Overview

Our project's goal is to obtain levan through a biosynthetic pathway and investigate the effects of different promoter sequences on levan expression. In the experimental design, we constructed an RFP (Red Fluorescent Protein) to identify which system is more effective based on the expression level of RFP and the final production of levan. Ultimately, through a large number of experiments, we confirmed the suitable promoter sequences.

 

Target T arget 1: Construction of plasmids 1)  Amplification of DNA fragments: PCR of zliES, SacB, RFP, plac, puv5. IPCR of plasmid 1 and plasmid 2 . 2)  Using enzyme digestion and enzyme ligation to construct our 6 plasmids.     Specifically as follows: Plasmid 1 : pUC-zliES Plasmid 2 : pUC-zli-SacB : C ompared to pUC-zliES , includ ing   the SacB sequence along with the promoter sequence naturally present in SacB itself. Plasmid 3 : pUC-zli-SacB-RFP :RFP was constructed into pUC-zli-SacB , however, this plasmid cannot express RFP as it lacks the corresponding promoter sequence. Plasmid 4 : pUC-zli-SacB-PRFPT : This part added a promoter to RFP, allowing RFP to express. Plasmid 5 : pUC-zli-plac-SacB-PRFPT : Plasmid 5, as compared to Plasmid 4, replaced the promoter sequence naturally carried by SacB with plac (a different promoter sequence), in the hope of achieving improved expression of SacB. Plasmid 6 : pUC-zli-puv-SacB-PRFPT :Plasmid 6, compared to plasmid 5, replaced the plac promoter with the puv promoter, altering the promoter, to compare the difference in expression of SacB.

Target 2: DNA extraction 1)  To separate the gel and our DNA fragment. 2)  Figure out the concentration of DNA we constructed.

Target 3: 1)  Testing the productivity of levan at different temperature. 2)  Compare the result brought by plasmid 4, plasmid 5 and plasmid 6.

 

Detailed results

 

Construction of Plasmid 1,2,3,4,5,6

 

 

Fig1. Work flow of construction of Plasmid 1,2,3,4,5,6

 

1)  We successfully obtained the target fragment, such as zliE-zliS, promoter, SacB, RFP, through PCR amplification. As shown in Figure 1, we confirmed their consistent sizes using DNA gel electrophoresis.

 

 

Fig 2.  Analysis of DNA gel electrophoresis for the identification of the target fragment

 

2)  Construction of plasmid 1(pUC-zliES)

In order to construct plasmid 1, we used PCR to amplify the EV(empty vector) pUC19 and zliES.

Then we used enzyme digestion to cut a same shape of the end of the DNA of pUC19 and zliES respectively. Finally, they were combined together by T4-DNA ligase.

 

Fig 3.1. Monoclonal cultivation

 

Fig 3.2. Sequencing results indicate the successful construction of pUC-zliES

3)  C onstruction of plasmid 2 (pUC-zli-SacB)

By using PCR to amplify SacB and IPCR to amplify plasmid 1, we had successfully obtaied recombiant plasmid. Afterwards, we still used the enzyme digestion to cut the same shape of the end of SacB and plasmid 1. Through Figures 4.1 and 4.2, we have successfully constructed pUC-zli-SacB.

 

 

Fig. 4.1 Monoclonal cultivation

 

Fig. 4.2   Identification of the SacB fragment after enzyme digestion through DNA gel electrophoresis

 

Fig.4.3 Sequencing results indicate the successful construction of pUC-zliES

 

 

4)  C onstruction of plasmid 3 (pUC-zli-SacB-RFP)

Similar as the previous sections, we used PCR to amplify plasmid 2 and RFP, while instead of using enzyme digestion and ligation, we use homologous recombination to combined plasmid 2 and RFP. As shown in Figures 5.1 and 5.2, we have successfully constructed pUC-Zli-SacB-RFP.

 

Fig.5.1 Identification of the RFP fragment after enzyme digestion through DNA gel electrophoresis

 

 

 

Fig 5.2 Sequencing results indicate the successful construction of pUC-Zli-SacB-RFP

 

 

5)  C onstruction of plasmid 4 (pUC-zli-SacB-PRFPT)

In order to add a promoter for RFP to help the plasmid to express its red fluorescence, we used PCR to amplify the promoter of RFP and IPCR to amplify plasmid 3. After that, we used enzyme digestion and T4-DNA ligase to combined them and construct plasmid 4. As shown in Figure 6, we have successfully obtained pUC-Zli-SacB-RFP-T.

 

Fig 6.1 Monoclonal cultivation

 

 

 

Fig.6.2 Identification of the promoter fragment after enzyme digestion through DNA gel electrophoresis, with a fragment size of approximately 300 bp.

 

 

Fig. 6.3 Sequencing results indicate the successful construction of pUC-Zli-SacB- P RFPT.

 

6)  Construction of plasmid 5 and plasmid 6 (pUC-zli-plac-SacB-PRFP and pUC-zli-puv-SacB-PRFP)

By using PCR, we successfully amplified plac, puv5 and plasmid 4. Then a series of enzyme digestion and enzyme ligation (to link plac with plasmid 4 and to link puv5 with plasmid 4 respectively.) were operated, and finally plasmid 5 and plasmid 6 were constructed . Figure 7.1 displays a monoclonal culture containing plasmid 5, Figure 7.2 displays a monoclonal culture containing plasmid 6, and Figures 7.3/7.4 are the respective sequencing identification results.

 

Fig7.1 Monoclonal culture containing plasmid 5

 

 

Fig7.2 Monoclonal culture containing plasmid 6

 

 

 

 

 

Fig7.3 Sequencing results indicate the successful construction of pUC-zli-plac-SacB-PRFP

 

 

 

Fig7.4 The sequencing results confirm the successful construction of pUC-zli-puv-SacB-PRFP

 

 

 

 

 

Functional Test  

 

1.  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 in Figure 8, which also confirmed that Plasmid 5 had higher values, indicating that SacB has a higher expression level in the Plasmid 5 system.

 

Fig 8.  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 9 demonstrates that in the Plasmid 5 system, there is also a higher level of RFP expression.

 

Fig.9.  Quantitative analysis of RFP expression levels in three different systems, with the vertical axis representing the fluorescence intensity of RFP.

 

 

 

 

2.  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 10-B/C/D. Additionally, we also tested levan production at different temperatures. In all three systems, levan production was higher at around 30 ° C.

 

Fig10. pUCzli-SacB-PRFP, pUCzli-PlacsacBRFP, pUCzli-PUVsac-BRFP plasmids ’ expression and functional identification.

 

 

 

Future plans

In the future, we hope to use the data from this experiment and the highest-yield plasmid 5 to mass-produce levan. Moreover, we hope to conduct more explorations into the production of levosan, making levosan more practical and allowing it to be used in various fields. For example, regarding cancer treatment, it is integrated into health products and skin care products to prevent aging.