Team:Aberdeen Scotland/Parts/ 0000

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<h1>Background to Parts Design</h1>
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<p>Antigen 43 (Ag43), the product of the </i>flu</i> gene, is a cell-surface autotransporter protein found in <i>Escherichia coli</i>. It is expressed at about 50,
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000 copies/cell and is initially synthesised as a precursor of 1039 amino acids. Upon removal of the signal peptide, the protein is transported to the cell surface
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and is composed of an α subunit (499 amino acids) at the N-terminus and a β subunit (488 amino acids) at the C-terminus. Ag43 is mainly known to induce cell-to-cell
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<h1>Our characterisation of existing BioBrick T9002: </h1>
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aggregation and be involved in biofilm formation. However, as the necessary information required for auto transportation resides in the protein itself, the main of
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<h1>GFP Producer Controlled by 3OC6HSL Receiver Device</h1>
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our project was to use it as a platform for displaying specific peptides on the surface of <i>E. coli</i>.</p>
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<img src="http://2014.igem.org/wiki/images/2/2e/Ag43.jpg" alt="Ag43">
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<h3>Aims and Rationale</h3>
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BioBrick BBa_T9002 on a pSB1C3 backbone is a composite part encoding a quorum sensing (QS) receiver driving expression of green fluorescent protein. T9002 was previously reported to exhibit increased GFP expression in the presence of AHL (N-acyl homoserine lactone).
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We aimed to confirm GFP-expression responsiveness and dependence of AHL, and how the physical proximity of this QS receiver to any QS sender (producer of homoserine lactone)  is facilitated by surface-binding affects quorum signalling.
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<h3>Materials and Methods</h3>
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T9002 <i>E. coli</i> transformants were grown in conjunction with other <i>E. coli</i> transformed with the AHL ‘Sender’ plasmid BBa_K1090000. BBa_K1090000 codes for AHL-synthesising enzymes, and also constitutively expresses RFP.
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<b><i>Poly-L-lysine Cell Adhesion</i></b>
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To investigate QS signalling, and how it is affected by binding of sender and receiver bacteria to a solid surface, we immobilised our cells on a surface coated with poly-lysine:
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1. 50μl 0.01% Poly-Lysine was added to each well of a 96 well glass-bottom Plate (Whatman), and incubated at room temperature for 2 hours
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2. Excess poly-lysine was removed and the plate washed three times with sterile water and allowed to dry at room temperature.
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3. <i>E. coli</i> cultures were grown overnight in a 37C shaking water bath, and diluted to an optical density-600nm(OD600) of 0.02.
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4. K1090000 ‘Sender’ transformants washed by centrifugation at 13000rpm for 1minute and resuspension in phosphate buffered saline (PBS), three times.
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5. Dual Sender-Receiver wells were diluted and combined in a colorimetric ratio to achieve a total OD600 of 0.02, with a range of 1:1 to 1:10,000,000 Sender:Receiver. 50μl total volume in Liquid Broth (LB) used per well. Incubated at 4C for 1 hour.
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6. Unbound cells removed by lightly shaking over waste and washed three times with Phosphate-Buffered Saline (PBS). 50μl fresh LB medium was added to each well.
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7. A FluoSTAR OPTIMA Fluoresence Plate Reader was used to measure Red(Excitation 544nm/Emission 612nm) and Green(Excitation 485nm/Emission 520nm) Fluorescence was recorded every 5 minutes for 7 hours; this incubates the samples at 37 degrees C and shakes (1mm double-orbital) for 30 seconds before each read.
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<h3>Results</h3>
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<b><i>K1090000 RFP expression</b></i> RFP expression of K1090000 transformed <i>E. coli</i> was compared against untransformed XL1-Blue <i>E. coli</i> negative control and an RFP pSB1C3 plasmid positive control. Analysis of red fluorescence using a fluorimeter plate reader clearly shows that K1090000 constitutively expressed moderate amounts of RFP (Figure 1).
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Revision as of 23:02, 17 October 2014

Team:Aberdeen Scotland/Parts - 2014.ogem.org




Our characterisation of existing BioBrick T9002:

GFP Producer Controlled by 3OC6HSL Receiver Device


Aims and Rationale

BioBrick BBa_T9002 on a pSB1C3 backbone is a composite part encoding a quorum sensing (QS) receiver driving expression of green fluorescent protein. T9002 was previously reported to exhibit increased GFP expression in the presence of AHL (N-acyl homoserine lactone).

We aimed to confirm GFP-expression responsiveness and dependence of AHL, and how the physical proximity of this QS receiver to any QS sender (producer of homoserine lactone) is facilitated by surface-binding affects quorum signalling.

Materials and Methods

T9002 E. coli transformants were grown in conjunction with other E. coli transformed with the AHL ‘Sender’ plasmid BBa_K1090000. BBa_K1090000 codes for AHL-synthesising enzymes, and also constitutively expresses RFP.

Poly-L-lysine Cell Adhesion
To investigate QS signalling, and how it is affected by binding of sender and receiver bacteria to a solid surface, we immobilised our cells on a surface coated with poly-lysine:

1. 50μl 0.01% Poly-Lysine was added to each well of a 96 well glass-bottom Plate (Whatman), and incubated at room temperature for 2 hours

2. Excess poly-lysine was removed and the plate washed three times with sterile water and allowed to dry at room temperature.

3. E. coli cultures were grown overnight in a 37C shaking water bath, and diluted to an optical density-600nm(OD600) of 0.02.

4. K1090000 ‘Sender’ transformants washed by centrifugation at 13000rpm for 1minute and resuspension in phosphate buffered saline (PBS), three times.

5. Dual Sender-Receiver wells were diluted and combined in a colorimetric ratio to achieve a total OD600 of 0.02, with a range of 1:1 to 1:10,000,000 Sender:Receiver. 50μl total volume in Liquid Broth (LB) used per well. Incubated at 4C for 1 hour.

6. Unbound cells removed by lightly shaking over waste and washed three times with Phosphate-Buffered Saline (PBS). 50μl fresh LB medium was added to each well.

7. A FluoSTAR OPTIMA Fluoresence Plate Reader was used to measure Red(Excitation 544nm/Emission 612nm) and Green(Excitation 485nm/Emission 520nm) Fluorescence was recorded every 5 minutes for 7 hours; this incubates the samples at 37 degrees C and shakes (1mm double-orbital) for 30 seconds before each read.

Results

K1090000 RFP expression RFP expression of K1090000 transformed E. coli was compared against untransformed XL1-Blue E. coli negative control and an RFP pSB1C3 plasmid positive control. Analysis of red fluorescence using a fluorimeter plate reader clearly shows that K1090000 constitutively expressed moderate amounts of RFP (Figure 1).