Difference between revisions of "Team:UMaryland/Interlab"

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<a href="https://2015.igem.org/Tracks/Measurement/Interlab_study">Interlab Requirements</a>

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<h1><b>Objective</b></h1>

<p style="font-size:20px">The iGEM interlab study embodies iGEM’s core values of collaboration, the development of an open community, the education of aspiring young scientists, and the advancement of synthetic biology. Thus, for the 2015 interlab study, the objective is for teams from around the globe to obtain fluorescence data from 3 different genetic devices. This aggregated data will not only aid the detailed characterization of these Biobricks, but also promote further joint scientific efforts of the iGEM community in synthetic biology. </p>

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<p style="font-size:20px">devices and controls here. </p>

<p style="font-size:20px">All of our devices were built in the PSB1C3 backbone. The Chassis for all of our devices was E. coli BL21. </p>

<p style="font-size:20px">Device 1: J23101+I13504 -> PSB1C3 </p>

<p style="font-size:20px">Device 2: J23106+I13504 -> PSB1C3 </p>

<p style="font-size:20px">Device 3: J23117+I13504 -> PSB1C3 </p>

<p style="font-size:20px"> Positive Control: BBa_I20270 -> PSB1C3 </p>

<h3>Construction of the Devices</h3>

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Our team acquired the necessary Biobricks for this interlab study from the distributed iGEM part kits. These Biobricks, each located in the PSB1C3 vector, were then transformed into E. coli K12 DH5-alpha following the standard transformation protocol. The transformed E. coli were then plated on chloramphenicol plates and incubated overnight. Individual colonies were then selected with a pipette tip. Overnight cultures with an individual colony were prepared with standard LB mixture in 10 mL culture tubes. These overnight cultures were then incubated overnight with the appropriate amount of chloramphenicol. The overnights were then miniprepped using Qiagen's mini prep kit. The efficiency of the minipreps was quantified with a Nanodrop. The isolated plasmids were digested the restriction enzymes EcoR1-HF and Pst1-HF using NEB's standard RE digest protocol for the enzymes. The digested plasmids were run on a 0.8% agarose gel and viewed on a transilluminator to confirm the size of the interlab parts. Primers were then designed to PCR amplify the promoters and GFP biobricks. The promoters along with the PSB1C3 backbone were each amplified with the PSB1C3 standard forward primer and a reverse primer that would amplify several bps at the 3' end of the promoter. This PCR reaction was designed to amplify and linearize the promoter in the PSB1C3 backbone. Primers were also designed to amplify the IL3504 out of the PSB1C3 backbone. The forward primer for this reaction had several bps at the 3' end of the primer that correspond to several bps at the 5' end of the GFP gene along with several bps at the 5' end of the primer that would include an overhanging segment to the 5' end of the primer when amplified. This overhanging sequence corresponded to the last few bps at the 3' end of the promoter of interest. As a result, this PCR amplification was run three times for the GFP that included a 3' end that would unique for each promoter. The reverse primer amplified several bps after the 3' end of the GFP gene. The Primer were designed following NEB's protocol for primer design. The PCR cycling conditions and annealing temperatures for each reaction were designed following NEB's standard PCR protocol for Q5 polymerase. The PCR products were then cleaned using Qiagen's PCR clean-up kit and the size of the PCR products was verified on an agarose gel. The linearized promoters + backbone were then each combined with the amplified IL3504 using the Gibson assembly protocol from NEB. The Gibson products were then transformed into BL21 and the completed plasmids were isolated following the aforementioned methods. These devices were then verified with gene sequencing through Genewiz. However, our team was unable to assemble the third device of the J23117 promoter and GFP. Thankfully, this DNA construct was graciously donated to us with iGEM approval from the William and Mary 2015 iGEM team. The controls for this experiment were transformed directly onto BL21. The plasmids were miniprepped and verified through gene sequencing.  

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Our team followed the 2015 InterLab protocol for testing that was posted by iGEM for this study. Three separate biological replicate for the verified constructs and controls were grown in 10 mL culture tubes with loose caps in an incubator overnight for 16-18 hours. The OD500 of the cells was then taken and the samples were diluted to within 5% of 0.5. 150 microliters of three biological replicates of each diluted sample and three technical replicates were transferred to a 96 well plate using a p-200 pipette. The fluorescence of the samples was then quantified using a microplater reader. The model that was used for testing was the SpectraMax M2 microplate reader. The excitation wavelength was set to 498 nm and the emission spectrum was set at 541 nm with the auto cutoff setting on. We are also very thankful to Dr. Bentley's lab on campus for allowing us to use their plate reader for testing. The results of the experiments were processed are reported below in arbitrary units.  

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<h1><b>Protocols</b></h1>

<p style="font-size:20px">Protocols for construction of devices here.</p>

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<a href = "http://parts.igem.org/Help:2015_DNA_Distribution">iGEM DNA distribution kit protocol</a>

<a href = "http://parts.igem.org/Help:Protocols/Transformation">iGEM transformation protocol</a>

<a href = "https://www.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510">NEB Gibson assembly protocol</a>

<a href = "http://www.hawaii.edu/microbiology/MO/docs/diversity/Qia-Miniprep.pdf">Qiagen miniprep protocol</a>

        <a href = "http://parts.igem.org/Help:Protocols/Transformation">Transformation</a>

                         <a href = "http://parts.igem.org/Help:Protocols/Restriction_Digest">iGEM RE digest protocol</a>

                         <a href = "http://parts.igem.org/Help:Primers/Design">iGEM primer design procedure</a>

                         <a href = "https://www.neb.com/protocols/2013/12/13/pcr-using-q5-high-fidelity-dna-polymerase-m0491">NEB PCR protocol</a>

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<h1><b>Results</b></h1>

<p style="font-size:20px">Results over here.</p>

<img width=500 height=400 src="https://static.igem.org/mediawiki/2015/d/df/UMD_interlab_data_1.png">

<img width=500 height=400 src="https://static.igem.org/mediawiki/2015/9/93/UMD_interlab_data_2.png">