Difference between revisions of "Part:BBa K2260002"

Latest revision as of 08:57, 2 October 2024

Phasin-HlyA

Phasin is an intracellular protein native to PHB-producing bacteria, and binds to PHB (polyhydroxybutyrate) deposits within the cell through electrostatic interaction. HlyA is a toxin produced by E. coli that is secreted via an endogenous, single-step type one secretion system. By adding the C-terminus of the HlyA gene to the end of the phasin molecule, the phasin is marked for secretion through the pathway designed for the HlyA. As a result, the phasin molecule binds to intracellular PHB granules and then is secreted due to the HlyA tag, effectively transporting PHB outside of the cell.

This part has been catered towards usage in E. coli, specifically in the strain BL21 DE3, as this strain has endogenous T7 polymerase to complement the T7 promoter we have for our gene. Thus, this sequence has been codon optimized to optimize success with E. coli. Furthermore, restriction sites have been altered in order to make this gene compatible with all RFC assembly standards, ensuring ease of access for future teams.

When designing our part, we used the part BBa_K2018024, designed by the SDU-Denmark 2016 team, as a template. It can be found at https://parts.igem.org/Part:BBa_K2018024.

Our assays showed that our part resulted in a 114% increase of secreted PHB found in the media, compared to PHB found in the media of cells without our part. More details can be found under the "experience" tab.

Sequence and Features

Concordia_Shanghai 2024's Characterization

Team: Concordia_Shanghai 2024

Authors: Amy Wang, David Doyle

Background

This part was successfully used for secretion of PHB in our system (BBa_K5165002). This part has been previously used by iGEM Calgary 2017 and iGEM Calgary 2022 to secrete PHB from E. coli cells. Here we again showed this capability while we also increased the uses of this part and opened up its use to a new field of research. By adding our new part (BBa_K5165000), which breaks down PHB into β-Hydroxybutyrate (BHB) via hydrolysis, our project, buy using BBa_K2260002 allowed for the accumulation of BHB in the extra cellular space of E. coli cells. BHB is a naturally forming ketone body which has applications in the field of nutritional science and is linked to improved athletic an cognitive performance. We used this part to accumulate BHB in the extra cellular environment and thus provide an opportunity for the industrial scale harvesting of this high value product. Our project’s primary goal was to produce β-Hydroxybutyrate (BHB); our main construct produces and secretes PHB which is then extracellularly depolymerized by PHAZ-TALFU into BHB. The produced PHB was marked for secretion into the extracellular environment by this Phasin-HlyA sequence.

Results

The high presence of extracellular BHB in our experiments indicates that PHB was successfully secreted using Phasin-HlyA and degraded, thereby confirming the functionality of the Phasin-HlyA sequence. We first conducted an assay to determine a standard curve for the concentration of BHB. The BHB colorimetric assay kit was purchased from Grace Biotechnology and standard curve was determined following the kit protocol. E.coli BL21 cells were successfully transformed with our designed plasmids. Colonies of each cell type from selection plates were grown overnight in liquid broth + ampicillin. The BHB colorimetric assay was carried out according to kit manufacturer's protocol where cells were separated from the growth media by centrifugation (12000rpm) for 10 minutes. Supernatant was collected and kit reagents added to each experimental group as per protocol.

The absorbance level of cells containing plasmid 1 (BBa_K5165002), plasmid 2 (BBa_K5165003), and wild type cells. Verifies the production of BHB (high absorbance indicating high levels of BHB production) and the functionality of Phasin-HlyA.]]

A high level of BHB was present in the extracellular fluid of cells containing the plasmid with BBa_K5165002, indicating endogenously produced AHL was up-regulating the expression of PHAZ_TALFU in comparison to trace amounts found the extracellular space of cells transformed with plasmid 2. Using the standard curve of BHB concentration (y = 0.4651x - 00221) we determined the average concentration of BHB in the extracellular space of cells transformed with plasmid 1 to be 1.347 umol/mL compared to just 0.138 umol/mL in the extracellular space of cells transformed with plasmid 2.

This means that the PHB produced has been secreted in relatively high amounts due to the Phasin-HlyA sequence. The resulting PHB has been extracellularly degraded into BHB through other components of our system.

The lack of extracellular BHB in Plasmid 2 is unrelated to Phasin-HlyA functionality; manual induction of this other system (BBa_K5165003) using AHL was likely unsuccessful due to errors in AHL preparation.

This demonstrates that an additional application of this Phasin-HlyA sequence is for BHB production after PHB has been secreted when linked to PHAZ_TALFU, such as in BBa_K5165002.

Figure 2. Qualitative data from β-Hydroxybutyrate colorimetric assay. Tube 1 contains kit reagents + a known quantity of standard solution (2 umol/mL). Tubes 2, 3, & 4 contains kit reagents + supernatant of cell environment for BL21 cells transformed with plasmid 1, plasmid 2, and wild type (untransformed cells) respectively. Expression of the system containing Phasin-HllyA resulted in high amounts of extracellular BHB, meaning the produced PHB was secreted by Phasin-HlyA and degraded by other components of our system. ]]