Part:BBa_K3552016
ptac-SccJ
We improved the part BBa_K180000 to improve its productivity. We reversed the Lac operon parts and changed to a reverse promoter, terminator and RBS. A new ribozyme, SccJ is added to the sequence. This part is in the part collection where we have 12 genes that code for the base generator of pilA.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Characterization
Our new ptac promoter consists of 7 components, the reverse lac terminator, lacI, RBS, lac promoter and normal ptac promoter, lacO optimized and SccJ. The new promoter comparing to the BBa_K180000 found in 2009 has a much larger gradient of changing when comparing to the concentration of IPTG. In our experiment, we add different concentration of IPTG to constant promoter and received the value of florescence. We compared the value to the negative control and acquire the stable points to compare the data from the old ptac promoter.
The main carbon source is the glycerol and the bacteria were cultured in M9 medium in order to simulate the expression of pilA. In this way, the result might be able to become a reference relating to our pilA production. The graph shows that after adding RiboJ, SccJ, and SarJ, the tangent of the line increased remarkably, referring to an ascent of the E.coli sensitivity of IPTG in low concentration. RiboJ has the best function on increasing the productivity of GFP among the five ribozymes.
We repeated the experiment in the culture medium with glucose as the source of carbon and gained similar results as the previous experiment. The data, in this manner, illustrate that the inducible promotor is robust in rising the yield in relatively low concentration. To our project, we have added RiboJ in front of our pilA genes to keep the gene in highly expression rate. In the following stage, we will apply these ribozymes on the generator transformation and better rise the production of pili generator. We hope this metamorphosis could generally increase the pili production.
| None |