Experiments: Secretion
Methods for Constructing BioBrick Parts
One of the objectives of this project was to create a library of (link to silver fusion wiki) Silver-fusion compatible BioBrick signal peptides and protein-coding parts for secretion studies. The Silver-fusion assembly method was used because the standard BioBrick prefix and suffix do not facilitate fusion of two parts. The scar that forms from the overlap of compatible restriction enzyme sites XbaI and SpeI is not conducive to fusion because it contains a stop codon and is 8 nucleotides long. Because the scar is not a multiple of three, the sequence after the scar will be read out-of-frame. The Silver-fusion assembly method retains compatibility with the standard BioBrick assembly method, but fusion is allowed. A single nucleotide is removed from the prefix and suffix of Silver-fusion BioBricks so that the scar that forms from the ligation of XbaI and SpeI sites does not contain a stop codon and is 6 nucleotides in length.
Five signal sequences were selected for this study based on the secretion pathway that they represent and their prominence in literature. The selected sequences are presented in Table X. Two protein coding regions were obtained: phasin and GFP. All of these sequences were designed for Silver-fusion compatibility. Four different promoters with an attached ribosome binding site were designed and then synthesized by DNA 2.0, followed by ligation into a BioBrick vector. Composite devices were assembled piecewise by cutting one part typically with EcoRI and XbaI, and the part to be inserted with EcoRI and SpeI. Analysis by PCR with the Primers VF2 and VR was used to qualitatively determine whether successful ligation had taken place. Once partially confirmed, samples were sequence at the Utah State University Center of Integrated Biotechnology.
Signal Peptides:
To construct the OmpA, PelB, and GeneIII sequences, complimentary forward and reverse oligonucleotides were synthesized by Eurofins Operon. These strands were then annealed together. The oligonucleotides were designed so that the silver fusion prefix and suffix sequences were appended onto the end of each sequence. These parts were then cut with EcoRI and SpeI and ligated into a BioBrick vector. Each of these parts were successfully constructed and sequenced.
The TorA and HlyA signal peptides were synthesized by DNA 2.0 because these sequences are longer than the other signal peptides, which made the complimentary oligonucleotides method not ideal. The Silver-fusion prefix and suffix was added to each of these constructs. EcoRI and SpeI were used to cut the part out of the commercial vector. The DNA was isolated by gel electrophoresis and ligated into a BioBrick compatible vector, pSB3K3.
Phasin:
The phasin (PhaP) sequence was isolated from the genomic DNA of Cupriavidus necator (also known as Ralstonia eutropha). There are four different phasin genes in the genomic DNA of this organism. This particular phasin was selected based on references in literature, although no information was acquired that indicated that one phasin gene would yield better production over another. The primers were designed so that the Silver-fusion prefix and suffix were overhanging, thereby resulting in a final product that is Silver-fusion compatible. The 579 bp phasin sequence was found to contain a PstI site. The PstI site was mutated using site-directed mutagenesis (LINK TO PROTOCOLS PAGE) (CTGCAG CTTCAG). Sequencing confirmed that this site was successfully removed.
GFP:
Near the beginning of this project, a Silver-fusion compatible GFP BioBrick (BBa_K125500) derived from BBa_E0040 by the Hawaii 2008 iGEM team was obtained. However, upon further analysis it was determined that this part was modified so that the start codon of the sequence was removed. Although this should not affect the expression of GFP in composite parts with a signal peptide prior to the sequence, it is not ideal for this particular project. The lack of a start codon requires N-terminal fusion of another protein or signal peptide, and a functional GFP control without a signal sequence would not be functional. This control is important in our study to compare with composite parts containing signal peptide-protein fusion to determine whether the produced GFP is being transported. Additionally, this part would not work with C-terminal signal peptide fusions. The HlyA signal peptide is recognized on the C-terminus of the target protein by the Type I secretion pathway (Mergulhão, 2005; Gentschev, 2003; Hess, 1990). The absence of the start codon inhibits study of this secretory pathway. Another disadvantage of this GFP part is its small Stokes shift (excitation 501 nm, emission 511 nm). An ideal GFP that fluorescence would have a shorter excitation wavelength so that GFP-positive samples can be detected visually using a UV transilluminator.
A new Silver-Fusion compatible GFP BioBrick part was constructed for this project via a similar mechanism as the phasin construct. This particular GFP was previously mutated for improved fluorescence photostability (Crameri, 1996). The excitation and emission wavelengths for this GFP are 395 nm and 501 nm, respectively. That being said, GFP-positive cells emit a bright green fluorescence when exposed to shorter-wavelength UV light, such as on a transilluminator. Primers were synthesized for isolation of the sequence and, like the phasin-specific primers, designed so that the Silver-fusion prefix and suffix were inserted on the ends of the sequence (see primers). Figure X shows GFP- Top10 E. coli colonies (left) and unfused GFP+ Top10 E. coli colonies (right). This figure shows that the GFP construct is functional and easily detectable.
Bioplastic Production:
A plasmid harboring the genes for PHB production (pBHR68) was used in these experiments. This plasmid contains the sequence for ampicillin resistance and contains a ColE1 origin of replication. E. coli harboring pBHR68 were cultured according to methods outlined by Kang et al (2008) and production of PHB was verified using 1H NMR analysis. The spectrum obtained from this experiment is given as Figure X. The observed peaks at 1.24 ppm, 2.54 ppm, and 5.2 ppm correspond with those observed in standard polyhydroxyalkanaote samples.
To maintain plasmid compatibility in E. coli transformed with both the pBHR68 and phasin plasmids, it was determined that the vector used for the phasin secretion device required a p15A ori site. BioBrick vector pSB3K3 was found suitable as the host for the secretion constructs. XL1-Blue E. coli were transformed with both a phasin device and the pBHR68 BioBrick plasmids, and these cells were cultured and tested for secretion.
SDS-PAGE Analysis
Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to analyze the protein content in transformed E. coli. As a positive control, E. coli containing the Lac/RBS/GFP/Terminator (BBa_K208045) construct were sonicated and centrifuged (see Figure X). Additionally, E. coli cells containing an individual BioBrick part (BBa_B0015) were analyzed as a negative control. The resulting gel was stained with coomassie blue and is shown as Figure X. The bright band at 27 kD in the GFP+ sample corresponds to the GFP protein (Bio-Rad). The absence of this band in the GFP- sample further reinforces the functionality of the GFP construct.
 The geneIII secretion signal sequence fused to the phasin protein was expressed in E. coli cells. The E. coli cells were grown overnight in LB growth media and centrifuged to pellet the cells. Supernatants (5ml) were then concentrated using a Centricon Centriplus concentrator (Amicon, Beverly MA). This process concentrated proteins that were larger than 10kDa and removed molecules smaller than 10kDa. Approximately 20ug of protein were then applied to a SDS polyacrylamide gel to separate the proteins according to size. The gel was then stained with coomassie blue for protein detection, as shown in Figure X. Following SDS polyacylamide gel electrophoresis (PAGE) and subsequent coomassie blue staining of the separated proteins, a protein with an approximate size of 22kDA is observed in the sample from the phasin-expressing E. coli cells that is not present in the control E. coli sample. The phasin protein has been reported by others to migrate on SDS PAGE from 14-28kDa (Pötter, 2002; York, 2002). These results indicate that the GeneIII::phasin expression construct is being produced by the E. coli cells and is being secreted outside the cell into the media.
 Western blotting with phasin-specific antibodies was performed to verify the observed band as phasin. Figure X shows the apparatus used to transfer proteins onto PVDF paper. Phasin antibody was kindly provided by Anthony J. Sinskey at Massachusetts Institute of Technology. The results of the western blotting were inconclusive. Non-specific binding to larger constructs was observed. Additional testing is required to further reinforce preliminary findings and confirm the secretion of phasin. The secretion of phasin would provide evidence that PHA recovery via phasin secretion is possible. Addtionally, this would reinforce that the constructed BioBricks are not only functional, but would be beneficial for use in other studies.
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