Difference between revisions of "Team:Newcastle/InterLab"
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<h7>Overview</h7> <br /> | <h7>Overview</h7> <br /> | ||
<!--- Why was this done? what problem is it addressing? How does it address that problem? How does it fit into the rest of the project? What were the aims for this? ---> | <!--- Why was this done? what problem is it addressing? How does it address that problem? How does it fit into the rest of the project? What were the aims for this? ---> | ||
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| + | <p> | ||
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The iGEM InterLab Study is the currently the largest interlaboratory study in synthetic biology [refs]. The interlab study investigates the replicability of data by providing all participating iGEM teams with protocols for measuring the fluorescence output of a set of genetic devices that have been designed to provide varying levels of Green Fluorescent Protein (GFP) expression. Team Newcastle 2017 followed the Interlab plate reader protocol, which can be found <a href='#'><u>here</u></a> (link protocol document). | The iGEM InterLab Study is the currently the largest interlaboratory study in synthetic biology [refs]. The interlab study investigates the replicability of data by providing all participating iGEM teams with protocols for measuring the fluorescence output of a set of genetic devices that have been designed to provide varying levels of Green Fluorescent Protein (GFP) expression. Team Newcastle 2017 followed the Interlab plate reader protocol, which can be found <a href='#'><u>here</u></a> (link protocol document). | ||
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<h7>InterLab Measurement Kit</h7><br /> | <h7>InterLab Measurement Kit</h7><br /> | ||
<!--- How was this stage designed? What was its sequence (if it's a part)? Was it modelled? What considerations were taken into account when making it? What challenges were there? How does the design link back to the rationale? (i.e. why does this design work). ---> | <!--- How was this stage designed? What was its sequence (if it's a part)? Was it modelled? What considerations were taken into account when making it? What challenges were there? How does the design link back to the rationale? (i.e. why does this design work). ---> | ||
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This year, teams were provided with the InterLab Measurement Kit containing fluorescein and LUDOX stocks, along with the devices stored in the Distribution kits. The devices are:<br /> | This year, teams were provided with the InterLab Measurement Kit containing fluorescein and LUDOX stocks, along with the devices stored in the Distribution kits. The devices are:<br /> | ||
- A positive control: BBa_I20270<br /> | - A positive control: BBa_I20270<br /> | ||
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- Test Device 6: BBa_J36005 (J23117+I13504)<br /> | - Test Device 6: BBa_J36005 (J23117+I13504)<br /> | ||
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<h7>Protocols</h7><br /> | <h7>Protocols</h7><br /> | ||
<!--- How was this part of the project implemented? How was it assembled if it is a part? How was it prepared for testing? What were the challenges? Why was it implemented in that way (e.g. why was that assembly method chosen)? How was it confirmed as having been implemented/assembled correctly? Include gels, images of plates, sequence data, preliminary information, etc. and refer to the specific sections of the lab-book which document this. ---> | <!--- How was this part of the project implemented? How was it assembled if it is a part? How was it prepared for testing? What were the challenges? Why was it implemented in that way (e.g. why was that assembly method chosen)? How was it confirmed as having been implemented/assembled correctly? Include gels, images of plates, sequence data, preliminary information, etc. and refer to the specific sections of the lab-book which document this. ---> | ||
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<h6>OD600 Reference Point</h6><br /> | <h6>OD600 Reference Point</h6><br /> | ||
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LUDOX-HS40 was used as a single point reference to obtain a radiometric conversion factor to convert absorbance data into a standard OD600 measurement. The Reference OD600 divided by the Abs600 from four replicates of LUDOX was used to obtain a correction factor for use against the cell based assays.<br /> | LUDOX-HS40 was used as a single point reference to obtain a radiometric conversion factor to convert absorbance data into a standard OD600 measurement. The Reference OD600 divided by the Abs600 from four replicates of LUDOX was used to obtain a correction factor for use against the cell based assays.<br /> | ||
| + | </p> | ||
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<h6>Fluorescein Standard Curve</h6><br /> | <h6>Fluorescein Standard Curve</h6><br /> | ||
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| + | <p> | ||
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A dilution series of fluorescein in four replicates was prepared and measured in the plate reader to obtain a standard curve of fluorescence for fluorescein concentration. This was used to correct cell-based readings to an equivalent fluorescein concentration, and to then convert this to a GFP concentration.<br /> | A dilution series of fluorescein in four replicates was prepared and measured in the plate reader to obtain a standard curve of fluorescence for fluorescein concentration. This was used to correct cell-based readings to an equivalent fluorescein concentration, and to then convert this to a GFP concentration.<br /> | ||
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| + | </p> | ||
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<h6>Plate Reader</h6><br /> | <h6>Plate Reader</h6><br /> | ||
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| + | <p> | ||
| + | Competent E. coli DH5α cells were transformed with each of the devices and plated onto LB+Chl agar, and two colonies from each transformation plate were grown overnight in 10 ml LB + Chl in 50 ml Falcon tubes. Protocols for making competent cells and cell transformation can be found here (link to protocol). Fluorescence was measured as specified in the InterLab protocol on a Synergy H1 plate reader. | ||
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| + | </p> | ||
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| − | <h7> | + | |
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| + | <h7>Results</h7> <br /> | ||
<!-- What experiments were performed? Refer to specific protocols, relevant sections of the lab-book, and provide enough detail that experiments could be carried out by anyone without prior knowledge of the project. Why was each experiment performed? How do they link back to the rationale/aims? What results were expected and what was actually observed? Explain the results with graphs/etc. (include legends) and what each result means. --> | <!-- What experiments were performed? Refer to specific protocols, relevant sections of the lab-book, and provide enough detail that experiments could be carried out by anyone without prior knowledge of the project. Why was each experiment performed? How do they link back to the rationale/aims? What results were expected and what was actually observed? Explain the results with graphs/etc. (include legends) and what each result means. --> | ||
| + | <p> | ||
| + | Raw results can be found <a href="#"><u>here</u></a>. <br /> | ||
| + | </p> | ||
| + | <p> | ||
| + | <b>Bacterial growth.</b> We found that the negative control, Tests 3, 5 and 6 grew the most in the 6 h experiment, with the positive control and Test 2 growing less so. Test 1 had the least growth. <br /> | ||
| + | </p> | ||
| + | <p> | ||
| + | <b>Fluorescence.</b> Test 2 showed the most fluorescence after 6 h, with Test 4 showing slightly less, followed closely by the positive control, then Tests 1 and 5. Tests 3 and 6 showed very little fluorescence.<br /> | ||
| + | </p> | ||
| + | <p> | ||
| + | <b>FL/OD.</b> Test 1 gave a significantly higher overall uM fluorescein/OD600 ratio compared to all other devices with values of 0.91 and 0.94 from the separate isolates, compared to a range of <0.01 and 0.18 from all other devices. <br /> | ||
| + | </p> | ||
| + | <p> | ||
| + | <b>Variability.</b> As part of the Interlab study, we analysed two separate bacterial transformants in quadruplicate. This allows an examination of the variability between replicates and between colonies. Between replicates, variation was minimal. However, between colonies containing the same device we saw a degree of variability in growth, fluorescence and FL:OD. Fluorescence and subsequently FL:OD are directly affected by growth, but factors including inaccuracies in growth set up and the user taking the samples could have also affected these. Also, plasmid copy number is not equal - we should add a comment to this effect here.<br /> | ||
| + | </p> | ||
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| − | <h7>Conclusions and Future Work</h7> | + | |
| + | <h7>Conclusions and Future Work</h7> <br /> | ||
<!--- Give a quick overview of what this section of the project was, the aims, and to what extent they were achieved. Discuss what further characterisation could be performed and whether anything (e.g. the design of a part) could be modified to improve it. Discuss what the next step would be (e.g. test more variants, repeats, etc.)--> | <!--- Give a quick overview of what this section of the project was, the aims, and to what extent they were achieved. Discuss what further characterisation could be performed and whether anything (e.g. the design of a part) could be modified to improve it. Discuss what the next step would be (e.g. test more variants, repeats, etc.)--> | ||
| + | <p> | ||
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| + | After carrying out the InterLab Study we decided to take a high, medium and low expressing device and analyse their sensitivity to changes in environmental conditions. We also looked into how automation could reduce variation between results. | ||
| + | </p> | ||
<br /> | <br /> | ||
Revision as of 13:17, 24 October 2017
Interlab Study Results |
Interlab Study: The ResultsBioBricks used: BBa_I20270, BBa_R0040, BBa_J36000, BBa_J36001, BBa_J36002, BBa_J36003, BBa_J36004, BBa_J36005
Diagrammatic Overview: This is a caption. This is a caption. This is a caption. This is a caption. This is a caption. This is a caption. The iGEM InterLab Study is the currently the largest interlaboratory study in synthetic biology [refs]. The interlab study investigates the replicability of data by providing all participating iGEM teams with protocols for measuring the fluorescence output of a set of genetic devices that have been designed to provide varying levels of Green Fluorescent Protein (GFP) expression. Team Newcastle 2017 followed the Interlab plate reader protocol, which can be found here (link protocol document).
This year, teams were provided with the InterLab Measurement Kit containing fluorescein and LUDOX stocks, along with the devices stored in the Distribution kits. The devices are: OD600 Reference Point
LUDOX-HS40 was used as a single point reference to obtain a radiometric conversion factor to convert absorbance data into a standard OD600 measurement. The Reference OD600 divided by the Abs600 from four replicates of LUDOX was used to obtain a correction factor for use against the cell based assays. Fluorescein Standard Curve
A dilution series of fluorescein in four replicates was prepared and measured in the plate reader to obtain a standard curve of fluorescence for fluorescein concentration. This was used to correct cell-based readings to an equivalent fluorescein concentration, and to then convert this to a GFP concentration. Plate ReaderCompetent E. coli DH5α cells were transformed with each of the devices and plated onto LB+Chl agar, and two colonies from each transformation plate were grown overnight in 10 ml LB + Chl in 50 ml Falcon tubes. Protocols for making competent cells and cell transformation can be found here (link to protocol). Fluorescence was measured as specified in the InterLab protocol on a Synergy H1 plate reader.
Raw results can be found here.
Bacterial growth. We found that the negative control, Tests 3, 5 and 6 grew the most in the 6 h experiment, with the positive control and Test 2 growing less so. Test 1 had the least growth.
Fluorescence. Test 2 showed the most fluorescence after 6 h, with Test 4 showing slightly less, followed closely by the positive control, then Tests 1 and 5. Tests 3 and 6 showed very little fluorescence.
FL/OD. Test 1 gave a significantly higher overall uM fluorescein/OD600 ratio compared to all other devices with values of 0.91 and 0.94 from the separate isolates, compared to a range of <0.01 and 0.18 from all other devices.
Variability. As part of the Interlab study, we analysed two separate bacterial transformants in quadruplicate. This allows an examination of the variability between replicates and between colonies. Between replicates, variation was minimal. However, between colonies containing the same device we saw a degree of variability in growth, fluorescence and FL:OD. Fluorescence and subsequently FL:OD are directly affected by growth, but factors including inaccuracies in growth set up and the user taking the samples could have also affected these. Also, plasmid copy number is not equal - we should add a comment to this effect here. After carrying out the InterLab Study we decided to take a high, medium and low expressing device and analyse their sensitivity to changes in environmental conditions. We also looked into how automation could reduce variation between results. |
Interlab Study Improvements: The ResultsBioBricks used: BBa_0123456 (New), BBa_7890123 (Team_Name 20XX)
Diagrammatic Overview: This is a caption. This is a caption. This is a caption. This is a caption. This is a caption. This is a caption.
|
