iGEM 2021 — Team BNDS_China

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# Team:BNDS China/Notebook

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Notebook

**Notebook**
In order to ensure the accuracy of experimental records, we designed the outline of experiments.

The experiment records are shown below.

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# Team:BNDS China/Engineering

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Title

EngineeringSuccess

To optimize the efficiency of rhamnolipid production in our host, Pseudomonas, we transcribed the plasmid originally used to manufacture rhamnolipid into E.coli by heterologous expression and then re-expressed the modified plasmid into Pseudomonas.
To begin, the lactose promoter, lactose operator, and lacI are fused to the C3591GF270 backbone (see features and sequence in BBa\_K3745043) with three unique enzymes that are responsible for synthesizing rhamnolipids via three different processes: glycolysis or gluconeogenesis, beta-oxidation, and fatty acid synthesis (see synthetic pathway for rhamnolipids on the main page of BBa\_K3745043) Additionally, IPTG is used in our research because it is a chemical that is similar to lactose. In other words, it can be used to induce Plac and further propel the production of rhamnolipids by activating rhlA, rhlB, and rhlC.

After successfully constructing our plasmid for synthesizing rhamnolipids, a quantification analysis of rhamnolipid production is carried out by using the HPLC-MS method. The particular quantification procedures are detailed on the BBa\_K3745043 design page. According to a prior study (Liu et al., 2014), since the peak of impurities and substances other than dirhamnolipids may occur in the time period of 6.5 minutes to 13.35 minutes due to the appearance of shorter fat chains (Rha2C10C10, Rha2C10C12-H2, and Rha2C10C8-H), HPLC-MS of rhamnolipids is measured in the time period between 13.937 minutes and 14.004 minutes for higher accuracy for quantitative analysis. The results are shown in the figure below: the high intensities at 503.0 and 677.7 Da correspond to pure rhamnolipid, monorhamnolipid with a C10-C12 tail, and dirhamnolipid with a C10-C10 tail, respectively. Thereby, the manufacture of rhamnolipids is successful.

Fig 1: HPLC-MS of culture induced with IPTG between13.937 min and 14.004 min

LIU, Y., ZHONG, H., LIU, Z., JIANG, Y., TAN, F., ZENG, G., LAI, M., & HE, Y. (2014). Purification and characterization of the biosurfactant rhamnolipid. Chinese Journal of Chromatography, 32(3), 248. https://doi.org/10.3724/sp.j.1123.2013.10026
Reference

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# Team:BNDS China/Experiments

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# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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# Team:BNDS China/Results

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Title

Result

Directed evolution
Random mutations are generated on the rhlABCto acquire multiple variants. This is achieved with EvolvR, by using the EvolvR plasmid, a enCas9 nickase guided by a sgRNA breaks a single strand on the double helix DNA and then the low fidelity polymerase adds mismatched nucleotides while repairing the strand. Unfortunately, our construction of the plasmid was unsuccessful.

DNA sequencing showed numerous mutations along the CDS of enCas9-Poli5M, possibly caused by the high GC% of the gene. And at the same time, the plasmid is up to 12000 bp large, and the enCas9-Poli5M itself consists of more than 7000 bp. So, the microbe has the tendency to silence the gene and release itself from the heavy burden. Gradually, these mutants with greater biological fitness became the majority of the population. In the end, due to the lack of time and money, we were unable to finish this part. However, it’s valuable experience and we would take more cautious measures in the future.

References
Halperin, S. O., Tou, C. J., Wong, E. B., Modavi, C., Schaffer, D. V., &Dueber, J. E. (2018). CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window. Nature, 560(7717), 248–252. https://doi.org/10.1038/s41586-018-0384-8 Han, L., Liu, P., Peng, Y., Lin, J., Wang, Q., & Ma, Y. (2014). Engineering the biosynthesis of novel rhamnolipids in escherichia coli for enhanced oil recovery. Journal of Applied Microbiology, 117(1), 139–150. https://doi.org/10.1111/jam.12515 LIU, Y., ZHONG, H., LIU, Z., JIANG, Y., TAN, F., ZENG, G., LAI, M., & HE, Y. (2014). Purification and characterization of the biosurfactant rhamnolipid. Chinese Journal of Chromatography, 32(3), 248. https://doi.org/10.3724/sp.j.1123.2013.10026

Figure 1: Sample tested with the paraffin method. Left: culture with induced with IPTG. Right: culture without IPTG added
The paraffin method relates the concentration of rhamnolipid to the diameter of the aqueous-phase mixture circle at the center. As rhamnolipid, a biosurfactant, is added to the culture with Paraffin solution of Sudan iii, the interfacial tension between the culture and paraffin is decreased due to the oriented adsorption of hydrophilic and hydrophobic groups on the opposite side of rhamnolipid molecule. This decreases the spreading resistance of the aqueous-phase mixture in the center circle, hence increase the final diameter of the inner circle. A significant increase in diameter of the aqueous-phase circle could be observed in the experiment group with IPTG-induced culture than the control group with IPTG-free culture. By comparing it to the standard curve that was created by rhamnolipid standard from 100 ug/L to 900ug/L (fig 2), the yield of rhamnolipid can be roughly estimate to be 50mg/L.

Figure 2: standard curve of diameter of inner circle/concentration of rhamnolipid, from 100 ug/L to 900ug/L, measured by paraffin method
Figure 3: HPLC-MS between 13.937 min and 14.004 min

Rhamnolipid is further tested using HPLC-MS. According to previous research (Liu et al, 2014), high intensity at 503.0 and 677.7 Da is observed in pure rhamnolipid, at 503.0 is monorhamnolipid with C10-C12 tail and at 677.7 is dirhamnolipid with C10-C10 tail. Therefore, the production of rhamnolipid is successful. Apart from rhamnolipid, many impurities can also be observed from the graph. This is anticipated due to the extraction method we adopted, it’s convenient yet can only eliminate some of the undesired molecules. For example, phospholipid that is common in cell debris have similar properties with rhamnolipid: hydrophilic head and hydrophobic tail. Therefore, when extracting rhamnolipid with ethanol and chloroform, these impurities can also be found in the organic phase and be detected in mass spectrometry.

Figure 4: Sequencing result of the EvolvR plasmid.
Although the EvolvR system is failed to be constructed, the designing and building of the biosensor utilized for screening positive mutants are successful.The concentration of rhamnolipid is then determined using a rhamnose promoter system, which is a reflection of the effectiveness of the new Rhamnolipid synthetase created by the mutant version of the gene. The resistance genesfor Ampicillin and Chloramphenicol are inserted into a plasmid with rhamnose-induced promoterthat can further activate therhamnosidase gene which is able to convert rhamnolipid to rhamnose using rhamnosidase. Thus, the absorbance of the solution reflects the concentration of bacteria in the solution, which indicates the host’s survival rate in the presence of these two antibiotics.Thereby, the host bacteria's survival rate should be proportional to the concentration of rhamnose.After conducting statistical analysis on the mathematical relationship between the absorbance of bacteria solution at various concentrations that induce the expression of antibiotic resistance genes, all the trials possess a positive slope, and the survival rate of bacteria increases as the concentration of rhamnose increases. Therefore, our biosensor system is reliable and successful for detecting the efficiency of rhamnolipids synthesis.
Fig. 5: Absorbance of bacteria solution with different rhamnose concentration inducing antibiotic resistance gene expression

Protein Scaffold
Our protein scaffold is composed of SH3, GBD, PDZ, and PDZ tags, as well as domains. Unfortunately, the plasmid: rhlABC, tags, and scaffolds on a pSB4C5 backbone was not successfully constructed. Our technique begins with the addition of tags to the end of each enzyme, which is subsequently substituted onto the pSB4C5 backbone and fused to the scaffolds using Gibson assembly. However, the genes encoding scaffold domains are linearized using PCR because scaffolds contain several repeated sequences that the primer must anneal to. As a result, this fragment PCR is time consuming, even more so when our desired segment is the shortest of several. By the time we had the fragment, we were unable to effectively complete the Gibson assembly on time.

Rhamnolipid synthesis
The rhlABC genes in Escherichia coli are successfully expressed and used to synthesize rhamnolipid. The genes were induced with 0.05 mM of IPTG in LB media with glucose as supplement. It’s fermented at 37° C, 220 rpm for 96 hours. After fermentation, the sample is pretested for rhamnolipid using the paraffin method, after positive result is gained, it’s put under HPLC-MS for confirmation and higher accuracy. (See protocol for exact method)

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