Engineering Success

Overview

 

Our team embarked on a rigorous scientific journey to enhance the visibility and yield of levan. Preliminary research indicates the potential of levan as an additive in pharmaceuticals, health supplements, and dermatological products due to its anti-aging and potential anti-carcinogenic properties. The prevailing challenge is the prohibitive cost of levan production and testing. Through a biosynthetic pathway, we have made contributions by altering the promoter sequences in the levan expression pathway to further enhance levan production. We look forward to inspiring and serving as a reference for future large-scale industrial production of levan.

 

Our D - B - T - L cycle

 

Fig.1 Overview of Designs

 

Cycle 1--- Enhancing Levan Visibility

Design: we planned to build 4 plasmids. At first, we only have a EV (empty vector) pUC19, so in order to construct a plasmid that can express itself by having fluorescence, we will have to add regulators and others. As a result, we planned that the first plasmid should be pUC-zliES, and then plasmid 2 pUC-zli-SacB (Part:BBa_K4873052) , plasmid 3 pUC-zli-SacB-RFP ((Part:BBa_K4873054)) , and finally plasmid 4 pUCzli-SacB-PRFPT ( Part:BBa_K4873055 ) . And we expected to see the fluorescence of the red color from plasmid 4.

 

 

Fig.2  The graph that illustrates the process of construction plasmids and transform  the plasmids into E.coli.

 

Build:   I n the lab , we conducted a substantial amount of experimental work. Finally ,  we generally used PCR, IPCR, enzyme digestion, enzyme ligation, transformation, sequencing identification, and homologous recombination to build up different plasmids. In the end, we successfully constructed a plasmid 4 capable of expressing RFP.

 

Fig. 3  The sequence map  of plasmid 4 (pUCzli-SacB-PRFPT)

 

 

 

 

Fig.4 Identification of the promoter fragment after enzyme digestion through DNA gel electrophoresis, with a fragment size of approximately 300 bp.

 

 

Fig.5 Sequencing results indicate the successful construction of pUC-Zli-SacB- P RFPT.

 

Test: To validate the successful expression of our RFP reporter gene in the system, we transformed Plasmid 4 into Escherichia coli for expression. After fluorescent imaging, we observed a significant red fluorescence in the RFP.

 

Fig.6  The recombinant plasmid containing RFP successfully expressed in Escherichia coli, exhibiting red color.

 

 

 

Cycle 2--- Evaluating the Efficiency of Various Promoters

 

Design: we planne d  to compare the effects of promoter plac and p uv on levan production. So, based on the plasmid 4 we had constructed, we will just have to use PCR and enzyme digestion and ligation to construct plasmid 5 （ pUC-zli-plac-SacB-PRFPT , BBa_K4873056 ） .  

 

Build ： I n the lab , we conducted a substantial amount of experimental work. Finally ,  we generally used PCR, IPCR, enzyme digestion, enzyme ligation, transformation, sequencing identification, and homologous recombination to build up different plasmids. In the end, we successfully constructed a plasmid 5 capable of expressing RFP.

 

 

 

Fig .7 Monoclonal culture containing plasmid 5

 

 

Fig .8  Sequencing results indicate the successful construction of pUC-zli-plac-SacB-PRFP

 

 

Test ：

In order to investigate the expression levels of SacB in different promoter systems (plasmid 4/5), we first compared the fluorescence intensity of RFP. We observed that in the Plasmid 5 system, SacB exhibited the highest red fluorescence intensity. Subsequently, we measured the fluorescence intensity values of RFP, as shown , which also confirmed that Plasmid 5 had higher values, indicating that SacB has a higher expression level in the Plasmid 5 system.

 

Fig.9   In the Plasmid 4/5 expression systems, when comparing the brightness of RFP, Plasmid 5 exhibits stronger RFP expression.

 

 

Cycle 3 --- Evaluating the Efficiency of Various Promoters  (II)

 

Design : To further investigate the impact of changes in promoter sequences on SacB expression, we replaced plac with the puv promoter, constructing Plasmid 6 (pUC-zli-puv-SacB-PRFPT, BBa_K4873057), using a series of genetic engineering methods.

 

Build ： Plasmid 6 has been successfully constructed. We validated our experiments through DNA gel electrophoresis and sequence comparison analysis.

 

 

Fig.10 Monoclonal culture containing plasmid 6

 

 

Fig.11  The sequencing results confirm the successful construction of pUC-zli-puv-SacB-PRFP

 

 

Test: In order to investigate the expression levels of SacB in different promoter systems (plasmid 4/5/6), we first compared the fluorescence intensity of RFP. We observed that in the Plasmid 5 system, SacB exhibited the highest red fluorescence intensity. Subsequently, we measured the fluorescence intensity values of RFP, as shown , which also confirmed that Plasmid 5 had higher values, indicating that SacB has a higher expression level in the Plasmid 5 system.

 

 

Fig.12 In the Plasmid 4/5/6 expression systems, when comparing the brightness of RFP, Plasmid 5 exhibits stronger RFP expression.

 

We conducted qualitative (RFP color brightness) and quantitative analyses of RFP expression levels in different plasmid systems. Figure 13 demonstrates that in the Plasmid 5 system, there is also a higher level of RFP expression.

 

 

Fig.13. Quantitative analysis of RFP expression levels in three different systems, with the vertical

 

In order to validate which biosynthetic pathway results in higher levan production, we compared the levan yields in the plasmid 4/5/6 systems. The average levan production was higher in the plasmid 5/6 systems, as shown in Figure 14-B/C/D. Additionally, we also tested levan production at different temperatures. In all three systems, levan production was higher at around 30 ° C.

 

 

 

Fig14. pUCzli-SacB-PRFP, pUCzli-PlacsacBRFP, pUCzli-PUVsac-BRFP plasmids ’ expression and functional identification.

 

Learn: In the construction of Parts, every modification to each component can have an impact on gene expression. In this design, we anticipated that the expression levels of Levan and RFP should be the highest in the Plasmid 6 system. However, experimental results showed higher expression in the Plasmid 5 system. This presents us with new challenges, which we will investigate further in future experiments .