Part:BBa_K5238001

Produce tetrahydrofolate to bind to target lilrb3 for the treatment of Alzheimer's disease

Tetrahydrofolate (THF) plays a crucial role as a coenzyme in one-carbon metabolism, which is essential for the proper functioning of the nervous system. Studies have indicated that THF has potential benefits in treating Alzheimer's disease (AD) and depression. It aids in the synthesis of neurotransmitters and DNA methylation, thereby improving cognitive function and mood regulation. Furthermore, THF can lower homocysteine levels, which helps reduce oxidative stress and neuroinflammation, providing therapeutic benefits for individuals with AD and depression[1][2].

In our research, we have demonstrated through molecular docking simulations and molecular interaction experiments that THF can effectively target the latest LilrB3 receptor, which is closely associated with APOE4 in AD and represents a pivotal target. By competitively inhibiting APOE4-LilrB3 receptor binding, this strategy addresses the root cause of amyloid protein generation and deposition, significantly advancing AD treatment and prevention and opening up new avenues for engineered bacterial therapy in AD.

Test: According to our experimental procedures, we successfully transformed plasmids into L168, confirmed through PCR and agarose gel electrophoresis. After culturing L168, we extracted RNA using bacteriophage and measured metabolites from the supernatant. We then conducted reverse transcription and quantitative PCR (qPCR) to quantify gene expression levels in L168, both with and without the pSIP403-P9-folKE plasmid. The experiment assessed the relative expression levels of two genes using qPCR. The results, shown in the figure 1, indicate that the expression of folK and folE was significantly higher in L168 containing the pSIP403-P9-folKE plasmid compared to the control group (Fig. 1).

Figure 1. Relative Expression Analysis of genes folK and folE by qPCR.

Moreover, we employed Liquid Chromatography-Mass Spectrometry (LC-MS) to analyze THF production in the supernatant of L168.The results clearly show that THF levels significantly increased in L168 with the pSIP403-P9-folKE plasmid compared to the control group, confirming that the folKE gene was effectively expressed in L168 and produced THF as anticipated (Fig. 2).

Figure 2. Relative levels of THF between two groups by LC-MS.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 906
  Illegal EcoRI site found at 1049
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 906
  Illegal EcoRI site found at 1049
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 906
  Illegal EcoRI site found at 1049
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 906
  Illegal EcoRI site found at 1049
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 906
  Illegal EcoRI site found at 1049
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 931