Team:Cambridge/Experiments

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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]]===
===[[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.
+
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 ]].
 +
 
 +
==Main Project - '<i>Bact<b>iridescence</b></i>'==
 +
 
 +
===Obtaining the Reflectin Sequence===
 +
====[[Team:Cambridge/Experiments/Squid_Dissection_and_Tissue_Sample | 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.
 +
 
 +
====[[Team:Cambridge/Experiments/Synthetic_Reflectin_PCR_and_Construction_of_GA1_to_6 | 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===
 +
====[[Team:Cambridge/Experiments/Protein_Purification | Over-Expression & Protein Purification]]====
 +
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]].
 +
 
 +
====[[Team:Cambridge/Experiments/Thin_Films | Making Thin Films]]====
 +
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.
-
==Main Project - 'Bactiridescence'==
+
All thin films were made in the Nanophotonics Centre, at the West Cambridge site.
-
===[[Team:Cambridge/Experiments/Squid_Dissection_and_Tissue_Sample | Amplification of Reflectin Genes from the Squid Genomic DNA - Part 1]]===
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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.
-
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 [[Team:Cambridge/Protocols/PCR|PCR]] reaction upon ''L. vulgaris'' [[Team:Cambridge/Protocols/Extraction of genomic DNA from squid| genomic DNA.]]
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===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 <i>in vitro</i> investigations.
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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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====[[Team:Cambridge/Experiments/Low_Level_Expression | 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.
-
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.
+
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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===[[Team:Cambridge/Experiments/Transformation_of_E.coli_with_Plasmids_from_Wendy | Amplification of Synthesised Reflectin Genes]]===
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====[[Team:Cambridge/Experiments/Periplasmic_Export | Periplasmic Export]]====
-
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.
+
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.
-
===[[Team:Cambridge/Experiments/Assembly_of_Reflectin_Constructs | Assembly of Reflectin Constructs]]===
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====[[Team:Cambridge/Project/Microscopy | Microscopy]]====
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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].
+
We performed microscopy on both squid cells taken from tissues known to contain reflectin and bacterial cells which were expressing our reflectin constructs.
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}

Latest revision as of 14:49, 21 September 2011

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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.