Team:Tacoma RAINmakers/Contribution
Team:ECUST/Lab/Notebook
Contributions
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.
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.
| AFUAVG±SD | 0HR | 3HR | 5HR |
|---|---|---|---|
| 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.
