Part:BBa_K4960021

Engineered Mitochondrial Uncoupler Pdp1NTD-EGFP-UCP1

Usage and Biology

Our project aims to design a system that can effectively deliver the uncoupling protein UCP1 into adipocytes via PVCs. For this, we are using BBa_K4960022 as the basic part to enables the delivery of UCP1 by serving as the payload for the PVC delivery system. UCP1 is a naturally occurring mitochondrial uncoupler protein found in brown adipose tissue of mammals. It works by transporting protons across the mitochondrial membrane, inducing a process of mitochondrial uncoupling that disconnects oxygen consumption from ATP synthesis. This uncoupling process results in the dissipation of energy in the form of heat, leading to an increase in energy expenditure and basal metabolic rate.

Special Design

During the testing of the BBa_K4960021 part, it was discovered that the interaction between the Pdp1NTD domain and UCP1 (as highlighted in the red box in Figure 1a) could potentially alter the local protein structure and impact the translocation and function of UCP1. To address this issue, we used AlphaFold2 to predict some of the possible structures and found that we could resolve this problem by swapping UCP1 and EGFP (as shown in Figure 1b). Consequently, we designed this part by connecting Pdp1NTD to the N-terminus EGFP and a UCP1 to the C-terminus (as depicted in Figure 1c).

Figure 1. Updated schematic diagram of design ideas. In the process of designing part, we switched the original sequence of EGFP and UCP1, and carried out the same experimental treatment as a new group of experimental groups, hoping to solve the problems encountered befor

Sequence and Feature

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Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1258
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 235
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Functional test

Methods

To validate the function of this part, we constructed pNC088, a CMV-driven Pdp1NTD-EGFP-UCP1 expressing plasmid. We transfected HEK-293T cells with pNC088 and observed the cellular localization of the fusion protein 48 hours post-transcription by widefield fluorescent microscopy and live-cell confocal imaging. We also analyzed the glucose consumption of the transfected cells by measuring the glucose levels in the culture medium. This analysis represented the level of cellular energy expenditure. We compared the results with those of the control group cells transfected with pcDNA3.1(+) vector only.

The results of both wide-field fluorescent imaging (Figure 2a) and live-cell confocal imaging (Figure 2b) indicated highly specific colocalization of Pdp1NTD-EGFP-UCP1 signal with mitochondria markers (MTS-mcherry, Figure 2c). Furthermore, cells that were transfected with pNC088 demonstrated a significantly higher glucose consumption rate when compared to cells transfected with the pcDNA3.1(+) vector (Figure 2c). This suggests that the energy consumption in these cells was significantly improved.

Figure 2a.Localization Pdp1NTD-EGFP-UCP1 in HEK-293T cells.Cells were transfected with pNC088 (PCMV-Pdp1NTD-EGFP-UCP1).

Figure 2b.Cells were co-transfected with MTS-mcherry and PNC088. Photos were taken 48 h post transfection, scale bar: 100μm for wide-field microscopy and 10 μm for confocal microscopy. Data are representative images of 3 independent experiments.

Figure 2c.Charactrization of cellular metabolism in HEK-293T cells transfected with either pNC088 or pcDNA3.1(+). Glucose concentration in the cell culture medium was measured 48 h after transfection; data shows mean±SD, n=3 independent experiments.

References

[1] Kolonin MG, Saha PK, Chan L, Pasqualini R, Arap W. Reversal of obesity by targeted ablation of adipose tissue. Nat Med. 2004 Jun;10(6):625-32.