Difference between revisions of "Part:BBa K4437502"

Revision as of 09:51, 12 October 2022 (view source) Mariangrell (Talk | contribs) ← Older edit		Latest revision as of 01:29, 14 October 2022 (view source) Mariangrell (Talk | contribs)
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	__TOC__		__TOC__
	===Usage and Biology===		===Usage and Biology===
−	<p>Phasin is an intracellular protein native to PHB-producing bacteria, which coats and stabilises polyhydroxybutyrate (PHB) granules in the bacterial cytoplasm, preventing the aggregation of the newly formed polymer [1]. By adding the C-terminus of the HlyA gene to the end of the phasin molecule, the resulting HlyA-tagged phasin molecule -- and the PHB granules to which it is bound -- can be secreted via the single-step Type I secretion system (TISS) in gram-negative bacteria [2]. Building upon the University of Calgary 2017 team’s BBa_K2260002, this part was produced by substituting the original RBS (BBa_B0034) for an RBS which results in only trace expression levels (BBa_K4437500). This part can be used as a negative control and as a means to test relative phasin-HlyA expression, and the subsequent yield of secreted PHB, of significantly stronger RBS alternatives.</p>	+	<p>Phasin is an intracellular protein native to PHB-producing bacteria, which coats and stabilises polyhydroxybutyrate (PHB) granules in the bacterial cytoplasm, preventing the aggregation of the newly formed polymer [1]. By adding the C-terminus of the HlyA gene to the end of the phasin molecule, the resulting HlyA-tagged phasin molecule -- and the PHB granules to which it is bound -- can be secreted via the single-step Type I secretion system (TISS) in gram-negative bacteria [2]. Building upon the University of Calgary 2017 team’s BBa_K2260002, this part was produced by substituting the original RBS (BBa_B0034) for the B0030 (BBa_B0030) RBS, which is relatively stronger. As such, this part can be used as a test to assess if increasing the strength of the RBS upstream of the FLAG-phasin-HlyA sequence, and the resulting expression levels of this protein, subsequently increases the yield of secreted PHB. </p>
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	===Design===		===Design===
−	<p> Using the original FLAG-phasin-HlyA part (BBa_K2260002) as a template, the B0034 (BBa_B0034) RBS was substituted with the DeadRBS (BBa_K4437500) RBS. The restriction enzyme cut sites for BstAPI and BstBI were also added, making the part compatible for digestion and ligation into the Tokyo Tech 2012 team’s BBa_K934001 plasmid for PHB expression assays. The FLAG protein allows for facilitated purification, and for characterization assays such as the Western blot. </p>	+	<p> Using the original FLAG-phasin-HlyA part (BBa_K2260002) as a template, the B0034 (BBa_B0034) RBS was substituted with the B0030 (BBa_B0030) RBS. The restriction enzyme cut sites for BstAPI and BstBI were also added, making the part compatible for digestion and ligation into the Tokyo Tech 2012 team’s BBa_K934001 plasmid for PHB expression assays. The FLAG protein allows for facilitated purification, and for characterization assays such as the Western blot. </p>
		+	[[Image:02constructmap.png|700px|thumb|center]]
−	===Sequence and Features===
−	<partinfo>BBa_K4437502 SequenceAndFeatures</partinfo>
		+	<p><i>Figure 1. </i> An overview of the BBa_K4437502 construct map.
−	===Characterization===
−	<p> We have successfully amplified the DeadRBS-FLAG-phasin-HlyA part, ligated it into the PHB (BBa_K934001) plasmid, and transformed and expressed this plasmid in TOP <i>E. coli</i> cells. These preliminary confirmations were performed with restriction digests and diagnostic gel electrophoresis. This recombinant DNA was also successfully transformed into BL21 (DE3) <E. coli> cells and characterized on an SDS-PAGE using anti-FLAG antibodies. Future directions for the characterization of this part include sequencing, and the <i>in-vitro</i> PHB production assays will be used to quantify resulting yields of secreted PHB.</p>
−		+	===Sequence and Features===
−	[[Image:DeadRBS-other-gel.png|700px|thumb|center]]	+	<partinfo>BBa_K4437502 SequenceAndFeatures</partinfo>
−		+
		+	===Characterization===
		+	<p> We have successfully amplified the B0030-FLAG-phasin-HlyA part, ligated it into the PHB (BBa_K934001) plasmid, and transformed and expressed this plasmid in TOP10 <i>E. coli</i> cells. These preliminary confirmations were performed with restriction digests and diagnostic gel electrophoresis. Future directions for the characterization of this part include the transformed of the recombinant DNA into BL21 (DE3) <i>E. coli</i> cells, the purification of this product, and the characterization of the desired FLAG-phasin-HlyA protein product on an SDS-PAGE using anti-FLAG antibodies. Additionally, this includes DNA sequencing, and the use of <i>in-vitro</i> PHB production assays to quantify resulting yields of secreted PHB.</p>
−	<p> <i>Figure 1.</i> Agarose electrophoresis of purified DNA. Lane 1 contains a 2log 1000bp ladder. Lanes 20-24 contain the DeadRBS-FLAG-phasin-HlyA insert in the BBa_K934001 plasmid, transformed in and purified from BL21 <i>E. coli</i>. Lane 20 indicates banding at the anticipated range of approximately 7100bp, indicating the successful insertion and transformation of the BBa_K4437502 insert.</p>
		+	[[Image:B0030gel.png|700px|thumb|center]]
−	[[Image:phasin-sds-page.png|700px|thumb|center]]
		+	<p> <i>Figure 2.</i> Agarose electrophoresis of purified DNA. Lane 1 contains a 2log 1000bp ladder. Lanes 2-4 contain the B0030-FLAG-phasin-HlyA insert in the BBa_K934001 plasmid, transformed in and purified from TOP10 <i>E. coli</i>. Lanes 2 and 4 indicate banding at the anticipated range of approximately 7100bp, indicating the successful insertion and transformation of the BBa_K4437502 insert.</p>
−	<p><i>Figure 2.</i>The expression of RBS-FLAG-phasin-hlyA with varying RBS strengths from the PHB construct (BBa_K934001) from BL21 (DE3) E.coli strain autoinduced for 24 hours. The process was visualised using 10% SDS-PAGE in 100V for 20 minutes and 180V for 40 minutes. The gel was stained with Coomassie blue. The gel was loaded as follows: (1) Ladder, (2) Lanmodulin positive control (BBa_K3945001), (3) Dead RBS upstream of phasin-Hlya in PHB construct (BBa_K4437501), (4) B0034 RBS upstream of phasin-Hlya in PHB construct (BBa_K226003), (5) B0035 upstream of phasin-Hlya in PHB construct (BBa_K4437502), (6) Tokyo 2012 PHB construct only (BBa_K934001). </p>

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Latest revision as of 01:29, 14 October 2022

B0030-FLAG-phasin-HlyA (E. coli)

Usage and Biology

Phasin is an intracellular protein native to PHB-producing bacteria, which coats and stabilises polyhydroxybutyrate (PHB) granules in the bacterial cytoplasm, preventing the aggregation of the newly formed polymer [1]. By adding the C-terminus of the HlyA gene to the end of the phasin molecule, the resulting HlyA-tagged phasin molecule -- and the PHB granules to which it is bound -- can be secreted via the single-step Type I secretion system (TISS) in gram-negative bacteria [2]. Building upon the University of Calgary 2017 team’s BBa_K2260002, this part was produced by substituting the original RBS (BBa_B0034) for the B0030 (BBa_B0030) RBS, which is relatively stronger. As such, this part can be used as a test to assess if increasing the strength of the RBS upstream of the FLAG-phasin-HlyA sequence, and the resulting expression levels of this protein, subsequently increases the yield of secreted PHB.

Design

Using the original FLAG-phasin-HlyA part (BBa_K2260002) as a template, the B0034 (BBa_B0034) RBS was substituted with the B0030 (BBa_B0030) RBS. The restriction enzyme cut sites for BstAPI and BstBI were also added, making the part compatible for digestion and ligation into the Tokyo Tech 2012 team’s BBa_K934001 plasmid for PHB expression assays. The FLAG protein allows for facilitated purification, and for characterization assays such as the Western blot.

Figure 1. An overview of the BBa_K4437502 construct map.

Sequence and Features

Characterization

<p> We have successfully amplified the B0030-FLAG-phasin-HlyA part, ligated it into the PHB (BBa_K934001) plasmid, and transformed and expressed this plasmid in TOP10 E. coli cells. These preliminary confirmations were performed with restriction digests and diagnostic gel electrophoresis. Future directions for the characterization of this part include the transformed of the recombinant DNA into BL21 (DE3) E. coli cells, the purification of this product, and the characterization of the desired FLAG-phasin-HlyA protein product on an SDS-PAGE using anti-FLAG antibodies. Additionally, this includes DNA sequencing, and the use of in-vitro PHB production assays to quantify resulting yields of secreted PHB.

Figure 2. Agarose electrophoresis of purified DNA. Lane 1 contains a 2log 1000bp ladder. Lanes 2-4 contain the B0030-FLAG-phasin-HlyA insert in the BBa_K934001 plasmid, transformed in and purified from TOP10 E. coli. Lanes 2 and 4 indicate banding at the anticipated range of approximately 7100bp, indicating the successful insertion and transformation of the BBa_K4437502 insert.

References

1. Brown B, Immethun C, Wilkins M, Saha R. Biotechnical applications of phasins: Small proteins with large potential. Renewable and Sustainable Energy Reviews. 2022 Apr 1;158:112129.

2. Thomas S, Holland IB, Schmitt L. The Type 1 secretion pathway — The hemolysin system and beyond. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2014 Aug 1;1843(8):1629–41.