Difference between revisions of "Team:Newcastle/InterLab"
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| − | <h4>BioBricks used: <a href="#"> | + | <h4>BioBricks used: <a href="#">BBa_I20270</a>, <a href="#">BBa_R0040</a>, <a href="#">BBa_J36000</a>, <a href="#">BBa_J36001</a>, <a href="#">BBa_J36002</a>, <a href="#">BBa_J36003</a>, <a href="#">BBa_J36004</a>, <a href="#">BBa_J36005 </a><br /></h4> |
<!--- Link all biobricks used. State whether they are our new bricks or current bricks and give the team name that originally submitted it. --> | <!--- Link all biobricks used. State whether they are our new bricks or current bricks and give the team name that originally submitted it. --> | ||
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| − | <h7> | + | <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? ---> | ||
| − | + | 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). | |
| − | + | ||
<br /> | <br /> | ||
| − | <h7> | + | <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). ---> |
| − | + | 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 negative control: BBa_R0040<br /> | ||
| + | - Test Device 1: BBa_J36000 (J23101.BCD2.E0040.B0015)<br /> | ||
| + | - Test Device 2: BBa_J36001 (J23106.BCD2.E0040.B0015)<br /> | ||
| + | - Test Device 3: BBa_J36002 (J23117.BCD2.E0040.B0015)<br /> | ||
| + | - Test Device 4: BBa_J36003 (J23101+I13504)<br /> | ||
| + | - Test Device 5: BBa_J36004 (J23106+I13504)<br /> | ||
| + | - Test Device 6: BBa_J36005 (J23117+I13504)<br /> | ||
<br /> | <br /> | ||
| − | <h7> | + | <h7>Protocols</h7><br /> |
| − | <!--- How was 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. ---> |
| + | <h6>OD600 Reference Point</h6><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 /> | ||
| + | <h6>Fluorescein Standard Curve</h6><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 /> | ||
| − | + | <h6>Plate Reader</h6><br /> | |
| − | < | + | 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. |
| − | < | + | |
| − | + | ||
Revision as of 15:15, 23 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: - A positive control: BBa_I20270 - A negative control: BBa_R0040 - Test Device 1: BBa_J36000 (J23101.BCD2.E0040.B0015) - Test Device 2: BBa_J36001 (J23106.BCD2.E0040.B0015) - Test Device 3: BBa_J36002 (J23117.BCD2.E0040.B0015) - Test Device 4: BBa_J36003 (J23101+I13504) - Test Device 5: BBa_J36004 (J23106+I13504) - Test Device 6: BBa_J36005 (J23117+I13504) OD600 Reference PointLUDOX-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 CurveA 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. |
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.
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