Team:NU Kazakhstan/Engineering

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The central aim of our project is to enhance the rhamnolipid production on the basis of the engineering modification of the Pseudomonas putida bacteria by adding pRGPDuo2 a  plasmid that has a dual expression system for the co-expression of two different inducible genes [1]. In our case we targeted the genes; nadE and rhlA/B genes as inserts for dual inducible co-expression. We hypothesized that the insertion of these genes should enhance the rhamnolipid synthesis under an electrofermentative system. The logic was based on the fact that a simultaneous expression of the respiratory gene nadE with rhamnolipid genes rhlA/B in an oil-challenged system could lead to improved remediation of the oil. This would merely be functional under the employment of electrofermentation to target elevated respiration of the bacterial system.

Throughout the project, we went through several iterations of the engineering design cycle, concurrently solving the issues that appeared during the engineering process. This engineering cycle is comprised of the following steps: Research, Imagine, Design, Build, Test, Learn, and Improve. Overall, we have 3 cycles.


Engineering cycle

Figure 1. The Engineering Design Cycle


Design cycle 1: P. putida engineering

Research: Characteristics of P. aeruginosa and P. putida, inserted genes and the influence of the electrical inducibility in the biosurfactant production.


The key product that our team wanted to obtain was rhamnolipid - a biological surfactant that reduces the surface tension between water and oil molecules, thereby increasing the oil degradation process performed by bacteria cells [3]. P. aeruginosa PAO1 is a bacteria that serves as a natural source of rhamnolipid production as it contains all the required genes for its synthesis [4]. However, the pathogenic characteristics of this bacteria encouraged us to think about the usage of safer biological strains which can be used in industrial applications [5]. Through the method of transferring genes of P. aeruginosa we decided to choose the similar Pseudomonas putida KT2440 strain which is well known for its frequent employment in bio-industrial applications [6].

  • We targeted to link the enhanced yield of rhamnolipid as a direct result of the increase in extracellular electron transfer orchestrated by nadE gene overexpression. nadE is a gene responsible for NAD synthetase production, which results in a greater amount of NAD+ cofactor making it available for recycling to NADH [7]. This is a very essential cofactor that is needed for numerous anabolic reactions which involve rhamnolipid synthesis too. Additionally, the electrofermentative conditions in which modified bacteria are going to live remarkably facilitate the reduction of NAD+ cofactor to the NADH which takes a part in anabolic pathways.
  • rhlA/B genes (initially not present in P. putida) are required in rhamnolipid production because they cooperate in the synthesis of rhamnolipid[8]. The rhlA gene’s activity and other metabolic pathways generate the substrate molecules - HAA - 3-(3-hydroxyalkanoyloxy)alkanoic acid ) that undergo reaction catalyzed by the rhlB gene’s expression product.
  • The insertion of nadE and rhlA/B genes into the P. putida will be performed by cloning them into the pRGPDuo2 plasmid provided by Dr. Rahul Gauttam, Research Fellow at Lawrence Berkeley National Laboratory (LBNL), USA. 
  • The supernatant of the centrifuged sample would be obtained and will be used in the bioremediation of crude oil samples due to the presence of rhamnolipid molecules in them.


Imagine: How to engineer P. putida


After reading the literature regarding the features of bacteria strains and the genes’ information we identified some aspects that we must follow:

  • The screening method is performed by a selective marker in the form of antibiotic kanamycin/gentamicin.
  • The intermediate species to which the pRGPDuo2 plasmid will be inserted is Escherichia coli.
  • Restriction Enzyme Cloning will be used to insert nadE and rhlA/B genes into two separate multiple cloning sites (MCS).



After analyzing the compartments of the series of pRGPDuo2 we have found that the Restriction Enzyme Cloning method would be the best choice for insertion of nadE and rhlA/B genes. By using the Polymerase Chain Reaction (PCR) technique, we planned to multiply the sequences of nadE and rhlA/B and with the help of restriction enzymes wanted to insert purified genes into pRGPDuo2 plasmids. In order to multiply the amount of plasmid and concurrently test the viability of the plasmid, we used Escherichia coli as an intermediate species to carry the plasmid. The antibiotic resistance sequences (tetR, KanR, GmR) in plasmids served as a selectable marker on the growing medium mixed with the corresponding antibiotic. Thus, we were able to screen individual colonies that have acquired modified plasmids.


You can find more specific information regarding the design part of our project on the Design page.


Test & Build

The building of the engineered P. putida strains was initiated by the extraction of P. aeruginosa DNA molecules from its other cellular compartments. These DNA molecules contained the rhlA/B and nadE target genes which in further steps have experienced the proliferation through the PCR technique.


Table 1. The Nanodrop measurements of P. aeruginosa extracted genomic DNA

Tube Concentration 260/280
1 50.44 ng/ul 1.55
2 30.14 ng/ul 1.54
3 58.52 ng/ul 1.85
4 150.1 ng/ul 1.6


We used PCR to multiply our target genes through the usage of early designed primers and extracted genomic DNA of P. aeruginosa. Our goal was to obtain expected visible bands of both genes on the agarose gel stained by ethidium bromide under exposure to UV light. While the results for nadE gene was proper, we did not see valid bands for rhlA/B genes.


Gel #1

Figure 2. The Gel electrophoresis of nadE and rhlA/B genes that undergone distinct PCR conditions



2 - DNA ladder
3 - rhl AB
4 - rhl AB (unsuccessful PCR)
5 - rhl AB (unsuccessful PCR)
6 - rhl AB (unsuccessful PCR)
7 - 15 nadE (different PCR conditions and DNA concentrations)

Approximate sizes of genes are the following: nadE gene - 883 bp, rhlA/B2244 bp (932 bp for rhlA and 1344 bp for rhlB)



After performing several trials of amplification of rhlA/B genes with different PCR conditions yielding low concentrations, which impeded ligation experiments, we identified that size of the rhlA/B sequence (2244 base pairs) might have been a source of difficulty in PCR amplification due to the high GC sequences of Pseudomonads, and therefore sought to singly amplify the genes. We also thought that permanent usage of the same reagents with repetitive freezing has negatively affected their performance in PCR. Therefore, we thought of different ways of proceeding with PCR and the usage of other reagents in it.


Design cycle 2: Elution of the target genes



We ended up designing new primers for the whole sequence and for individual genes (rhlA and rhlB). In addition, we ordered new dNTPs, Q5 High-Fidelity DNA Polymerase, Q5 reaction buffer, and Q5 GC enhancer. Such adjustments benefited us in several ways:

  • The use of new reagents might help in better PCR performance.
  • The separate amplification of distinct rhlA and rhlB genes might result in better PCR amplification.
  • The newly designed primers allow usage of newly arrived restriction enzymes (SacI, SalI, BmtI, and Nhe1) that create sticky ends for cloning to the prepared plasmids.



After using the recently arrived primers and other reagents the desired genes showcased their respective bands on gel electrophoresis.


Gel #2

Figure 3. The Gel electrophoresis of nadE and separated rhlA and rhlB genes



  • A - rhlAB (old primers)
  • B - rhlAB (new primers)
  • C - rhlA 
  • D - rhlB
  • E - nadE


We proceeded with the laboratory work on the elution process. We have used Quick Gel Extraction Kits to do so. However, due to the destructive effect of ethidium bromide on DNA molecules, we divided the gel into two identical halves one of which was stained with ethidium bromide while the other one was left intact. Both halves were loaded with similar PCR samples for gel electrophoresis in the same manner. The image of bands on the first half was used as a reference for the cut of the desired DNA bands. Even with this method, the Nanodrop Spectrophotometer measured a low concentration of DNA molecules.


As we were unsuccessful in the elution of our rhlA/B genes with the methods described above, we assumed the following explanation to be responsible for this:

  • The procedure was made with the old primers and without aid from assistance reagents. Therefore, the elution with new reagents should be performed again.
  • The way of cutting the gel in half and using one of them as a reference to another is not accurate enough and probably resulted in the mistake during the cutting of the band part. Therefore, it is better to find another staining reagent that would allow the DNA band to be cut under its exposure.


Design cycle 3: Challenging bioremediation performance of modified P. aeruginosa


The ethidium bromide application for staining agarose gel was unsuccessful when we wanted to elute DNA molecules of genes. Therefore, we have changed the staining compound to the GelRed, which was advantageous for several reasons:

  • GelRed is a safer reagent, as it is neither carcinogenic nor mutagenic [9]. 
  • GelRed does not have any influence on the DNA molecules, which makes it perfect for elution.



Nanodrop of elution #1

Nanodrop of elution #2

Figure 4. The Nanodrop Spectroscopy measurements regarding eluted DNA samples


The concentrations measured by the Nanodrop spectrophotometer (in ng/μl) are the following:

  • C1 - rhlA
  • D1 - rhlB
  • G1 - nadE
  • H1 - pRGPDuo2

Afterward, we sought to insert purified gene sequences into the plasmids and perform electrotransformation in order for our bacteria to acquire modified plasmids. As it was stated earlier plasmid contains antibiotic resistance sequences that allow them to grow on the medium with the corresponding antibiotics. Unfortunately, we were not able to obtain transformation of P. putida due to the gene uptake efficiency of our strains and therefore we decided to prepare engineered P. aeruginosa as model strains through which we tested the electro fermentative activity and yield of rhamnolipid production:

  • Wild-type P. aeruginosa (as control 1)
  • P. aeruginosa with pRGPDuo2 empty plasmid (as control 2)
  • P. aeruginosa with pRGPDuo2 plasmid + nadE
  • P. aeruginosa with pRGPDuo2 plasmid + rhlA
  • P. aeruginosa with pRGPDuo2 plasmid + rhlB


plate #1

Figure 5. The wild type P. aeruginosa


plate #2

Figure 6. The P. aeruginosa with pRGPDuo2


plate #3

Figure 7. The P. aeruginosa with pRGPDuo2 and nadE


plate #4

Figure 8. The P. aeruginosa with pRGPDuo2 and rhlA


plate #5

Figure 9. The P. aeruginosa with pRGPDuo2 and rhlB


After inserting the genes into P. aeruginosa and growing in the media (LB agar) with antibiotic (kanamycin), we extracted DNA to test with nanodrop. And we obtained the results as shown in the figure below. The ratio of absorbance at 260 nm and 280 nm for all samples indicate that the DNA samples are pure (higher than or equal to 1.80).


nanodrop of extraction #2

Figure 10. The Nanodrop Spectroscopy measurements regarding extracted DNA molecules


  • B1 - pRGPDuo2 plasmid
  • C1 - pRGPDuo2 plasmid + nadE gene
  • E1 - pRGPDuo2 plasmid + rhlB gene
  • F1 - pRGPDuo2 plasmid + rhlA gene


In order to be sure that the P. aeruginose cells have received pRGPDuo2 (both modified with genes and unmodified), we performed the gel electrophoresis of double digested DNA samples extracted from abovementioned modified strains of P. aeruginose.


Gel #3

Figure 11. The Gel electrophoresis of double digested plasmids



  • A - pRGPDuo2 plasmid undigested 
  • B - pRGPDuo2 digested with SacI and SalI (digestion at 37° for 1 hour)
  • C - pRGPDuo2 + nadE digested with SacI and SalI (digestion at 37° for 1 hour)
  • D - pRGPDuo2 + rhlA digested with SacI and SalI (digestion at 37° for 1 hour)
  • E - pRGPDuo2 + rhlB digested with SacI and SalI (with digestion at 37° for 1 hour)
  • F - pRGPDuo2 digested with SacI and SalI (digested at 37° for 15 min)
  • G - pRGPDuo2+ nadE digested with SacI and SalI (digested at 37° for 15 min)
  • H - pRGPDuo2+ rhlA digested with SacI and SalI (digested at 37° for 15 min)
  • I - pRGPDuo2+ rhlB digested with SacI and SalI (digested at 37° for 15 min)


Then we grew them in minimal salt media (MgSO4.7H20 (1 g/L), KH2PO4 (2 g/L), NaNO3 (6 g/L), ammonium chloride (0.6 g/L) and crude oil (2 %v/v)) and tested for oil samples remediation. We had 5 bacteria samples to test:


  • P.aeruginosa wild-type (control 1)
  • P. aeruginosa with pRGPDuo2 empty plasmid
  • P. aeruginosa with pRGPDuo 2 plasmid + nadE
  • P. aeruginosa with pRGPDuo 2 plasmid + rhlA
  • P. aeruginosa with pRGPDuo 2 plasmid + rhlB




Figure 12.Bioremediation treatment of crude oil contaminated soils with crude biosurfactants from the various production set-ups (electrofermentation) using the different P. aeruginosa strains


A: Treatment with P. aeruginosa Wild type crude biosurfactant (from conventional fermentation)

B: Untreated control

C: Treatment with P. aeruginosa nadE crude biosurfactant (electrofermentation)

D: Treatment with P. aeruginosa rhlA crude biosurfactant (electrofermentation)

E: Treatment with P. aeruginosa rhlB crude biosurfactant (electrofermentation)


Afterward, we tried to determine the oil remediation abilities of each group. If you want to learn more about our results or even more specific aspects of our engineering process, have a look at our Results and Design page




Reference List:

Gauttam, R., Mukhopadhyay, A., Simmons, B. A., & Singer, S. W. (2021). “Development of dual‐inducible duet‐expression vectors for tunable gene expression control and CRISPR interference‐based gene repression in Pseudomonas putida KT2440”. Microbial Biotechnology. doi:10.1111/1751-7915.13832

GelRed Nucleic Acid Stain, SDS 

Muthaliar, Arularasu & Sevakumaran, Vigneswari & Bhubalan, Kesaven. (2019). PRODUCTION AND TOXICITY EVALUATION OF RHAMNOLIPIDS PRODUCED BY Pseudomonas STRAINS ON L6 AND HepG2 CELLS. Malaysian Applied Biology. 48. 149-156. 

R. M. Maier and G. Soberón-Chávez, “P. aeruginosa
rhamnolipids: Biosynthesis and potential applications,” Applied Microbiology and Biotechnology, vol. 54, no. 5, pp. 625–633, 2000. 

C. I. Kang, S. H. Kim, H. B. Kim, S. W. Park, Y. J. Choe, M. Oh, E. C. Kim, and K. W. Choe, “ P. aeruginosa bacteremia: Risk factors for mortality and influence of delayed receipt of effective antimicrobial therapy on clinical outcome,” Clinical Infectious Diseases, vol. 37, no. 6, pp. 745–751, 2003. 

R. Gauttam, A. Mukhopadhyay, and S. W. Singer, “Construction of a novel dual-inducible duet-expression system for gene (over)expression in Pseudomonas putida,” Plasmid, vol. 110, p. 102514, 2020. 

Yong, Xiao-Yu; Feng, Jiao; Chen, Yi-Lu; Shi, Dong-Yan; Xu, Yu-Shang; Zhou, Jun; Wang, Shu-Ya; Xu, Lin; Yong, Yang-Chun; Sun, Yong-Ming; Shi, Chen-Lu; OuYang, Ping-Kai; Zheng, Tao (2014). Enhancement of bioelectricity generation by cofactor manipulation in microbial fuel cell. Biosensors and Bioelectronics, 56(), 19–25. doi:10.1016/j.bios.2013.12.05

Wittgens, A., Kovacic, F., Müller, M.M. et al.“Novel insights into biosynthesis and uptake of rhamnolipids and their precursors”. Appl Microbiol Biotechnol 101, 2865–2878 (2017).

Gelred; SDS No. 41001 [Online]; Biotium, Inc; 46117 Landing Parkway, Fremont, CA 94538, USA, Sep 7, 2021. (accessed October 19th, 2021 )



Kabanbay batyr av., 53, Nur-Sultan, Kazakhstan