Part:BBa_K4144004

Codon-optimized Pet8p for S-adenosyl methionine transportation

Background and purpose

Pet8p is one transporter of mitochondrial carrier protein family of Saccharomyces cerevisiae. It is also called Sam5p, since it functions to transport S-adenosyl methionine (SAMe). We WHU-China iGEM planed to utilize such transporter to transport SAMe from engineered bacterial cytosol to extracellular environment. Exogenous expression of such an eukaryotic protein Pet8p in prokaryotic cells has been achieved (Fig. 1 Left). Since it is a bidirectional transporter, the flow of SAMe depends on its concentration difference between outside and inside. So we think that the direction of integration of Pet8p might not matter, which means whether the N-terminus directs outside or inside will not affect its transport function (Fig. 1 Right).

Figure. 1 Expression of Pet8p in E. coli and transportation efficiency measurement in liposome(Marobbio CM, Agrimi G, 2003)

Design of opPet8p

Since our engineered bacteria are purposed to be gram-positive, to improve Sam2 expression efficiency, we used a codon optimization tool ExpOptimizer to optimize and gain opPet8p. And we constructed it into a shuttle vector pBE2 to express opSam2 in Bacillus subtilis. We insert XylR which wiill constitutively express, and then we insert Pxyl-6xHis::Mistic::opPet8p::sfGFP whose regulation can be regulated by Xylose-binding XylR (Fig. 2).
To facilitate the membrane localization of opPet8p, we utilized a signal peptide Mistic in Bacillus subtilis, which was optimized to be opMistic by us.
To verify its expression, we used sfGFP as fluorescent and antigen tag. Besides, we utilized 6xHis tag as another antigen tag for Western Blot.

Figure. 2 Graph description of construction of recombined plasmid pBE-XylR-opPet8p.

Result

We have successfully verified the expression of opPet8p in both E. coli and B. subtilis (Fig. 3). We can observe green fluorescence after UV exciting (Fig. 3A) and under visual light (Fig. 3B). We observe the existence of green signal in fluorescent microscopy (Fig 3C). After the standradizing total protein concentration (Fig 3D), we performed Western blot against N-terminus His tag (Fig 3F) and against C-terminus sfGFP (Fig 3E). Both can proved that our target protein was expressed successfully.

Figure. 3 Expression verification of opPet8p in E. coli and B. subtilis.

To further characterize membrane localization ability of our recombined protein, we firstly analyzed the membrane integration pattern using Phobius (Fig 4A). And then we used fluorescent microscopy with higher magnification times to observe the distribution (Fig 4B). So according to the distribution of fluorescent signal, we supposed that the recombined proteins might target to the bacterial membrane. However, if the expression rate is too high to conduct the proper folding so that they might form the particle-like inclusion body, they might gather together to precipitate.

Figure. 4 Membrane Insertion Pattern Analysis of CDS and Fused opPet8p.

Reference
[1]Marobbio CM, Agrimi G, Lasorsa FM, Palmieri F. Identification and functional reconstitution of yeast mitochondrial carrier for S-adenosylmethionine. EMBO J. 2003 Nov 17;22(22):5975-82. doi: 10.1093/emboj/cdg574. PMID: 14609944; PMCID: PMC275433.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 184
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 54
  Illegal BamHI site found at 682
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]