Difference between revisions of "Part:BBa K4724083"

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<p>Fig. 8 SDS-PAGE of recombinant protein expression products in the supernatant of fermentation broth under induction conditions of 20°C for 19h
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<p>Fig. 4 SDS-PAGE of recombinant protein expression products in the supernatant of fermentation broth under induction conditions of 20°C for 19h
(M: Marker; lane 1: 20 ℃ supernatant of LSPET primordial cells crushed; lane 2: 20 ℃ precipitation solution of LSPET primordial cells crushed; lane 3: 20 ℃ supernatant of DsbA-LSPET fermentation broth; lane 4: 20 ℃ supernatant of OmpA-LSPET fermentation broth)
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(M: Marker; lane 1: 20 ℃ supernatant of LSPET primordial cells crushed; lane 2: 20 ℃ precipitation solution of LSPET primordial cells crushed; lane 3: 20 ℃ supernatant of DsbA-LSPET fermentation broth; lane 4: 20 ℃ supernatant of OmpA-LSPET fermentation broth) </p>
From Figure 8, after induced expression, the LSPET enzyme with the signal peptide added at the N-terminus was not directed into the fermentation broth. Reviewing the literature again we guessed that the recombinant target protein was most likely directed into the periplasmic space of <i>E.coli</i>.
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We therefore subsequently fragmented the fermentation broth after centrifugation of the fermentation broth and analyszd it again by SDS-PAGE.</p>
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<p>As depicted in Figure 4, it was observed that upon induction of expression, the LSPETase enzyme, when equipped with the N-terminal signal peptide, did not exhibit localization within the fermentation broth. Upon revisiting the relevant literature, it was hypothesized that the recombinant target protein was likely directed to the periplasmic compartment of <i>E. coli</i>. Consequently, to further investigate this phenomenon, the fermentation broth was subjected to centrifugation, followed by fragmentation, and subsequent analysis using SDS-PAGE. </p>
  
 
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<p>Fig. 9 SDS-PAGE of the bacteria connected to the signal peptide as well as the induced expression proteins of the original bacteria
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<p>Fig. 5 SDS-PAGE of the LSPETase and LSPETase fused with the signal peptide after induction at 20 ℃ for 19h
(M: Marker; lane 1: 20 ℃ ISPET primordial cells broken supernatant; lane 2: 20 ℃ LSPET primordial cells broken precipitate solution; lane 3: 20 ℃ OmpA-LSPET cells broken supernatant; lane 4: 20 ℃ OmpA-LSPET cells broken precipitate solution; lane 5: 20 ℃ DsbA-LSPET cells broken precipitate solution). LSPET cells; lane 6: 20 ℃ DsbA-LSPET cells precipitation solution).
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(M: Marker; lane 1: Supernatant of LSPETase slurry; lane 2: Precipitate of LSPETase slurry; lane 3: Supernatant of OmpA-LSPETase slurry; lane 4: Precipitate of OmpA-LSPETase slurry; lane 5: Supernatant of DsbA-LSPETase slurry; lane 6: Precipitate of DsbA-LSPETase slurry.) </p>
The target gene is known to express a protein length of 30.2 kDa, 32.3 kDa with the addition of the signal peptide DsbA and 32.2 kDa with the addition of the fusion tag OmpA.
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From Fig. 9, after induced expression, the soluble expression of LSPET enzyme protein after the addition of the signal peptide was greatly enhanced, and the comparison of the two signal peptides revealed that DsbA-LSPET significantly reduced the insoluble expression of LSPET enzyme in the precipitate, and the effect of OmpA-LSPET, although it also reduced the insoluble expression of LSPET enzyme in the precipitate, was not as obvious. The protein expression of OmpA-LSPET in the supernatant was significantly higher than that of LSPET primordia and DsbA-LSPET bacteria. It can be concluded that OmpA signal peptide can increase the soluble expression of LSPET enzyme by increasing the soluble expression of LSPET enzyme, but the effect is weaker than that of DsbA signal peptide in reducing the insoluble expression of LSPET enzyme. Both signal peptides were effective in the soluble expression of LSPET enzymes.
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<p>The target gene is known to express a protein length of 30.2 kDa, 32.3 kDa with the addition of the signal peptide DsbA and 32.2 kDa with the addition of the fusion tag OmpA.</p>
Further optical density analysis was performed on protein gels at induction conditions of 20°C for 19 h to quantify the increase in protein expression, and the results were analyzed as follows:</p>
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<p>As shown in Fig. 5, the soluble expression of LSPETase enzyme protein after the addition of the signal peptide was greatly enhanced. The comparison of the two signal peptides revealed that DsbA-LSPETase significantly reduced the insoluble expression of LSPETase enzyme in the precipitate, and the effect of OmpA-LSPETase, although it also reduced the insoluble expression of LSPETase enzyme in the precipitate, was not as obvious. The protein expression of OmpA-LSPETase in the supernatant was significantly higher than that of LSPETase and DsbA-LSPETase. It can be concluded that OmpA signal peptide can increase the soluble expression of LSPETase enzyme by increasing the soluble expression of LSPETase enzyme, but the effect is weaker than that of DsbA signal peptide in reducing the insoluble expression of LSPETase enzyme. Both signal peptides were effective in soluble expression of LSPETase. </p>
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<p>Further optical density analysis was performed on protein gels at induction conditions of 20°C for 19 h to quantify the increase in protein expression, and the results were analyzed as follows:</p>
  
 
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<p>Fig. 10 Histogram of the optical density analysis data of protein bands of the bacteria after linking the signal peptide as well as the original bacteria</p>
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<p>Fig. 6 Histogram of the optical density analysis data of supernatant and precipitated protein bands from the SDS-PAGE gel of the bacterial cell breakage solution before and after the attachment of the signal peptides </p>
  
 
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<p>Fig. 11 Percentage comparison of optical density analysis data of the supernatant and precipitated protein bands of the cell shattering solution of the bacteria after linking the signal peptide as well as the original bacteria</p>
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<p>Fig. 7 Percentage comparison of optical density analysis data of the supernatant and precipitated protein bands of the bacterial cell breakage solution before and after the attachment of the signal peptides</p>
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<p>An analysis of Figure 6 and Figure 7 showed that under the specified induction conditions, the protein solubility of DsbA-LSPETase and OmpA-LSPETase was increased by 1.7-fold and 1.4-fold respectively compared to the original LSPETase. The density values of the protein bands in the precipitate also decreased. This confirms the conclusion that the addition of signal peptides effectively facilitates the solubility of the target protein. </p>
  
An analysis of Figure 10 and Figure 11 shows that under the induction conditions, the protein solubility of DsbA-LSPET and OmpA-LSPET, which are connected signal peptides, is increased by 1.7-fold and 1.4-fold respectively compared to the original LSPET. The density values of the protein bands in the precipitate also decreased. This confirms the conclusion that the addition of signal peptides has a good effect in promoting the solubility of the target gene protein.
 
  
 
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Revision as of 08:36, 12 October 2023


DsbA-LSPETase

Insert the signal peptide DsbA gene sequence into the vector pET-22b (+) - LSPET-12, and construct a recombinant plasmid using Gibson assembly method to promote the soluble expression of the original target fragment LSPET protein.

Characterize the results

Fig. 4 SDS-PAGE of recombinant protein expression products in the supernatant of fermentation broth under induction conditions of 20°C for 19h (M: Marker; lane 1: 20 ℃ supernatant of LSPET primordial cells crushed; lane 2: 20 ℃ precipitation solution of LSPET primordial cells crushed; lane 3: 20 ℃ supernatant of DsbA-LSPET fermentation broth; lane 4: 20 ℃ supernatant of OmpA-LSPET fermentation broth)

As depicted in Figure 4, it was observed that upon induction of expression, the LSPETase enzyme, when equipped with the N-terminal signal peptide, did not exhibit localization within the fermentation broth. Upon revisiting the relevant literature, it was hypothesized that the recombinant target protein was likely directed to the periplasmic compartment of E. coli. Consequently, to further investigate this phenomenon, the fermentation broth was subjected to centrifugation, followed by fragmentation, and subsequent analysis using SDS-PAGE.

Fig. 5 SDS-PAGE of the LSPETase and LSPETase fused with the signal peptide after induction at 20 ℃ for 19h (M: Marker; lane 1: Supernatant of LSPETase slurry; lane 2: Precipitate of LSPETase slurry; lane 3: Supernatant of OmpA-LSPETase slurry; lane 4: Precipitate of OmpA-LSPETase slurry; lane 5: Supernatant of DsbA-LSPETase slurry; lane 6: Precipitate of DsbA-LSPETase slurry.)

The target gene is known to express a protein length of 30.2 kDa, 32.3 kDa with the addition of the signal peptide DsbA and 32.2 kDa with the addition of the fusion tag OmpA.

As shown in Fig. 5, the soluble expression of LSPETase enzyme protein after the addition of the signal peptide was greatly enhanced. The comparison of the two signal peptides revealed that DsbA-LSPETase significantly reduced the insoluble expression of LSPETase enzyme in the precipitate, and the effect of OmpA-LSPETase, although it also reduced the insoluble expression of LSPETase enzyme in the precipitate, was not as obvious. The protein expression of OmpA-LSPETase in the supernatant was significantly higher than that of LSPETase and DsbA-LSPETase. It can be concluded that OmpA signal peptide can increase the soluble expression of LSPETase enzyme by increasing the soluble expression of LSPETase enzyme, but the effect is weaker than that of DsbA signal peptide in reducing the insoluble expression of LSPETase enzyme. Both signal peptides were effective in soluble expression of LSPETase.

Further optical density analysis was performed on protein gels at induction conditions of 20°C for 19 h to quantify the increase in protein expression, and the results were analyzed as follows:

Fig. 6 Histogram of the optical density analysis data of supernatant and precipitated protein bands from the SDS-PAGE gel of the bacterial cell breakage solution before and after the attachment of the signal peptides

Fig. 7 Percentage comparison of optical density analysis data of the supernatant and precipitated protein bands of the bacterial cell breakage solution before and after the attachment of the signal peptides

An analysis of Figure 6 and Figure 7 showed that under the specified induction conditions, the protein solubility of DsbA-LSPETase and OmpA-LSPETase was increased by 1.7-fold and 1.4-fold respectively compared to the original LSPETase. The density values of the protein bands in the precipitate also decreased. This confirms the conclusion that the addition of signal peptides effectively facilitates the solubility of the target protein.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1186
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 322
    Illegal NgoMIV site found at 442
    Illegal AgeI site found at 529
  • 1000
    COMPATIBLE WITH RFC[1000]