Part:BBa_K577895:Experience

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UNIQ939ebff8076b6f80-partinfo-00000000-QINU UNIQ939ebff8076b6f80-partinfo-00000001-QINU

• WashU_StLouis

The 2015 WashU iGEM conducted a series of induction experiments to look at the performance of this part. The results obtained suggest that the part isn’t inducible. The part either fluoresced at a constant, high level or didn’t fluoresce at all when tested. The team transformed the part into E coli DH10B twice. The colonies of the first transformation looked like this: Most of the cells were red. The WashU iGEM team then conducted an induction experiment to test how the cells fluoresced at different concentrations of aTc. The team pipetted cell culture into different aTc concentrations serially diluted across 8 wells. Results from this induction experiment are shown below. While the part had a 1.3x increase between the lowest and highest inducer concentrations, the fluorescence increase was not uniform across all concentrations; fluorescence decreased at certain concentrations when it should’ve been increased. The team proceeded with a second transformation to test the part even further. Colonies from that transformation are shown below. Cells were largely colorless, with few red colored colonies to be found. Using the same procedure from the first experiment, the team ran a second induction experiment on these colonies to observe whether red fluorescence increased with aTc concentration. The results from that experiment are shown below. The colonies exhibited no remarkable fluorescence regardless of the aTc concentration. At this point in time, the team hypothesized that the colonies from the first transformation were simply different than the colonies from the second transformation, perhaps as a result of homologous recombination. The team decided to run a third induction experiment, with the same protocol used in the previous two experiments, to determine the validity behind this assertion. Two colonies were picked from the first transformation; one colony was picked from the second transformation. The results from this experiment are shown below. Values for each colony are consistent with what was obtained in their respective experiments. While the team didn’t sequence the parts in each colony, it suspects that two separate strains had been produced, in which the part had been mutated in some manner. Regardless, the team holds little confidence in the effectiveness of this part. Methods- Induction -Pipette 4 mL sterile LB/Antibiotic into appropriate culture tubes. For the plasmid containing part BBa_K577895, pipette 4 mL sterile LB/CM. For wild type negative controls, pipette 4 mL of LB. -Place the cap on the culture tubes to the first position to allow aeration. -Place the tubes into the incubator and allow them to grow overnight (16 hours) at 37 deg C and 250 RPM Next day... -Make frozen stock for all cultures grown -Measure OD at 600 nm in cuvettes for each culture (OD O/N) (900 uL water and 100 uL culture well mixed) -Dilute cultures to OD of 0.2 in a total of 4 mL fresh LB/antibiotic (just LB for the wild type negative controls) in new 14 mL culture tubes -Volume of overnight culture = (0.2*4)/(OD overnight) -Volume of LB/antibiotic (just LB if negative control) = 4mL - V overnight -Incubate each culture tube for 2 hours at 37 deg C and 250 rpm -While waiting for culture tubes to complete incubation, prepare 96 deep well plates for induction -Set up induction such that each consecutive well’s aTc concentration is 5x diluted from the well next to it, such that the total volume is 0.6 mL. It’s important to note that 0.1 mL of this volume is cell culture, which must be taken into account when doing calculations -For example, one would pipette 1.875 uL of 105 ng/uL aTc concentration into 648.125 uL of LB/Anti mixture, so that when 150 uL of that mixture is taken and added to the well next to it, the well will have a concentration that when diluted to 600 uL through addition of cell culture, possesses the target concentration -Once wells are prepared, add cell culture to appropriate rows -Cover the deep well plates with breathable membranes. -Incubate 6-8 hours at 37°C and 250 rpm. To measure... -Centrifuge at 3000 g for 15 minutes. Remember to balance with another plate. -Discard supernatant. -Resuspend all cells in 200 μL 1x PBS. -Transfer 200 μL of wells to a black 96-well plate. -Measure with a Tecan plate reader set to 580 nm excitation and 610 nm emission for mRFP, absorbance at 600 nm and the gain set to 100. -Divide fluorescence by absorbance per well. -Subtract average fluorescence/absorbance of corresponding wild type strain from the fluorescence/absorbance of the strains with the fluorescent protein. -Calculate average fluorescence/absorbance and standard deviation based on the entire population for each strain at each inducer concentration. -Plot average fluorescence/absorbance versus inducer concentration for each strain.