Proof of Concept
Integrated Human Practices
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.
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),
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–
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://
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
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.
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)