Part:BBa_K2471005
Expressible glycosyltransferase family 2 protein from Bacillus subtilis
This BioBrick™ is a brand new addition to the Parts Registry, it contains the necessary genetic circuitry to constitutively express the team's new epsE gene (BBa_K2471014), that when expressed, results in the production of the epsE enzyme, and thus, biofilm formation is promoted whilst inhibiting motility.
A chassis with the T7 polymerase gene is needed for the successful expression of this BioBrick™.
Usage and Biology
The epsE gene, in the same way, constitutes a fundamental part of the project, since it eliminates cell motility by promoting the separation of the flagellum from the motor proteins located in the cell membrane. The BioBrick® presented in this section is a new and functional addition to iGEM's Parts Registry, since both's codifying genes' sequences are different from the ones in the catalog (and are confirmed to codify for their respective proteins, unlike the parts that are already in the Parts Registry, which is reported as being putative) and were synthesized thanks to the sponsorship granted by IDT®. This new BioBrick® was transformed into Escherichia coli BL21(DE3) (both) and Erwinia amylovora. After performing polyacrylamide gel electrophoresis, it was concluded that our new aiiA BioBrick is functional because the protein was expressed in both bacterial types that it was transformed into; on the other hand, the epsE BioBrick didn't work, since no protein expression was found in Escherichia coli BL21(DE3).
Characterization of the epsE gene
This BioBrick, as BBa_K2471004, has a T7 promoter (BBa_J64997), RBS (BBa_B0034), a gene of interest (glycosyltransferase family 2 protein; BBa_K2471014) and two terminators, T1 (BBa_B0010) and T7 (BBa_B0012); this flanked by the iGEM prefix and suffix on the 5' and 3' end respectively. As this part was synthesized thanks to the IDT® sponsorship, it came in the pUCIDT vector with ampicillin resistance; the change to the pSBC13 backbone was carried out through enzymatic digestion and ligation. SnapGene® indicated that this BioBrick has a length of 3,095 base pairs, agarose gel (1%) electrophoresis was performed to corroborate that the obtained product had the expected molecular weight.
Figure 1. (A) SnapGene® map of BBa_K2471005. (B) Agarose gel (1%) electrophoresis of BBa_K2471005 compared with NEB Quick-Load® Purple 2-Log DNA Ladder (0.1 - 10.0 kb), where the highlighted bands correspond to approximately 3,000 bp.
Escherichia coli BL21(DE3) was transformed with this new BioBrick, as for the characterization of BBa_K2471004, electrophoresis in polyacrylamide gel (12%) was performed to corroborate that the protein was being expressed. The methodology followed was the same as the one previously mentioned. To calculate the molecular weight of the protein, the peptidic sequence provided by NCBI (NCBI Reference Sequence: WP_032722561.1) was taken and analyzed in ExPASy - ProtParam tool, calculating a molecular weight of 32.17317 kDa.
Figure 2. (A) SDS-PAGE (12%) of Escherichia coli BL21(DE3) transformed with BBa_K2471005. (B) SDS-PAGE (12%) of Escherichia coli BL21(DE3) that hasn't undergone transformation.
A band of the same relative intensity can be seen at the approximate weight of 32 kDa from the third hour onwards in Figure 2.A and from the second hour onwards in Figure 2.B. Based on this, we can affirm that our epsE protein is not being expressed, since banding of approximately equal relative intensity is found in both the transformed bacteria and in the one that hasn't undergone transformation, this means that both express whatever protein is responsible for that banding. Bibliographic research shows that this protein it's only been reported to work in Bacillus sp.; based on this, it can be said that the part was characterized because this protein does not express in Escherichia coli BL21(DE3).
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]
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