 and 1B on low-copy plasmids [placeholder].
Protein Purification - First attempt
Bacteria expressing his-tagged reflectin were lysed, and the protein was purified using a his-trap column and a denaturing protocol in order to solubilise reflectin. The isolated protein was then precipitated by dialysis and vacuum centrifugation.
Constructs
A list of plasmid constructs made during the competition.
