Difference between revisions of "Part:BBa K4632004"
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'''What we have done? (SCAU-China 2023)''' | '''What we have done? (SCAU-China 2023)''' | ||
| − | <P>Building upon the previous research by Huijiao Lin et al. (2007), our team aimed to achieve the ultimate goal of a toxin working in conjunction with a protease inhibitor to inhibiting the activity of serine protease in the intestine of the fourth instar larvae of ''Solenopsis invicta(''S. invicta'')''. We selected ''Escherichia coli''(''E.coli'') as the host organism. To enable extracellular protein | + | <P>Building upon the previous research by Huijiao Lin et al. (2007), our team aimed to achieve the ultimate goal of a toxin working in conjunction with a protease inhibitor to inhibiting the activity of serine protease in the intestine of the fourth instar larvae of ''Solenopsis invicta(''S. invicta'')''. We selected ''Escherichia coli''(''E.coli'') as the host organism. To enable extracellular protein excretion, we attempted to use the signal peptide ZnuA, which was selected based on its high compatibility with ''E. coli'' according to SignalP (https://services.healthtech.dtu.dk/services/SignalP-5.0/)</P><P>'''However''', after more than two months of repeated attempts, we found that the TIA protease inhibitor gene, with or without the ZnuA signal peptide, could not be expressed in ''E. coli''. We concluded that ''E. coli'' might not be suitable for the expression of the TIA protease inhibitor gene. Therefore, we decided to switch to a new protease inhibitor, Cowpea Trypsin Inhibitor (CPTI).</P> |
| − | + | ||
| − | '''However''', after more than two months of repeated attempts, we found that the TIA protease inhibitor gene, with or without the ZnuA signal peptide, could not be expressed in ''E. coli''. We concluded that ''E. coli'' might not be suitable for the expression of the TIA protease inhibitor gene. Therefore, we decided to switch to a new protease inhibitor, Cowpea Trypsin Inhibitor (CPTI). | + | |
===Sequence and Features=== | ===Sequence and Features=== | ||
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===Construction and Characterization=== | ===Construction and Characterization=== | ||
---- | ---- | ||
| − | <p><strong> Verifying the Expression and | + | <p><strong> Verifying the Expression and Excretion Proficiency of TIA</strong></p> |
"We transformed the pET28b-ZnuA-TIA plasmid into ''E. coli'' BL21, induced expression with IPTG for 4 hours, and then centrifuged to obtain supernatant and pellet fractions. The supernatant was concentrated using ultrafiltration tubes, while the pellet was subjected to lysis with a lysis buffer. After lysis, the sample was centrifuged again to separate the lysed supernatant from the lysed pellet. The lysed pellet was resuspended in lysis buffer. SDS-PAGE and Western Blot analyses were performed on the aforementioned samples. '''However''', the results indicated that the TIA protein was not expressed in ''E. coli'' (as shown in Figures 1 and 2)." | "We transformed the pET28b-ZnuA-TIA plasmid into ''E. coli'' BL21, induced expression with IPTG for 4 hours, and then centrifuged to obtain supernatant and pellet fractions. The supernatant was concentrated using ultrafiltration tubes, while the pellet was subjected to lysis with a lysis buffer. After lysis, the sample was centrifuged again to separate the lysed supernatant from the lysed pellet. The lysed pellet was resuspended in lysis buffer. SDS-PAGE and Western Blot analyses were performed on the aforementioned samples. '''However''', the results indicated that the TIA protein was not expressed in ''E. coli'' (as shown in Figures 1 and 2)." | ||
''https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/part-3-2.png'' | ''https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/part-3-2.png'' | ||
| − | <p><strong>Figure 1:</strong> SDS-PAGE analysis of TIA expression | + | <p><strong>Figure 1:</strong> SDS-PAGE analysis of TIA expression</P> |
| − | Lane 1: Total extract of pET-28(b)(+IPTG); Lane2 -8 were ZnuA-TIA. Lane 2 : precipitate without IPTG; Lane 3: precipitate with IPTG; Lane 4: supernatant without IPTG; Lane 5: supernatant with IPTG; Lane 6: concentrated supernatant without IPTG; Lane 7: concentrated supernatant with IPTG; Lane 8: whole cell extract without IPTG;Lane 9: Total extract of pET-28(b)-ZnuA-TIA(+IPTG)</p> | + | <P>Lane 1: Total extract of pET-28(b)(+IPTG); Lane2 -8 were ZnuA-TIA. Lane 2 : precipitate without IPTG; Lane 3: precipitate with IPTG; Lane 4: supernatant without IPTG; Lane 5: supernatant with IPTG; Lane 6: concentrated supernatant without IPTG; Lane 7: concentrated supernatant with IPTG; Lane 8: whole cell extract without IPTG;Lane 9: Total extract of pET-28(b)-ZnuA-TIA(+IPTG)</p> |
''https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/part-3-3.png'' | ''https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/part-3-3.png'' | ||
| − | <p><strong>Figure 2:</strong>Western blot analysis of TIA expression | + | <p><strong>Figure 2:</strong>Western blot analysis of TIA expression</P> |
| − | Lane1-6 were TIA. Lane1:concentrated supernatant with IPTG;Lane2:concentrated supernatant without IPTG;Lane3:supernatant with IPTG;Lane4:supernatant without IPTG;Lane5:precipitate with IPTG;Lane6:precipitate without IPTG;Lane7:supernatant of pht254-LpfA(positive control);Lane8:supernatant of pET28(b) with IPTG | + | <P>Lane1-6 were TIA. Lane1:concentrated supernatant with IPTG;Lane2:concentrated supernatant without IPTG;Lane3:supernatant with IPTG;Lane4:supernatant without IPTG;Lane5:precipitate with IPTG;Lane6:precipitate without IPTG;Lane7:supernatant of pht254-LpfA(positive control);Lane8:supernatant of pET28(b) with IPTG</P> |
===References=== | ===References=== | ||
Latest revision as of 14:30, 12 October 2023
Serine protease inhibitor(TIA) fused with ZnuA signal peptide
Description
It has been demonstrated that transgenic Enterobacter cloacae GEI-a, using the transferred Trypsin Inhibitor of Glycine max L (Tia gene), can produce a serine protease inhibitor (TIA) with inhibitory activity against the midgut trypsin of red imported fire ant larvae (Huijiao Lin et al., 2007).
What we have done? (SCAU-China 2023)
Building upon the previous research by Huijiao Lin et al. (2007), our team aimed to achieve the ultimate goal of a toxin working in conjunction with a protease inhibitor to inhibiting the activity of serine protease in the intestine of the fourth instar larvae of Solenopsis invicta(S. invicta). We selected Escherichia coli(E.coli) as the host organism. To enable extracellular protein excretion, we attempted to use the signal peptide ZnuA, which was selected based on its high compatibility with E. coli according to SignalP (https://services.healthtech.dtu.dk/services/SignalP-5.0/)
However, after more than two months of repeated attempts, we found that the TIA protease inhibitor gene, with or without the ZnuA signal peptide, could not be expressed in E. coli. We concluded that E. coli might not be suitable for the expression of the TIA protease inhibitor gene. Therefore, we decided to switch to a new protease inhibitor, Cowpea Trypsin Inhibitor (CPTI).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 416
Illegal EcoRI site found at 532 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 416
Illegal EcoRI site found at 532 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 416
Illegal EcoRI site found at 532 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 416
Illegal EcoRI site found at 532 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 416
Illegal EcoRI site found at 532 - 1000COMPATIBLE WITH RFC[1000]
Construction and Characterization
Verifying the Expression and Excretion Proficiency of TIA
"We transformed the pET28b-ZnuA-TIA plasmid into E. coli BL21, induced expression with IPTG for 4 hours, and then centrifuged to obtain supernatant and pellet fractions. The supernatant was concentrated using ultrafiltration tubes, while the pellet was subjected to lysis with a lysis buffer. After lysis, the sample was centrifuged again to separate the lysed supernatant from the lysed pellet. The lysed pellet was resuspended in lysis buffer. SDS-PAGE and Western Blot analyses were performed on the aforementioned samples. However, the results indicated that the TIA protein was not expressed in E. coli (as shown in Figures 1 and 2)."
Figure 1: SDS-PAGE analysis of TIA expression
Lane 1: Total extract of pET-28(b)(+IPTG); Lane2 -8 were ZnuA-TIA. Lane 2 : precipitate without IPTG; Lane 3: precipitate with IPTG; Lane 4: supernatant without IPTG; Lane 5: supernatant with IPTG; Lane 6: concentrated supernatant without IPTG; Lane 7: concentrated supernatant with IPTG; Lane 8: whole cell extract without IPTG;Lane 9: Total extract of pET-28(b)-ZnuA-TIA(+IPTG)
Figure 2:Western blot analysis of TIA expression
Lane1-6 were TIA. Lane1:concentrated supernatant with IPTG;Lane2:concentrated supernatant without IPTG;Lane3:supernatant with IPTG;Lane4:supernatant without IPTG;Lane5:precipitate with IPTG;Lane6:precipitate without IPTG;Lane7:supernatant of pht254-LpfA(positive control);Lane8:supernatant of pET28(b) with IPTG
References
[1] Huijiao Lin, Richou Han, Xue Qiu, Xun Hongyan. Transgene enterobacter cloacae CEI-a and its construction method and use: CN101144067B[P] 2011-08-10.