Part:BBa_K1331001

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:

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

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