Part:BBa_K2471004
Expressible N-acyl homoserine lactonase from Bacillus sp. A24
This BioBrick™ is a brand new addition to the Parts Registry, it contains the necessary genetic circuitry to constitutively express the team's new aiiA gene (BBa_K2471013), that when expressed, results in the production of the aiiA enzyme, and thus, the quenching of quorum sensing thanks to the decrease in AHLs.
A chassis with the T7 polymerase gene is needed for the successful expression of this BioBrick™.
Usage and Biology
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 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 (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.
Characterization of the aiiA gene by Tec-Chihuahua
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.
Figure 1. (A) SnapGene® map of BBa_K2471004. (B) Agarose gel (1%) electrophoresis of BBa_K2471004 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.
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 a 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.
Figure 2. (A) SDS-PAGE (12%) of Escherichia coli BL21(DE3) transformed with BBa_K2471004. (B) SDS-PAGE (12%) of Escherichia coli BL21(DE3) that hasn't undergone transformation.
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.
Figure 3. SDS-PAGE (12%) of Erwinia amylovora transformed with BBa_K2471004.
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.
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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