Team:AQA Unesp/Parts



iGEM AQA_Unesp

parts

We submitted four parts to the Registry (see list below). Unfortunately, some parts that we’ve worked with are under patent, like SCI-57 and PenShuf, and though we had no legal problems working with it in the lab, we could not submit or make any profits and have credit for these parts.
We also had problem cloning some parts and we were not able to submit them: the usp45 promoter and the signal peptide from Lactococcus lactis, and the cell-penetrating peptide penetratin. However, we have submitted interesting and novel parts to the Registry:

  • BBa_K2270010 (basic part): a super-folding GFP codon-optimized that exhibits strong fluorescence in L. lactis. We’ve characterized this part, providing future teams an useful reporter to be used in circuits with L. lactis as chassis;

  • BBa_K2270005 (basic part): the regulatory promoter from the gal operon of L. lactis repressed by the presence of glucose and can be useful for many future circuits;

  • BBa_K2270006 (basic part): a synthetic small RNA that regulates the gene expression at posttranscriptional level and can be used in different circuits, under the control of different promoters and controlling the gene expression of any gen designed with the 5’ sequence complementary to the sRNA sequence. Thus, we provided a new alternative to regulate the gene expression in bacteria;

  • BBa_K2270008 (composite part): a device with BBa_K2270005 and BBa_K2270006 that works as a double inverter gate and regulates the gene expression by a glucose input. This part is particularly interesting and useful for other teams interested in building a circuit regulated by glucose in gram-positive bacteria.

Our parts help to fill the lack of Lactococcus lactis parts that we found in the Registry and can also be useful to stimulates other teams to work with L. lactis as chassis, that has been recently used for many applications as a platform for protein expression and therapeutics.

Furthermore, we have improved the srfA promoter (long variant) from Bacillus subtilis (BBa_K305008) that we’ve used in one of our constructions. We have characterized this promoter and showed that it’s stronger that the veg promoter, considerate a strong B. subtilis promoter. Thus, we’ve contributed to improve the Bacillus subtilis collection in the Registry and we offer future teams a new option when choosing a constitutive strong promoter for their devices.

Loading.....

improve

In the characterization of this promoter, we made a comparison between srfA and Pveg promoter, which is recognized as being a strong one and is already featured in the registry (BBa_K143012), and we show a relationship of cell growth and expression of GFP. As a tool for measuring the activity of promoters, reporter vectors can be required. Therefore, we chose the luciferase reporter (pBs3Clux-PsrfA), and green fluorescence protein reporter (pBs1C-PsrfA-GFP). For luciferase reporter, we did the percentage of luminescence making the ratio by the point of greatest luminescence and multiplying by 100, Figure 1 and 2. Already by GFP, we measured at 535nm for emission and 485nm for excitation, Figure 3. The readings were performed on the Infinite® M200 (Tecan).

Figure 1. Growth curve of Bacillus subitilis for about 7 hour, 37°C and 220 rpm.


Figure 2: Percentage of Luminescence of PsrfA and Pveg; We use for negative control the plasmid containing pBs3Clux without promoter. The growth has occurred for about 7 hour, 37°C and 220 rpm.


As shown graphically, we report that luminescence induced with pveg is the frist to grow, but the srfa induction grows more than pveg promoter does. To show the relationship of cell growth to the reporter promoter response, we used GFP as can be seen in the figure 3.

Figure 3: Correlation of GFP expression (pBs1C-srfA-GFP) and cell growth as a function of time for about 8 hour, 37°C and 220 rpm.


In this case, GFP production follows cell growth. With luciferase and GFP as reporter genes, we show that the srfA promoter behaves the same no matter the downstream gene athached
Team: AQA_Unesp