Team:Baltimore BioCrew/Contribution
Characterization
Aims
In order to provide more insight into the characterization of promoters that are widely used in iGEM, we, the Baltimore Biocrew, decided to characterize 5 different strength promoters: J23100, J23101, J23103, J23105, J23118. Our first step was to find a diverse group of strengths of promoters, shown in the table below. In addition, the RFP in au by iGEM2006_Berkeley measured their strength via protein while we used RNA. We used RNA because other teams have previously used protein so we wanted to diversify the characterization of promoters.| Promoter Name | Strength | RFP in AU measured by iGEM2006_Berkeley | Plasmid Backbone |
|---|---|---|---|
| BBa_J23100 | 1 | 2547 | BBa_J61002 |
| BBa_J23101 | 0.70 | 1791 | BBa_J61002 |
| BBa_J23103 | .01 | 17 | BBa_J61002 |
| BBa_J23105 | .24 | 623 | BBa_J61002 |
| BBa_J23108 | .56 | 1429 | BBa_J61002 |
Details
- 1. On 6\15\19 we chose 5 consecutive Anderson promoter-RFP constructs that were available in our distribution kit as existing parts to characterize.
- 2. On 6\22\19 we re-suspended the DNA of our select parts from the distribution kit and did transformations, resulting in our DNA being in on bacteria on plates.
- 3. On 6\29\19 we repeated a transformation for J23101 because the plate was broken with few if any colonies and only one of the two liquid cultures grew cells.
- 4. On 7\12\19, three colonies from the plates were selected to grow in liquid culture overnight at 37 degrees Celsius.
- 5. On 7\13\19 extracted NA from bacteria (duplicates of each five promoter-RFP composite parts) and stored in -80c.
- 6. On 7\20\19 we measured the concentration of the RNA with the Nanodrop and made cDNA through reverse transcription with. Invitrogen superscript III kit.
- 7. On 7\20\19, we designed primers for RFP for real time PCR and ordered those as well as primers for an endogenous reference gene, rrsD( ribosomal RNA,16S).