Team:Cambridge/Experiments

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(Microscopy)
 
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=Experiments=
 
Details of the experiments carried out throughout the project are linked from this page.
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.
These experiments should also be linked to from the appropriate blog entry.
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====[[Team:Cambridge/Experiments/PreliminaryExercise|Training Exercise]]====
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===[[Team:Cambridge/Experiments/PreliminaryExercise|Training Exercise]]===
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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. Find out what we got up to on the [[Team:Cambridge/Blog | blog ]].
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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. Find out what we got up to on the [[Team:Cambridge/Blog | blog ]]
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==Main Project - '<i>Bact<b>iridescence</b></i>'==
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==Main Project - 'Bactiridescence'==
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===Obtaining the Reflectin Sequence===
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====[[Team:Cambridge/Experiments/Squid_Dissection_and_Tissue_Sample | Genomic DNA Extraction Attempt]]====
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===Genomic DNA Extraction Attempt===
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We designed primers to amplify reflectin genes directly from DNA extracted from [http://en.wikipedia.org/wiki/Loligo ''Loligo''] tissue. Various combinations of Loligo, primers and DNA extraction protocol were used, ultimately with no success.
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====[[Team:Cambridge/Experiments/Squid_Dissection_and_Tissue_Sample | Amplification of Reflectin Genes from the Squid Genomic DNA - Part 1]]====
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We designed primers to amplify reflectin genes 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 [[Team:Cambridge/Protocols/PCR|PCR]] reaction upon ''L. vulgaris'' [[Team:Cambridge/Protocols/Extraction of genomic DNA from squid| genomic DNA.]]
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Synthesised reflectin sequences were generously donated by Wendy Crookes-Goodson, author of many of the papers on reflectin.
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See [[Team:Cambridge/Blog/Week_4 | week four]] of our blog for more on this!
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====[[Team:Cambridge/Experiments/Synthetic_Reflectin_PCR_and_Construction_of_GA1_to_6 | Synthetic Gene Amplification & Plasmid Construction]]====
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In anticipation that our genomic DNA extraction might fail, 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.
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====[[Team:Cambridge/Experiments/Squid_Dissection_and_Tissue_Sample_Improved_Protocol | Amplification of Reflectin Genes from the Squid Genomic DNA - Part 2]]====
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We extracted the DNA, transfected cells and grew up these plasmids, then used their reflectin sequences to assemble constructs with reflectin A1 with and without a his tag, each on high and low copy plasmids. In addition, we put Reflectins A2 and 1B on low-copy plasmids and created Reflectin A1 : GFP translational fusions.
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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.
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===In Vitro Experiments===
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====[[Team:Cambridge/Experiments/Protein_Purification | Over-Expression & Protein Purification]]====
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Using our reflectin constructs, we over expressed reflectin and then tried a number of techniques to purify the protein, including [[Team:Cambridge/Protocols/Protein_Purification | HIS trap purification]] and an [[Team:Cambridge/Protocols/Inclusion_Body_Prep | inclusion body prep]]. We verified our protein by running an [[Team:Cambridge/Protocols/Gel_Electrophoresis_of_Protein | SDS PAGE protein gel]].
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===Assembly of Constructs and Protein Purification===
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====[[Team:Cambridge/Experiments/Thin_Films | Making Thin Films]]====
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====[[Team:Cambridge/Experiments/Transformation_of_E.coli_with_Plasmids_from_Wendy | Amplification of Synthesised Reflectin Genes]]====
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The culmination of the <i>in vitro</i> work was the production of thin films of reflectin which demonstrate iridescence. We tried a number of different combinations of protein purification protocol and thin films coating method, and produced numerous thin films.
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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.
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====[[Team:Cambridge/Experiments/Assembly_of_Reflectin_Constructs | Assembly of Reflectin Constructs]]====
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All thin films were made in the Nanophotonics Centre, at the West Cambridge site.
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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].
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====[[Team:Cambridge/Experiments/Protein_Purification | His-Trap Protein Purification - First attempt]]====
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We found protein purity to be a major hindrance in progress, with either formation of crystal structure formation or wetting and solvent evaporation problems.
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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.
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====[[Team:Cambridge/Experiments/Protein_Purification2 | His-Trap Protein Purification - Second attempt]]====
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===In Vivo Experiments===
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As before bacteria expressing his-tagged reflectin were lysed and the protein was purified using a his-trap column but this time the isolated protein was then precipitated by ethanol or acetone precipitation.
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We wanted to investigate reflectin's effect on E.coli when expressed at a low level. Up until this point, researchers had focussed on using E.coli in order to manufacture large amounts of reflctin for <i>in vitro</i> investigations.
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===Making Thin Films===
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====[[Team:Cambridge/Experiments/Low_Level_Expression | Low Level Expression]]====
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====[[Team:Cambridge/Experiments/Reflectin_Thin_Films_I | Reflectin Thin Films I - First films with unfiltered protein]]====
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Various tests were done on E.coli with reflectin expressed on a low copy plasmid under an arabinose induced promoter (pBAD). We tested both normal E.coli cells, and ones with a titratable arabinose response.
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A sub team visited the Nanophotonics Centre Thin Films and Interfaces lab to work with Dr Matthew Hawkeye. We spincoated and flowcoated our purified reflectin samples in addition to several control solutions. We recorded our results using reflected light bright field microscopy and an ocean optics spectrometer.
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====[[Team:Cambridge/Experiments/Reflectin_Thin_Films_II | Reflectin Thin Films II - Improved films with centrifuged reflectin-solvent solution]]====
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While we found that reflectin is surprisingly non-toxic to E.coli, we did not find any evidence that the reflectin had folded correcty or that it had self assembled into stacks as it does in squid. However, by using our reflectin-GFP fusion on a low copy number plasmid as a control, we found that reflectin did not form inclusion bodies as it does under a high copy plasmid, but was distributed throughout the cell.
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We refined our methods in order to produce better thin films. We recorded the reflectance spectra of some of the thin films, ran a control using the protein bovine serum albuimin and heat cured the films.
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====[[Team:Cambridge/Experiments/Reflectin_Thin_Films_III | Reflectin Thin Films III - Reflectin Controls]]====
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====[[Team:Cambridge/Experiments/Periplasmic_Export | Periplasmic Export]]====
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In addition to making some more thin films various controls were implemented in order to verify that the origin of iridescence we were observing were due to the reflectin protein and not due to impurities which may have originated in the elution buffer or at other stages of the purification . The results suggest iridescence is due to the presence of reflectin.
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We attempted to see what would become of reflectin once exported to the periplasm. Our GFP control suggested that our initial attempt at export had failed. There were several possible problems - one being that we were likely expressing reflectin too strongly and the export machinery was becoming saturated - however due to the unprecedented time constraints of the competition, we simply didn't have the time to try again.
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===Part Characterization===
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====[[Team:Cambridge/Project/Microscopy | Microscopy]]====
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====[[Team:Cambridge/Experiments/Characterization_Inclusion_Bodies | Characterization of Parts -- Inclusion Body Formation]]====
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We performed microscopy on both squid cells taken from tissues known to contain reflectin and bacterial cells which were expressing our reflectin constructs.
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Details of the process for characterizing components of our reflectin-producing system.
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{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}

Latest revision as of 14:49, 21 September 2011

Loading...
OVERVIEW
home
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.

Contents

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. Find out what we got up to on the blog .

Main Project - 'Bactiridescence'

Obtaining the Reflectin Sequence

Genomic DNA Extraction Attempt

We designed primers to amplify reflectin genes directly from DNA extracted from [http://en.wikipedia.org/wiki/Loligo Loligo] tissue. Various combinations of Loligo, primers and DNA extraction protocol were used, ultimately with no success.

Synthesised reflectin sequences were generously donated by Wendy Crookes-Goodson, author of many of the papers on reflectin.

Synthetic Gene Amplification & Plasmid Construction

In anticipation that our genomic DNA extraction might fail, 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.

We extracted the DNA, transfected cells and grew up these plasmids, then used their reflectin sequences to assemble constructs with reflectin A1 with and without a his tag, each on high and low copy plasmids. In addition, we put Reflectins A2 and 1B on low-copy plasmids and created Reflectin A1 : GFP translational fusions.

In Vitro Experiments

Over-Expression & Protein Purification

Using our reflectin constructs, we over expressed reflectin and then tried a number of techniques to purify the protein, including HIS trap purification and an inclusion body prep. We verified our protein by running an SDS PAGE protein gel.

Making Thin Films

The culmination of the in vitro work was the production of thin films of reflectin which demonstrate iridescence. We tried a number of different combinations of protein purification protocol and thin films coating method, and produced numerous thin films.

All thin films were made in the Nanophotonics Centre, at the West Cambridge site.

We found protein purity to be a major hindrance in progress, with either formation of crystal structure formation or wetting and solvent evaporation problems.

In Vivo Experiments

We wanted to investigate reflectin's effect on E.coli when expressed at a low level. Up until this point, researchers had focussed on using E.coli in order to manufacture large amounts of reflctin for in vitro investigations.

Low Level Expression

Various tests were done on E.coli with reflectin expressed on a low copy plasmid under an arabinose induced promoter (pBAD). We tested both normal E.coli cells, and ones with a titratable arabinose response.

While we found that reflectin is surprisingly non-toxic to E.coli, we did not find any evidence that the reflectin had folded correcty or that it had self assembled into stacks as it does in squid. However, by using our reflectin-GFP fusion on a low copy number plasmid as a control, we found that reflectin did not form inclusion bodies as it does under a high copy plasmid, but was distributed throughout the cell.

Periplasmic Export

We attempted to see what would become of reflectin once exported to the periplasm. Our GFP control suggested that our initial attempt at export had failed. There were several possible problems - one being that we were likely expressing reflectin too strongly and the export machinery was becoming saturated - however due to the unprecedented time constraints of the competition, we simply didn't have the time to try again.

Microscopy

We performed microscopy on both squid cells taken from tissues known to contain reflectin and bacterial cells which were expressing our reflectin constructs.