Team:Northeastern Boston/measurement
INTERLAB STUDY
As participants of 2015’s Interlab study, we compared the relative abundance of Green Fluorescent Protein (GFP) under three promoters. These promoters, part of a collection colloquially known as the “Anderson promoters,” are unique because of their well-characterized difference in expression. Promoters regulate transcription by facilitating the binding of RNA polymerase. The Anderson promoters have variations in their nucleotide sequence—particularly between the 1st and 6th position and 26th and 30th—altering the binding potential of RNA polymerase and resulting in unique levels of GFP. We measured these GFP levels in replicate to determine the difference in promoter strength.
Workflow
July 17: K823005, K823008, K823013, and I13504 were reconstituted and transformed into TOP10 E. coli (Invitrogen) by heat shock (45 seconds at 42°C). Transformed cells were plated with glass beads onto either Ampicillin (I13504) or Chloramphenicol (all others) LB-agar plates. All plates were put in a 37°C incubator overnight.
July 18: All 4 strains were subcloned into 5mL of LB broth with their respective antibiotic.
July 19: K823005, K823008, K823013, and I13504 were miniprepped (Thermo Fischer) and then nanodropped to determine DNA concentration. I13504 was digested with XbaI and PstI (1x Tango Buffer, 2x PstI, 1x XbaI, 37°C, 1 hour). K823005, K823008, and K823013 were digested with SpeI and PstI (1x Tango Buffer, 2x PstI, 1x SpeI, 37°C, 1 hour). K823005 and I13504, with their respective restriction sites, are shown above.
Digested plasmids were run on a 1% Agarose gel with loading dye and 1kbp ladder at 100V for 60 minutes. The gel was stained for 10 minutes with ethidium bromide. The band in I13504’s lane, at 875bp, was cut and placed in a centrifuge tube. Bands in the lanes of K823005, K823008, and K823013 were cut at 2100bp and placed in centrifuge tubes. All band-containing-centrifuge-tubes were placed in a 4°C fridge overnight.
July 20: Bands were purified by column purification (Thermo Fischer). K823005, K823008, and K823013 were each (independently) ligated with I13504 in PCR tubes with T4 Ligase (Life Technologies).
The resulting plasmids, with reconstituted I20270 and R0040 in parallel, were transformed into heat-shocked TOP10 E. coli (45 seconds at 42°C) and plated onto LB-agar plates containing Chloramphenicol. All plates were put in a 37°C incubator.
July 21: Colonies were visualized through a blue-light, dissecting scope to check for the expression of GFP. Individual colonies were restreaked onto new plates; 3 biological replicates per strain. One representative colony from each plate was subcloned to check plasmids by restriction digest.
July 22: 150uL of LB-broth containing Chloramphenicol was aliquoted into 51 wells of a 96 well plate. 1 colony from each of the biological repeats, for each strain, was picked up and subcloned into 3 wells. Each strain had a row of 9 wells, 3 technical replicates per biological replicate. 3 wells were left blank, 3 were subcloned with E. coli from R0040. The 96 well plate was placed in a 37°C incubator overnight. Cultures were miniprepped and digested with EcoRI (plus PstI as a double digest for J23101, J23106, and J23117).
July 23: 16 hours after subcloning, OD600 was read on a BioTek Synergy 2 Multi-Mode Reader. Using the 3 R0040 control wells for a dilution curve, wells were diluted with LB-broth such that all wells fell within 5% of 0.5 OD600 (0.475 to 0.525). Fluorescence was measured with an excitation of 485nm and emission of 528nm. Values were recorded for further analysis.
Analysis
Fluorescence values at 485/528nm were averaged between technical replicates to determine means of the biological replicates, A, B and C. Standard deviation was determined for each triplet of technical replicates. The standard deviations of the technical replicates were propagated to determine the standard deviation of the biological means.
J23101 shows the strongest expression of GFP, followed by J23106, J23117, I20270 (Positive), and then R0040 (Negative). The relative fluorescence of each strain was determined (table). The resulting numbers, with the notable exception of J23117, closely correlate with those provided on the Anderson Promoter page. From a practical standpoint these promoters could be used in biomachines that require precise proportional expression of genes with little room for variations in flux.