Our team acquired the necessary Biobricks for this interlab study from the distributed iGEM part kits. These Biobricks in PSB1C3 were then transformed into E. coli K12 DH5-alpha following the standard transformation protocol. The E. coli were then plated on chloramphenicol plates and incubated overnight. Individual colonies were then selected and overnight cultures with an individual colony and a 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 extracted plasmids were digested with restriction enzymes and run on a agarose gel to confirm the size of the interlab parts. Primers were then designed to PCR amplify the promoters and GFP biobricks. The promoters with the PSB1C3 backbone were each amplified with the PSB1C3 standard forward primer and a reverse primer that would amplify several bps after the 3' end of the promoter. Primers were also designed to amplify the IL3504 out of the PSB1C3 backbone. The primers were designed to amplify several bps after 3' end of the gene and include an overhang of several bps at the 5' end that would have 10-15 bps that correspond to the last 10-15 bps of the 3' of the respective promoter. The PSB1C3 backbone was also linearized with PCR and the standard forward and reverse primers. The PCR cycling conditions and annealing temperatures were designing 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 promoters were then each combined with IL3504 and the linearized backbone 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.

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. The fluorescence of the samples was then quantified 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.  

DNA Kit Plate Instructions  (http://parts.igem.org/Help:2015_DNA_Distribution)

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Transformation (http://parts.igem.org/Help:Protocols/Transformation)

 

• Prepare culture in a 10 mL culture tube with a loose top

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• Add 5uL of 1000x antibiotic (Chloramphenicol.)

• Pick a distinct colony using a pipette tip and eject tip into tube.

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