Before going on to the oscillations, we wanted to characterize the promoter strengths of our newly submitted promoters, since a proper characterization for these parts did not exist. Therefore, we set up experiments in a plate reader, for the 5 promoters used in the Murray et al 5n1 system, of which 2 were also a part of the square wave generating oscillator.
Parts containing the fluorescent GFP reporter (BBa_E0040) under the 5 repressible promoters (pLac, pTet, pCI, pPhlF, pSrpR) were cloned and created, and grown overnight in LB. The cultures were then diluted 1:50 and 1:200 in LB, and put into a plate reader, with 4 replicates. Growth was observed via absorbance at 600 nm, and fluorescence was observed by excitation at 485 nm and emission at 511 nm.
We see in Fig. 1 that the decreasing order of strengths for the five repressible promoters is pLac, pcI, pPhlF, pTet and pSrpR. This is also evident in Fig. 2 when all the 5 promoters settle to a steady state. Fig. 3 depicts the protein production rates for the promoters. pLac has the largest production rate reaffirming its highest strength. However, despite dominating completely in terms of protein production rate, we see in Figs. 2 and 4 that in the initial transient phase, pLac has lower relative strength compared to pTet and pcI. This is due to the highest dilution rate for pLac in this regime, which can be seen in Figs. 5 and 6. Similar arguments can be made about pcI.
Fig 1. Relative Strength for the five repressible promoters, including the novel promoterspPhlF and pSrpR, used in the construction and analysis of 5n1. The decreasing order of relative strength is pLac>pcI>pPhlF>pTet>pSrpR. Rlative strengths have been averaged over 4 trials.
Fig 2. Relative Strength over time for the five repressible promoters, including the novel promoterspPhlF and pSrpR, used in the construction and analysis of 5n1. The plots have been averaged over 4 trials.
Fig 3. Protein production rate over time for the five repressible promoters, including the novel promoterspPhlF and pSrpR, used in the construction and analysis of 5n1. The plots have been averaged over 4 trials.
Fig 4. Relative Strength versus OD over time for the five repressible promoters, including the novel promoterspPhlF and pSrpR, used in the construction and analysis of 5n1. The plots have been averaged over 4 trials.
Fig 5. Dilution rate over time for the five repressible promoters, including the novel promoterspPhlF and pSrpR, used in the construction and analysis of 5n1. The plots have been averaged over 4 trials.
Fig 6. OD over time for the five repressible promoters, including the novel promoters pPhlF and pSrpR, used in the construction and analysis of 5n1. The plots have been averaged over 4 trials.
As shown in Fig. 2, pLac and pcI have three distinct regions of behavior in terms of relative strength over time. In the initial transient phase, dilution rate is higher than the protein production rate, thus we see that relative strength per OD decreases over time. The middle corresponds to overcompensation phase, where protein production rate surpasses the dilution rate. Finally, the relative strength settles a steady state value, entering an exact compensation between protein production and dilution rates. Contrary to pLac and pcI, the other promoters don’t exhibit the overcompensation phase.
