Team:Tec-Chihuahua/Silver
Silver
Overview
The aiiA gene was of such relevance to the project's development because of its function since it's responsible for the inhibition of two of Erwinia amylovora's main virulence factors: the type III secretion system and the production of exopolysaccharides. Quorum sensing regulates, among many other cell functions, both of these; therefore, by disrupting the quorum sensing, the bacteria will lose these virulence factors. 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. Each 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®. These two new BioBricks (aiiA: BBa_K2471004 and epsE: BBa_K2471005) were transformed into Escherichia coli BL21(DE3) (both) and Erwinia amylovora (just aiiA). 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).
New BioBrick Parts in the Registry, Their Role in our Project and its Characterization
Two of the three bioparts critical to our project, aiiA (BBa_C0060) and epsE (BBa_K143032), are reported as being putative; this means that these sequences are classified as those genes based on empirical evidence, it hasn't been completely proven that they codify for their supposed proteins, it’s purely hypothetical. Because of this, we synthesized two new BioBricks with sequences that are reported to code for our proteins of interest; both have the genetic circuitry necessary to correctly express their confirmed genes. The first one, BBa_K2471004, codifies for aiiA, an N-acyl homoserine lactonase from Bacillus sp. A24 (NCBI GenBank: AF397400.1) and has a molecular weight of 29.45340 kDa; the other one, BBa_K2471005, codes for a glycosyltransferase family 2 protein from Bacillus subtilis (NCBI Reference Sequence: WP_032722561.1) with a molecular weight of 32.17317 kDa. This decision was taken to reduce the chances of problems during the expression of said enzymes.
BBa_K2471004This BioBrick counts with a T7 promoter (BBa_J64997), RBS (BBa_B0034), the gene of interest (N-acyl homoserine lactonase; BBa_K2471013), 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. This part was synthesized thanks to the IDT® sponsorship. Thus, 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 3,070 base pairs, agarose gel (1%) electrophoresis was performed to corroborate that the obtained product had the expected molecular weight.
Both Escherichia coli BL21(DE3) and Erwinia amylovora were transformed, by heat shock and electroporation respectively, with this new BioBrick. In the same way, as for the characterization of BBa_C0060 (in the bronze section of results), electrophoresis in polyacrylamide gel (12%) was performed to corroborate that our protein of interest was indeed being expressed. To calculate the molecular weight of the protein, the sequence provided by NCBI (GenBank: AF397400.1) was taken and introduced in ExPASy - Translate tool, which generated the open reading frame in amino acids. This peptidic sequence was then analyzed in ExPASy - ProtParam tool, where a molecular weight of 29.45340 kDa was calculated.
Upon examination of figure 2, an appreciable band can be seen at the approximate weight of 30 kDa from the second hour onwards in figure 2.A, while none can be seen in 2.B. From this, it can be concluded that our aiiA protein is being expressed in the transformed Escherichia coli BL21(DE3). In the same way, the total protein extraction was carried out better on the bacteria transformed with our BioBrick than in the one that wasn't. Based on this, we can state that our enzyme is not only being expressed, but it's also performing its function; which is quorum quenching, as previously mentioned in the bronze section of results. One of the effects of this transformation is the disruption in the production of exopolysaccharides, one of the main components of Escherichia coli's biofilm. The deficient production of these molecules translates into deficient biofilm formation; thus, the cell lysis is performed to a greater degree, yielding a better protein extraction.
As mentioned before, the bacteria Erwinia amylovora was also transformed with this new BioBrick. Electrophoresis in polyacrylamide gel (12%) to corroborate that the protein of interest was being expressed. A band at the approximate weight of 30 kDa can be seen in figure 3; thus, from what is observed in both figure 2 and 3, we can infer that our new BioBrick (BBa_K2471004) indeed codes for our aiiA gene and expresses its enzyme in both types of bacteria transformed with it.
BBa_K2471005
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.
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.
A band of the same relative intensity can be seen at the approximate weight of 32 kDa from the third hour onwards in figure 5.A and from the second hour onwards in figure 5.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).
Keep exploring our results!
Follow us on social media:
Facebook: facebook.com/igemcuu/
Twitter: @iGEM_TecChih
Email: igemtecchihuahua@gmail.com
We thank our sponsors:
