Part:BBa_K317998
RhlA
Pseudomonas aeruginosa produces rhamnose-containing glycolipid biosurfactants called rhamnolipids. Rhamnolipid is composed of β-D-(β-D-hydroxyalkanoyloxy)alkanoic acids(HAA) derivatized with one- or two-rhamnose sugars.In rhamnolipid biosynthesis, RhlA is required for the formation of the HAA portion of the molecule and RhlB plays a role of the rhamnosyl transferase. When RhlA and RhlB are expressed in E.coli BL21(DE3) strain, rhamnolipid indicating emulsification ability will be produced. This part includes 1 PstI site.
Contribution
Team: iGEM21_NU_Kazakhstan Author: Arsen Orazbek iGEM21_NU_Kazakhstan developed the RemiDuET project where we considered incorporating nadE and rhlAB 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:
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:
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
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 720
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 720
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 69
Illegal BamHI site found at 629
Illegal XhoI site found at 805 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 720
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 720
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 294
Illegal BsaI.rc site found at 478
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