Parts overview
For the construction of Biobricks, we used parts from the iGEM registry and also made use of the IDT synthesis offer. Since our aim was to develop a bacterial QR code with inducible properties, we focused on characterization of the promoters and hence worked on screening synthetic promoter libraries to obtain the best fit of promoter.
From the beginning, Vibrio natriegens was our foremost organism of interest due to its fast growth rate, that would result in a quick readout of the QR code. We designed a basic part comprising of V. natriegens native P1 promoter (BBa_K3171171) and a composite part P1-mCherry fusion protein (BBa_K3171172). The strength of the P1 promoter was measured in the P1-mCherry construct, which was designed by 3A assembly method.
Another basic part was constructed using a synthetic promoter library on pTet (BBa_R0040) in order to enhance function and inducibility (BBa_K3171173). We optimized tetracycline-inducibility in E. coli by creating randomizations in the sequence to obtain arbitrary promoters. The screening was done based on quantifications of the fluorescence intensity measured. The best version of the promoter was determined based on fold change in the fluorescence intensity and low basal level. This promoter is also used in the composite part with mCherry fusion protein (BBa_K3171175).
Furthermore, we experimented with CRISPR with the aim to make a histidine auxotroph by knocking out HisD in both E. coli and V. natriegens. Specific gRNA targets were selected for knockout of the HisD gene. We designed 3 homologous parts (BBa_K3171177, BBa_K3171178, BBa_K3171179) and 3 gRNA target sites (BBa_K3171180, BBa_K3171181, BBa_K3171182) which all fall under the category of basic parts. We hope these constructs would be beneficial in the future in a way to combine our basic parts with different expression levels or produce a unique composite part.
Table 1. BioBrick Parts submitted by University of Groningen iGEM 2019 team. All BioBricks were constructed by the team members engaged in the wet lab; Sander de Weerd, Michelle Scharte, Nurul Wirusanti, Yashika Venkatesh, Dasha Vedernikova, Sophie Schretlen, Lieke van Iersel.
| # |
Name |
Type |
Description |
Designer |
| 1 |
BBa_K3171171 |
Basic |
V. natriegens native P1 promoter |
RUG, 2019 |
| 2 |
BBa_K3171172 |
Composite |
V. natriegens native promoter P1-mCherry |
RUG, 2019 |
| 3 |
BBa_K3171173 |
Basic |
Improved pTet promoter in E. coli |
RUG, 2019 |
| 4 |
BBa_K3171175 |
Composite |
pTet-mCherry optimized in E. coli |
RUG, 2019 |
| 5 |
BBa_K3171177 |
Basic |
HisD 3000bp Homologous part 1 |
RUG, 2019 |
| 6 |
BBa_K3171178 |
Basic |
HisD 1500bp Homologous part 2 |
RUG, 2019 |
| 7 |
BBa_K3171179 |
Basic |
HisD 600bp Homologous part 3 |
RUG, 2019 |
| 8 |
BBa_K3171180 |
Basic |
HisD gRNA 1 |
RUG, 2019 |
| 9 |
BBa_K3171181 |
Basic |
HisD gRNA 2 |
RUG, 2019 |
| 10 |
BBa_K3171182 |
Basic |
HisD gRNA 3 |
RUG, 2019 |
