Difference between revisions of "Part:BBa K1331001"
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<partinfo>BBa_K1331001 short</partinfo>
 
<partinfo>BBa_K1331001 short</partinfo>
  
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This is an improvement of [https://parts.igem.org/Part:BBa_K317998 Part:BBa_K317998] designed by iGEM10_Tokyo-NoKoGen.
 
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A mutation has been done to remove the PstI restriction site (at 720) to meet BioBrick RFC[10] requirement without changing the amino acid sequence. The sequence we provide here includes the whole coding sequence and a part of downstream noncoding sequence of rhlA.
 
  
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RhlA together with rhlB encodes rhamnosyltransferase I for mono-rhamnolipids biosynthesis in ''Pseudomonas aeruginosa''.This part encodes rhamnosyltransferase I subunit A, and the other part [https://parts.igem.org/Part:BBa_K1331004 rhlB] encodes subunit B.
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This is an improvement of [https://parts.igem.org/Part:BBa_K317998 BBa_K317998] designed by iGEM10_Tokyo-NoKoGen. A mutation has been done to remove the PstI restriction site (at 720) to meet BioBrick RFC[10] requirement without changing the amino acid sequence.
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===Usage and Biology===
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The sequence we provide here includes the whole coding sequence and a part of downstream noncoding sequence of <em>rhlA</em>.
 +
 
 +
<em>RhlA</em> together with <em>rhlB</em> encodes rhamnosyltransferase I for mono-rhamnolipids biosynthesis in ''<em>Pseudomonas aeruginosa</em>''. This part encodes rhamnosyltransferase I subunit A, and the our part [https://parts.igem.org/Part:BBa_K1331004 BBa_K1331004] encodes subunit B.
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The noncoding sequence included in this part is for composition with rhlB. See our composite part <em>rhlAB</em> ([https://parts.igem.org/Part:BBa_K1331006 BBa_K1331006]).
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===Contribution from iGEM21_NU_Kazakhstan===
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Team: iGEM21_NU_Kazakhstan
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Author: Arsen Orazbek
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iGEM21_NU_Kazakhstan developed the RemiDuET project where we considered incorporating <em>nadE</em> and <em>rhlA/B</em> genes with pRGPDuo2 plasmid for enhanced rhamnolipid production in electrofermentative conditions. The plasmid was aquired from Gauttam, R. [1] Our team extracted <em>rhlA</em> gene by using these primers: https://parts.igem.org/Part:BBa_K4083014, https://parts.igem.org/Part:BBa_K4083015. Obtained genes were amplified in a PCR machine. Then, these PCR products were analyzed in the gel electrophoresis experiment:
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https://static.igem.org/mediawiki/parts/f/f2/RhlA_emhasized.jpg
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<em><strong>Figure 2.</strong> Gel electrophoresis of PCR products.</em>
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It can be observed that <em>rhlA</em> genes were properly extracted as their bands are located near the 1kbp which is near the actual size of the nadE gene (932bp). The smears in each well can result from the high concentration of primers, we learned from our mistake and tried to lower the concentration.
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Next, these gels were eluted, and collected genes were inserted into the pRGPDuo2 plasmid. To incorporate <em>rhlA</em> genes, we digested plasmids with NheI, SacI, SalI restrictases, and T4 ligase. These plasmids with incorporated <em>nadE</em> gene were electroporated into <em>Pseudomonas putida</em> and <em>Pseudomonas aeruginosa</em>. Unfortunately, due to the lack of time from the COVID-19 situation and late reagents delivery, we were not able to properly insert our genes into <em>P. putida</em>. However, we managed to cultivate <em>P. aeruginosa</em> in kanamycin in LB agar. Then, we extracted these engineered plasmids, and double digested them by SacI and SalI restrictases:
 +
 
 +
https://static.igem.org/mediawiki/parts/6/69/RhlA%2Bplasmid_emhasized.jpg
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<em><strong>Figure 3.</strong> Gel Electrophoresis of extracted plasmids with genes</em>
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In this picture,  E well contains pRGPDuo2+<em>rhlB</em> which was double digested. The base-pair length corresponds to the actual length of pRGPDuo2 and <em>rhlA</em>.
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More about our project you can visit this page: https://parts.igem.org/Part:BBa_K4083007
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<strong>Reference</strong>
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[1] Gauttam, R., Mukhopadhyay, A., & Singer, S. W. (2020). Construction of a novel dual-inducible duet-expression system for gene (over)expression in Pseudomonas putida. Plasmid, 110. https://doi.org/10.1016/j.plasmid.2020.102514
  
 
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Latest revision as of 01:51, 22 October 2021

Modified rhlA coding sequence from Pseudomonas aeruginosa SQ6


This is an improvement of BBa_K317998 designed by iGEM10_Tokyo-NoKoGen. A mutation has been done to remove the PstI restriction site (at 720) to meet BioBrick RFC[10] requirement without changing the amino acid sequence.

Usage and Biology

The sequence we provide here includes the whole coding sequence and a part of downstream noncoding sequence of rhlA.

RhlA together with rhlB encodes rhamnosyltransferase I for mono-rhamnolipids biosynthesis in Pseudomonas aeruginosa. This part encodes rhamnosyltransferase I subunit A, and the our part BBa_K1331004 encodes subunit B.

The noncoding sequence included in this part is for composition with rhlB. See our composite part rhlAB (BBa_K1331006).

Contribution from iGEM21_NU_Kazakhstan

Team: iGEM21_NU_Kazakhstan

Author: Arsen Orazbek

iGEM21_NU_Kazakhstan developed the RemiDuET project where we considered incorporating nadE and rhlA/B genes with pRGPDuo2 plasmid for enhanced rhamnolipid production in electrofermentative conditions. The plasmid was aquired from Gauttam, R. [1] Our team extracted rhlA gene by using these primers: https://parts.igem.org/Part:BBa_K4083014, https://parts.igem.org/Part:BBa_K4083015. Obtained genes were amplified in a PCR machine. Then, these PCR products were analyzed in the gel electrophoresis experiment:

RhlA_emhasized.jpg

Figure 2. Gel electrophoresis of PCR products.


It can be observed that rhlA genes were properly extracted as their bands are located near the 1kbp which is near the actual size of the nadE gene (932bp). The smears in each well can result from the high concentration of primers, we learned from our mistake and tried to lower the concentration.

Next, these gels were eluted, and collected genes were inserted into the pRGPDuo2 plasmid. To incorporate rhlA genes, we digested plasmids with NheI, SacI, SalI restrictases, and T4 ligase. These plasmids with incorporated nadE gene were electroporated into Pseudomonas putida and Pseudomonas aeruginosa. Unfortunately, due to the lack of time from the COVID-19 situation and late reagents delivery, we were not able to properly insert our genes into P. putida. However, we managed to cultivate P. aeruginosa in kanamycin in LB agar. Then, we extracted these engineered plasmids, and double digested them by SacI and SalI restrictases:

RhlA%2Bplasmid_emhasized.jpg

Figure 3. Gel Electrophoresis of extracted plasmids with genes


In this picture, E well contains pRGPDuo2+rhlB which was double digested. The base-pair length corresponds to the actual length of pRGPDuo2 and rhlA.

More about our project you can visit this page: https://parts.igem.org/Part:BBa_K4083007

Reference

[1] Gauttam, R., Mukhopadhyay, A., & Singer, S. W. (2020). Construction of a novel dual-inducible duet-expression system for gene (over)expression in Pseudomonas putida. Plasmid, 110. https://doi.org/10.1016/j.plasmid.2020.102514


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 69
    Illegal BamHI site found at 629
    Illegal XhoI site found at 805
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 294
    Illegal BsaI.rc site found at 478