Team:Tacoma RAINmakers/Contribution

Team:TacomaRAINmakers/Notebook -



Part Characterization

BBa_J23100, BBa_J23101, BBa_J23102, BBa_J23105, BBa_J23106, BBa_J23112

We collected characterization data on the relative RFP expression rates of Anderson Promoters BBa_J23100, BBa_J23101, BBa_J23102, BBa_J23105, BBa_J23106, and BBa_J23112 in the RFP expression plasmid BBa_J61002 (samples provided in the 2019 iGEM Distribution Kit).

Our protocols were adapted from the 2018 Interlab protocol. Based on the results of past Interlab studies, we know that there are sometimes variability in expression that occur between labs. We chose to characterize a select number of Anderson promoters because they are frequently used by iGEM teams and we wanted to contribute data that would help teams select the right promoter strength for their needs. We were also interested in better understanding promoter strengths for our own work when we were planning our project. As our team did not have access to calibration standards such as Texas Red or Rhodamine B, this data was analyzed relative to BBa_J23100 expression. The comparison against BBa_J23100 is consistent with the original Anderson characterization.

When analyzing arbitrary fluorescence units (AFU), we found that our data were comparable to that collected by the Anderson Lab after 3 hours of growth (Table 1). BBa_J23100 showed the strongest expression followed by BBa_J23102, BBa_J23101, BBa_J23106, BBa_J23105, and BBa_J23112 showing the weakest expression. At hour 5, our data presented a slightly different order of strength. There was increased expression of BBa_J23101 over BBa_J23102. However, after normalizing the data against BBa_J23100, the expression levels of BBa_J23101 and BBa_J23102 were comparable. The OD660 values were still increasing from 3HR and 5HR, which indicates the cultures were still growing. Allowing the cultures to grow further would allow us a better determination regarding the difference in promoter strength of BBa_J23101 and BBa_J23102.

Characterization Protocol

Characterization Data

Table 1. Summarized Anderson lab data for comparison.

Grown to saturation
BBa_J23100 1.00
BBa_J23102 0.86
BBa_J23101 0.70
BBa_J23106 0.47
BBa_J23105 0.24
BBa_J23112 0.00

Table 2. Average arbitrary fluorescence units of Anderson promoters.

J23100 116.5±6.2 159.4±9.2 210.6±6.0
J23101 103±5.9 143.0±3.2 178.7±15.9
J23102 97±6.9 151.1±3.4 157.8±15.5
J23105 82±11.6 101.2±7.3 123.8±23.4
J23106 80±8.2 118.8±9.5 131.9±8.6
J23112 67±4.0 86.3±5.1 83.0±8.7
Neg 57.4±2.4 65.3±5.5 80.2±3.3

Figure 1. Graph of fluorescence using Anderson promoters from Table 2.

Table 3. Optical density of cultures measured at Abs600nm.

OD660 AVG±SD Hour 0 Hour 3 Hour 5
J23100 0.006±0.006 0.123±0.018 0.294±0.011
J23101 0.009±+0.006 0.206±0.016 0.395±0.028
J23102 0.007±+0.007 0.152±0.015 0.354±0.018
J23105 0.017+0.005 0.250±0.013 0.436±0.010
J23106 0.008±0.004 0.198±0.010 0.376±0.010
J23112 0.0198±0.009 0.247±0.012 0.415±0.013
Neg -0.009±0.004 0.007±0.012 -0.011±0.003

Figure 2. Graph of OD660 of cultures from Table 3.

Table 4. Relative strengths with AFU normalized to OD, as compared to J23100

Hour 0 Hour 3 Hour 5
J23100 1.00 1.00 1.00
J23102 0.60 0.54 0.62
J23101 0.77 0.77 0.62
J23106 0.27 0.31 0.40
J23105 0.56 0.47 0.49
J23112 0.18 0.27 0.28

Figure 3. Graph of relative promoter strength from Table 4.