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<tr> | <tr> | ||
− | <td><a href="#K2020002" style="color:#0000EE;">K2020002</td> | + | <td><a href="#K2020002" style="color:#0000EE;">K2020002</a></td> |
<td>expression system for subtilisin E in <i>E. coli</i></td> | <td>expression system for subtilisin E in <i>E. coli</i></td> | ||
</tr> | </tr> | ||
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This expression system consists of the promoter <a href=”http://parts.igem.org/Part:BBa_R0011”><u style="color:#0000EE;">BBa_R0011</u></a>, the ribosome binding site <a href=”http://parts.igem.org/Part:BBa_B0034”><u style="color:#0000EE;">BBa_B0034</u></a>, the newly created BioBrick part <a href=”http://parts.igem.org/Part:BBa_K2020001”><u style="color:#0000EE;">BBa_K2020001</u></a> and the terminator <a href=”http://parts.igem.org/Part:BBa_B0010”><u style="color:#0000EE;">BBa_B0010</u></a>. BioBrick <a href=”http://parts.igem.org/Part:BBa_K2020001”><u style="color:#0000EE;">K2020001</u></a> is a composite part itself and includes the secretion tag pelB (<a href=”http://parts.igem.org/Part:BBa_J32015”><u style="color:#0000EE;">BBa_J32015</u></a>) and a subtilisin E gene optimized for <i>Escherichia coli</i> codon usage (<a href=”http://parts.igem.org/Part:BBa_K2020000”><u style="color:#0000EE;">BBa_K2020000</u></a>). Once introduced into <i>E. coli</i>, this BioBrick is able to produce subtilisin E, an alkaline serine protease which non-specifically digest proteins, and simultaneously secret the enzyme into the periplasm of the cell. Caused by the lacI regulated promoter <a href=”http://parts.igem.org/Part:BBa_R0010”><u style="color:#0000EE;">BBa_R0010</u></a>, the expression system can be induced by addition of IPTG. </br> | This expression system consists of the promoter <a href=”http://parts.igem.org/Part:BBa_R0011”><u style="color:#0000EE;">BBa_R0011</u></a>, the ribosome binding site <a href=”http://parts.igem.org/Part:BBa_B0034”><u style="color:#0000EE;">BBa_B0034</u></a>, the newly created BioBrick part <a href=”http://parts.igem.org/Part:BBa_K2020001”><u style="color:#0000EE;">BBa_K2020001</u></a> and the terminator <a href=”http://parts.igem.org/Part:BBa_B0010”><u style="color:#0000EE;">BBa_B0010</u></a>. BioBrick <a href=”http://parts.igem.org/Part:BBa_K2020001”><u style="color:#0000EE;">K2020001</u></a> is a composite part itself and includes the secretion tag pelB (<a href=”http://parts.igem.org/Part:BBa_J32015”><u style="color:#0000EE;">BBa_J32015</u></a>) and a subtilisin E gene optimized for <i>Escherichia coli</i> codon usage (<a href=”http://parts.igem.org/Part:BBa_K2020000”><u style="color:#0000EE;">BBa_K2020000</u></a>). Once introduced into <i>E. coli</i>, this BioBrick is able to produce subtilisin E, an alkaline serine protease which non-specifically digest proteins, and simultaneously secret the enzyme into the periplasm of the cell. Caused by the lacI regulated promoter <a href=”http://parts.igem.org/Part:BBa_R0010”><u style="color:#0000EE;">BBa_R0010</u></a>, the expression system can be induced by addition of IPTG. </br> | ||
With the iGEM promoter <a href=”http://parts.igem.org/Part:BBa_R0011”><u style="color:#0000EE;">BBa_R0011</u></a>, which was integrated in our sequence at first, it was not possible to successfully express subtilisin E due to fatal mutations inside the expression system in all analyzed colonies. Either there have been single base deletions or insertions in the pro-peptide, which led to a frameshift of the whole protein, or a 23 base pair deletion in the promoter. Both types of mutations result in an incorrect expression system, so that an expression of the protease is impossible. Since the promoter <a href=”http://parts.igem.org/Part:BBa_R0011”><u style="color:#0000EE;">BBa_R0011</u></a> is leaky and induce the expression even without addition of IPTG, it can be assumed that subtilisin E is toxic for E. coli. </br> | With the iGEM promoter <a href=”http://parts.igem.org/Part:BBa_R0011”><u style="color:#0000EE;">BBa_R0011</u></a>, which was integrated in our sequence at first, it was not possible to successfully express subtilisin E due to fatal mutations inside the expression system in all analyzed colonies. Either there have been single base deletions or insertions in the pro-peptide, which led to a frameshift of the whole protein, or a 23 base pair deletion in the promoter. Both types of mutations result in an incorrect expression system, so that an expression of the protease is impossible. Since the promoter <a href=”http://parts.igem.org/Part:BBa_R0011”><u style="color:#0000EE;">BBa_R0011</u></a> is leaky and induce the expression even without addition of IPTG, it can be assumed that subtilisin E is toxic for E. coli. </br> | ||
− | Hence, we exchanged the promoter against <a href=”http://parts.igem.org/Part:BBa_R0010”><u style="color:#0000EE;">BBa_R0010</u></a>. For achieving this, we fulfilled a Polymerase Chain Reaction to extract everything but the promoter and the RBS and simultaneously extend the remaining DNA sequence with the pre-fix of iGEM. Afterwards, we assembled it with the BioBrick <a href=”http://parts.igem.org/Part:BBa_J04500”><u style="color:#0000EE;">J04500</u></a> and in parallel cloned it into the vector pSB1C3 - by cutting RFP out of the BioBrick <a href=”http://parts.igem.org/Part:BBa_J04450”><u style="color:#0000EE;">J04450</u></a>. The implemented BioBrick <a href=”http://parts.igem.org/Part:BBa_J04500”><u style="color:#0000EE;">J04500</u></a> itself contains another IPTG inducible promoter (<a href=”http://parts.igem.org/Part:BBa_R0010”><u style="color:#0000EE;">BBa_R0010</u></a>) and the same RBS (<a href=”http://parts.igem.org/Part:BBa_B0034”><u style="color:#0000EE;">BBa_B0034</u></a>). | + | Hence, we exchanged the promoter against <a href=”http://parts.igem.org/Part:BBa_R0010”><u style="color:#0000EE;">BBa_R0010</u></a>. For achieving this, we fulfilled a Polymerase Chain Reaction to extract everything but the promoter and the RBS and simultaneously extend the remaining DNA sequence with the pre-fix of iGEM. Afterwards, we assembled it with the BioBrick <a href=”http://parts.igem.org/Part:BBa_J04500”><u style="color:#0000EE;">J04500</u></a> and in parallel cloned it into the vector pSB1C3 - by cutting RFP out of the BioBrick <a href=”http://parts.igem.org/Part:BBa_J04450”><u style="color:#0000EE;">J04450</u></a>. The implemented BioBrick <a href=”http://parts.igem.org/Part:BBa_J04500”><u style="color:#0000EE;">J04500</u></a> itself contains another IPTG inducible promoter (<a href=”http://parts.igem.org/Part:BBa_R0010”><u style="color:#0000EE;">BBa_R0010</u></a>) and the same RBS (<a href=”http://parts.igem.org/Part:BBa_B0034”><u style="color:#0000EE;">BBa_B0034</u></a>). An expression with the newly integrated promoter <a href=”http://parts.igem.org/Part:BBa_R0010”><u style="color:#0000EE;">BBa_R0010</u></a> led in a colony with the correct sequence in opposition to our trial of gaining a positive clone while working with the first promoter <a href=”http://parts.igem.org/Part:BBa_R0011”><u style="color:#0000EE;">BBa_R0011</u></a>.</br> |
− | An expression with the newly integrated promoter <a href=”http://parts.igem.org/Part:BBa_R0010”><u style="color:#0000EE;">BBa_R0010</u></a> led in a colony with the correct sequence in opposition to our trial of gaining a positive clone while working with the first promoter <a href=”http://parts.igem.org/Part:BBa_R0011”><u style="color:#0000EE;">BBa_R0011</u></a>. | + | We continued our experiments by performing a skim milk assay on agar plates. Therefore, we poured LB skim milk agar plates containing IPTG and the needed antibiotic and streaked the <i>E. coli</i> BL21 cells containing the plasmid with the expression system.</br> |
− | + | ||
+ | <center><img src="https://static.igem.org/mediawiki/2016/f/fb/T--Aachen--labbook_ecoli_skim_milk_native.png" style="width:600px;"/></center> | ||
+ | |||
+ | <figcaption style="text-align:center; font-size: 15px; padding-left:2cm;padding-right:2cm; "><b>Figure 1: Skim milk plates assay.</b> Cells containing the empty backbone (left) and cells containing the expression system for native subtilisin E (right) after incubation for 3 days at 30°C.</figcaption></br> | ||
+ | |||
+ | <p align="justify" style="padding-left: 1.0cm; padding-right: 1.0cm; font-size:16px;"> | ||
+ | Comparing the clearance of the skim milk plates, a proteolytic activity could be proven for the cells containing the expression system for native subtilisin E. As a results, we concluded that within three days these cells are able to produce the native protease which will then digest the skim milk in the agar plates, resulting in a clearance.</br> | ||
+ | In conclusion, we were able to express subtilisin E in <i>E. coli</i> and to prove its proteolytic activity via skim milk assay.</br> | ||
+ | All details about this part can be seen on its <a href=”http://parts.igem.org/Part:BBa_K2020002”><u style="color:#0000EE;">registry page</u></a>.</br></br> | ||
</div> | </div> | ||
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<a name="K2020000" class="anchor" style="color:#005C04;"><big><b>K2020000</b></big> – subtilisin E gene, optimized for <i>E. coli</i> codon usage</a> | <a name="K2020000" class="anchor" style="color:#005C04;"><big><b>K2020000</b></big> – subtilisin E gene, optimized for <i>E. coli</i> codon usage</a> | ||
The gene of this BioBrick can be used to express subtilisin E, which is an alkaline serine protease, which non specifically digest proteins, in <i>Escherichia coli</i>. To use this part a suitable leader sequence has to be placed in front as the sequence of this BioBrick does not contain a start codon. | The gene of this BioBrick can be used to express subtilisin E, which is an alkaline serine protease, which non specifically digest proteins, in <i>Escherichia coli</i>. To use this part a suitable leader sequence has to be placed in front as the sequence of this BioBrick does not contain a start codon. | ||
− | Its sequence was originally obtained from a wild type <i>Bacillus subtilis</i> but was codon optimized for <i>E. coli</i>.</br></br> | + | Its sequence was originally obtained from a wild type <i>Bacillus subtilis</i> but was codon optimized for <i>E. coli</i>.</br> |
+ | All details about this part can be seen on its <a href=”http://parts.igem.org/Part:BBa_K2020000”><u style="color:#0000EE;">registry page</u></a> and in the section of the related BioBrick <a href="#K2020002" style="color:#0000EE;">K2020002</a>.</br></br> | ||
<a name="K2020001" class="anchor" style="color:#005C04 ;"><big><b>K2020001</b></big> – subtilisin E gene, optimized for <i>E. coli</i> codon usage, with leader sequence pelB</a> | <a name="K2020001" class="anchor" style="color:#005C04 ;"><big><b>K2020001</b></big> – subtilisin E gene, optimized for <i>E. coli</i> codon usage, with leader sequence pelB</a> | ||
Line 244: | Line 253: | ||
To generate a functional coding sequence that can be expressed in <i>E. coli</i> the leader sequence pelB, which begins with a start codon, was placed in front of the subtilisin E gene.</br> | To generate a functional coding sequence that can be expressed in <i>E. coli</i> the leader sequence pelB, which begins with a start codon, was placed in front of the subtilisin E gene.</br> | ||
Caused to subtilisin E’s partly toxicity for <i>E. coli</i>, this BioBrick should be cloned into an expression system with an inducible promoter. This has to hinder the organism in the expression of the protease while its growing period. With the iGEM promoter <a href=”http://parts.igem.org/Part:BBa_R0011”><u style="color:#0000EE;">BBa_R0011</u></a> it was not possible to successfully express subtilisin E. Hence, we exchange the promoter against <a href=”http://parts.igem.org/Part:BBa_R0010”><u style="color:#0000EE;">BBa_R0010</u></a>.</br> | Caused to subtilisin E’s partly toxicity for <i>E. coli</i>, this BioBrick should be cloned into an expression system with an inducible promoter. This has to hinder the organism in the expression of the protease while its growing period. With the iGEM promoter <a href=”http://parts.igem.org/Part:BBa_R0011”><u style="color:#0000EE;">BBa_R0011</u></a> it was not possible to successfully express subtilisin E. Hence, we exchange the promoter against <a href=”http://parts.igem.org/Part:BBa_R0010”><u style="color:#0000EE;">BBa_R0010</u></a>.</br> | ||
− | All details about this part can be seen on its <a href=”http://parts.igem.org/Part:BBa_K2020001”><u style="color:#0000EE;">registry page</u></a>.</br></br> | + | All details about this part can be seen on its <a href=”http://parts.igem.org/Part:BBa_K2020001”><u style="color:#0000EE;">registry page</u></a> and in the section of the related BioBrick <a href="#K2020002" style="color:#0000EE;">K2020002</a>.</br></br> |
<span style="color:#005C04;"><big><b>K2020002</b></big> – expression system for subtilisin E in <i>E. coli</i></span></br> | <span style="color:#005C04;"><big><b>K2020002</b></big> – expression system for subtilisin E in <i>E. coli</i></span></br> | ||
− | Details about this part can be seen above in the section <a href="#composite" style="color:#0000EE;">“Best New Composite Part”</a> and on | + | Details about this part can be seen above in the section <a href="#composite" style="color:#0000EE;">“Best New Composite Part”</a> and on its <a href=”http://parts.igem.org/Part:BBa_K2020002”><u style="color:#0000EE;">registry page</u></a>.</br> |
Line 299: | Line 308: | ||
<span style="color:#005C04;"><big><b>K2020042</b></big> – tRNA specific for tyrosine and UAG codon in <i>E. coli</i></span></br> | <span style="color:#005C04;"><big><b>K2020042</b></big> – tRNA specific for tyrosine and UAG codon in <i>E. coli</i></span></br> | ||
− | Details about this part can be seen above in the section <a href="#basic" style="color:#0000EE;">“Best Basic Part”</a> and on | + | Details about this part can be seen above in the section <a href="#basic" style="color:#0000EE;">“Best Basic Part”</a> and on its <a href=”http://parts.igem.org/Part:BBa_K2020042”><u style="color:#0000EE;">registry page</u></a>.</br></br> |
<span style="color:#005C04;"><big><b>K2020043</b></big> – tRNA synthetase specific for the ncAA AzF and UAG codon in <i>E. coli</i> → AzF-RS</span></br> | <span style="color:#005C04;"><big><b>K2020043</b></big> – tRNA synthetase specific for the ncAA AzF and UAG codon in <i>E. coli</i> → AzF-RS</span></br> | ||
− | Details about this part can be seen above in the section <a href="#collection" style="color:#0000EE;">“Parts Collection”</a> and on | + | Details about this part can be seen above in the section <a href="#collection" style="color:#0000EE;">“Parts Collection”</a> and on its <a href=”http://parts.igem.org/Part:BBa_K2020043”><u style="color:#0000EE;">registry page</u></a>.</br></br> |
<span style="color:#005C04;"><big><b>K2020045</b></big> – tRNA synthetase specific for the ncAA NitroY and UAG codon in <i>E. coli</i> → NitroY-RS</span></br> | <span style="color:#005C04;"><big><b>K2020045</b></big> – tRNA synthetase specific for the ncAA NitroY and UAG codon in <i>E. coli</i> → NitroY-RS</span></br> | ||
− | Details about this part can be seen above in the section <a href="#collection" style="color:#0000EE;">“Parts Collection”</a> and on | + | Details about this part can be seen above in the section <a href="#collection" style="color:#0000EE;">“Parts Collection”</a> and on its <a href=”http://parts.igem.org/Part:BBa_K2020045”><u style="color:#0000EE;">registry page</u></a>.</br></br> |
<span style="color:#005C04;"><big><b>K2020046</b></big> – tRNA synthetase specific for the ncAA CNF and UAG codon in <i>E. coli</i> → CNF-RS</span></br> | <span style="color:#005C04;"><big><b>K2020046</b></big> – tRNA synthetase specific for the ncAA CNF and UAG codon in <i>E. coli</i> → CNF-RS</span></br> | ||
− | Details about this part can be seen above in the section <a href="#collection" style="color:#0000EE;">“Parts Collection”</a> and on | + | Details about this part can be seen above in the section <a href="#collection" style="color:#0000EE;">“Parts Collection”</a> and on its <a href=”http://parts.igem.org/Part:BBa_K2020046”><u style="color:#0000EE;">registry page</u></a>.</br></br> |
<span style="color:#005C04;"><big><b>K2020050</b></big> – tRNA synthetase specific for tyrosine and UAG codon in <i>E. coli</i> → Y-RS</span></br> | <span style="color:#005C04;"><big><b>K2020050</b></big> – tRNA synthetase specific for tyrosine and UAG codon in <i>E. coli</i> → Y-RS</span></br> | ||
− | Details about this part can be seen above in the section <a href="#collection" style="color:#0000EE;">“Parts Collection”</a> and on | + | Details about this part can be seen above in the section <a href="#collection" style="color:#0000EE;">“Parts Collection”</a> and on its <a href=”http://parts.igem.org/Part:BBa_K2020050”><u style="color:#0000EE;">registry page</u></a>.</br></br> |
<span style="color:#005C04;"><big><b>K2020051</b></big> – mutated tRNA synthetase specific for tyrosine and UAG codon in <i>E. coli</i> → Y-RS with Y32G</span></br> | <span style="color:#005C04;"><big><b>K2020051</b></big> – mutated tRNA synthetase specific for tyrosine and UAG codon in <i>E. coli</i> → Y-RS with Y32G</span></br> | ||
− | Details about this part can be seen above in the section <a href="#collection" style="color:#0000EE;">“Parts Collection”</a> and on | + | Details about this part can be seen above in the section <a href="#collection" style="color:#0000EE;">“Parts Collection”</a> and on its <a href=”http://parts.igem.org/Part:BBa_K2020051”><u style="color:#0000EE;">registry page</u></a>.</br></br> |
<span style="color:#005C04;"><big><b>K2020052 to K2020061</b></big> - tRNA synthetase specific for DMNB-serine and UAG codon in <i>E. coli</i> → version 1 to 10</span></br> | <span style="color:#005C04;"><big><b>K2020052 to K2020061</b></big> - tRNA synthetase specific for DMNB-serine and UAG codon in <i>E. coli</i> → version 1 to 10</span></br> | ||
− | Details about these parts can be seen above in the section <a href="#collection" style="color:#0000EE;">“Parts Collection”</a> and on | + | Details about these parts can be seen above in the section <a href="#collection" style="color:#0000EE;">“Parts Collection”</a> and on their <a href=”http://parts.igem.org/Part:BBa_K2020052”><u style="color:#0000EE;">registry pages</u></a>.</br></br> |
Revision as of 20:49, 18 October 2016
Parts
BioBrick | Description |
K2020000 | subtilisin E gene, optimized for E. coli codon usage |
K2020001 | subtilisin E gene, optimized for E. coli codon usage, with leader sequence pelB |
K2020002 | expression system for subtilisin E in E. coli |
K2020003 | mutated expression system for subtilisin E in E. coli (S221Y) |
K2020004 | mutated expression system for subtilisin E in E. coli (S221X) |
K2020005 | mutated expression system for subtilisin E in E. coli (Y77W) |
K2020006 | mutated expression system for subtilisin E in E. coli (Y77X) |
K2020026 | leader sequence MFalpha (different version of the biobrick K792002) |
K2020040 | screening plasmid for incorporation of non-canonical amino acids → pRXG (twin pFRY) |
K2020041 | screening plasmid for incorporation of non-canonical amino acids → pRYG (twin pFRYC) |
K2020042 | tRNA specific for tyrosine and UAG codon in E. coli |
K2020043 | tRNA synthetase specific for the ncAA AzF and UAG codon in E. coli → AzF-RS |
K2020045 | tRNA synthetase specific for the ncAA NitroY and UAG codon in E. coli → NitroY-RS |
K2020046 | tRNA synthetase specific for the ncAA CNF and UAG codon in E. coli → CNF-RS |
K2020050 | tRNA synthetase specific for tyrosine and UAG codon in E. coli → Y-RS |
K2020051 | mutated tRNA synthetase specific for tyrosine and UAG codon in E. coli → Y-RS with Y32G |
K2020052 | tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 1 |
K2020053 | tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 2 |
K2020054 | tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 3 |
K2020055 | tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 4 |
K2020056 | tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 5 |
K2020057 | tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 6 |
K2020058 | tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 7 |
K2020059 | tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 8 |
K2020060 | tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 9 |
K2020061 | tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 10 |
K2020002 – expression system for subtilisin E in E. coli This expression system consists of the promoter BBa_R0011, the ribosome binding site BBa_B0034, the newly created BioBrick part BBa_K2020001 and the terminator BBa_B0010. BioBrick K2020001 is a composite part itself and includes the secretion tag pelB (BBa_J32015) and a subtilisin E gene optimized for Escherichia coli codon usage (BBa_K2020000). Once introduced into E. coli, this BioBrick is able to produce subtilisin E, an alkaline serine protease which non-specifically digest proteins, and simultaneously secret the enzyme into the periplasm of the cell. Caused by the lacI regulated promoter BBa_R0010, the expression system can be induced by addition of IPTG. With the iGEM promoter BBa_R0011, which was integrated in our sequence at first, it was not possible to successfully express subtilisin E due to fatal mutations inside the expression system in all analyzed colonies. Either there have been single base deletions or insertions in the pro-peptide, which led to a frameshift of the whole protein, or a 23 base pair deletion in the promoter. Both types of mutations result in an incorrect expression system, so that an expression of the protease is impossible. Since the promoter BBa_R0011 is leaky and induce the expression even without addition of IPTG, it can be assumed that subtilisin E is toxic for E. coli. Hence, we exchanged the promoter against BBa_R0010. For achieving this, we fulfilled a Polymerase Chain Reaction to extract everything but the promoter and the RBS and simultaneously extend the remaining DNA sequence with the pre-fix of iGEM. Afterwards, we assembled it with the BioBrick J04500 and in parallel cloned it into the vector pSB1C3 - by cutting RFP out of the BioBrick J04450. The implemented BioBrick J04500 itself contains another IPTG inducible promoter (BBa_R0010) and the same RBS (BBa_B0034). An expression with the newly integrated promoter BBa_R0010 led in a colony with the correct sequence in opposition to our trial of gaining a positive clone while working with the first promoter BBa_R0011. We continued our experiments by performing a skim milk assay on agar plates. Therefore, we poured LB skim milk agar plates containing IPTG and the needed antibiotic and streaked the E. coli BL21 cells containing the plasmid with the expression system.
![](https://static.igem.org/mediawiki/2016/f/fb/T--Aachen--labbook_ecoli_skim_milk_native.png)
Comparing the clearance of the skim milk plates, a proteolytic activity could be proven for the cells containing the expression system for native subtilisin E. As a results, we concluded that within three days these cells are able to produce the native protease which will then digest the skim milk in the agar plates, resulting in a clearance. In conclusion, we were able to express subtilisin E in E. coli and to prove its proteolytic activity via skim milk assay. All details about this part can be seen on its registry page.
K2020043 – tRNA synthetase specific for the ncAA AzF and UAG codon in E. coli → AzF-RS K2020045 – tRNA synthetase specific for the ncAA NitroY and UAG codon in E. coli → NitroY-RS K2020046 – tRNA synthetase specific for the ncAA CNF and UAG codon in E. coli → CNF-RS K2020050 - tRNA synthetase specific for tyrosine and UAG codon in E. coli → Y-RS K2020051 - mutated tRNA synthetase specific for tyrosine and UAG codon in E. coli → Y-RS with Y32G K2020052 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 1 K2020053 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 2 K2020054 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 3 K2020055 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 4 K2020056 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 5 K2020057 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 6 K2020058 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 7 K2020059 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 8 K2020060 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 9 K2020061 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 10
Expression in E. coli
Basic Building Blocks
K2020000 – subtilisin E gene, optimized for E. coli codon usage The gene of this BioBrick can be used to express subtilisin E, which is an alkaline serine protease, which non specifically digest proteins, in Escherichia coli. To use this part a suitable leader sequence has to be placed in front as the sequence of this BioBrick does not contain a start codon. Its sequence was originally obtained from a wild type Bacillus subtilis but was codon optimized for E. coli. All details about this part can be seen on its registry page and in the section of the related BioBrick K2020002. K2020001 – subtilisin E gene, optimized for E. coli codon usage, with leader sequence pelB This composite part consists of the leader sequence pelB (BBa_J32015) and the subtilisin E gene (K2020000) from our first BioBrick. It can be used to express subtilisin E in Escherichia coli and simultaneously secret the enzyme into the periplasm of the cell. Subtilisin E is an alkaline serine protease which non-specifically digests proteins. To generate a functional coding sequence that can be expressed in E. coli the leader sequence pelB, which begins with a start codon, was placed in front of the subtilisin E gene. Caused to subtilisin E’s partly toxicity for E. coli, this BioBrick should be cloned into an expression system with an inducible promoter. This has to hinder the organism in the expression of the protease while its growing period. With the iGEM promoter BBa_R0011 it was not possible to successfully express subtilisin E. Hence, we exchange the promoter against BBa_R0010. All details about this part can be seen on its registry page and in the section of the related BioBrick K2020002. K2020002 – expression system for subtilisin E in E. coli Details about this part can be seen above in the section “Best New Composite Part” and on its registry page.
Mutated Versions
K2020003 – mutated expression system for subtilisin E in E. coli (S221Y) The named BioBrick K2020002 above for the expression of subtilisin E in E. coli is the basic concept of this new part. Therefore it consists of the promoter BBa_R0010, the ribosome binding site BBa_B0034, the leader sequence pelB BBa_J32015, the newly created BioBrick part BBa_K2020000 and the terminator BBa_B0010 like the expression system itself. The whole expression is based on the usage in E. coli, so the sequence of the subtilisin E gene from a wild type Bacillus subtilis was optimized for E. coli codon usage. The sequence was partly ordered from IDT (BBa_K2020001 + BBa_B0010) and then cloned into BBa_J04500, a protein expression backbone which already carries the LacI promoter BBa_R0010 and the ribosome binding site BBa_B0034. Afterwards, a mutation in the active site of the enzyme was introduced by performing a site-directed mutagenesis. The codon AGC of serine221 was substituted with TAC which codes for tyrosine. By performing a site-directed mutagenesis, serine in the catalytic triade of the enzyme was exchanged against tyrosine. Therefore, the alkaline serine protease looses its proteolytic activity of non-specifically digesting proteins. Caused to its leader sequence pelB BBa_J32015, the BioBrick secrets an inactive version of subtilisin E into the periplasm of the cell as soon as it is introduced into E. coli. K2020004 – mutated expression system for subtilisin E in E. coli (S221X) K2020005 – mutated expression system for subtilisin E in E. coli (Y77W) K2020006 – mutated expression system for subtilisin E in E. coli (Y77X)
Expression in S. cerevisiae
Improvement of an Existing Part
K2020026 – leader sequence MFalpha (different version of the biobrick K792002) This part is a different version of the leader sequence MFalpha (BBa_K792002) for Saccharomyces cerevisiae. It can be cloned directly in front of the protein one wants to be secreted. Therefore, it will be relocated into the medium and the tag will be cleaved off during this process. This alpha mating factors secretion tag is naturally occurring in the genome of S. cerevisiae. Futhermore, the sequence contained an illegal restriction site, which was corrected via PCR by us.
Evolution of a New Synthetase
Screening System
K2020040 – screening plasmid for incorporation of non-canonical amino acids → pRXG This part is an improvement of the existing BioBrick K1416004, called pFRY by the iGEM Team Austin Texas 2014. K2020041 – screening plasmid for incorporation of non-canonical amino acids → pRYG This part is an improvement of the existing BioBrick K1416003, called pFRYC by the iGEM Team Austin Texas 2014.
tRNA and Synthetases
K2020042 – tRNA specific for tyrosine and UAG codon in E. coli Details about this part can be seen above in the section “Best Basic Part” and on its registry page. K2020043 – tRNA synthetase specific for the ncAA AzF and UAG codon in E. coli → AzF-RS Details about this part can be seen above in the section “Parts Collection” and on its registry page. K2020045 – tRNA synthetase specific for the ncAA NitroY and UAG codon in E. coli → NitroY-RS Details about this part can be seen above in the section “Parts Collection” and on its registry page. K2020046 – tRNA synthetase specific for the ncAA CNF and UAG codon in E. coli → CNF-RS Details about this part can be seen above in the section “Parts Collection” and on its registry page. K2020050 – tRNA synthetase specific for tyrosine and UAG codon in E. coli → Y-RS Details about this part can be seen above in the section “Parts Collection” and on its registry page. K2020051 – mutated tRNA synthetase specific for tyrosine and UAG codon in E. coli → Y-RS with Y32G Details about this part can be seen above in the section “Parts Collection” and on its registry page. K2020052 to K2020061 - tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 1 to 10 Details about these parts can be seen above in the section “Parts Collection” and on their registry pages.