Part:BBa_K4873056

pUCzli-PlacsacBRFP

Composite Part: BBa_K4873056 (pUCzli-PlacsacBRFP)

Construction Design

BBa_K4873056 (pUCzli-PlacsacBRFP) is composed of BBa_K4873008 (Plac) and BBa_K4873055 (pUCzli-SacB-PRFPT). We planned to compare the effects of the promoter plac and puv on levan production. So, based on the plasmid 4 (pUCzli-SacB-PRFPT), we had constructed, we will just have to use PCR and enzyme digestion and ligation to construct plasmid 5 (pUC-zli-plac-SacB-PRFPT).

Fig.1 The plasmid map of pUC-zli-plac-SacB-PRFPT

Engineering Principle

In prokaryotes, levansucrase (SacB) can decompose sucrose into glucose and fructose. This process is completed by levansucrase1. Levansucrase SacB is an important protein that decomposes sucrose to produce levan. Levansucrase SacB, derived from Zymomonas mobilis, is an important protein that decomposes sucrose to produce levan. The expression and transport of this enzyme are regulated by the functional genes zliE and zliS. We constructed pUCzli-SacB, which is composed of ZliE-zliS and SacB2-3. On this basis, we added RFP to use the fluorescence intensity of RFP to characterize the feasibility of the system. The levansucrase gene was fused with the RFP reporter gene, and the fluorescence of RFP was combined with the yield of levan to characterize the colorless metabolites without reliable screening phenotype2-3. The promoter pzliE was added to the plasmid pUCzli-SacB-RFPT to allow RFP expression. Then, the original promoter of sacB was replaced by the Plac promoter and the PlacUV5 promoter. We planned to compare the effects of promoter plac and puv on levan production.

Fig.2 Overview of Designs

Experimental Approach

1. PCR, IPCR, enzyme digestion, enzyme ligation, transformation, sequencing identification, and homologous recombination: In 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.3 Monoclonal culture containing plasmid 5

   

   Fig.4 Sequencing results indicate the successful construction of pUC-zli-plac-SacB-PRFP

Characterization/Measurement

1. Determination of RFP fluorescence intensity: 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 5, which also confirmed that Plasmid 5 had higher values, indicating that SacB has a higher expression level in the Plasmid 5 system.

   

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

   

   Fig.6. Quantitative analysis of RFP expression levels in three different systems, with the vertical

2. Determination of fructan content: 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 7-B/C/D. Additionally, we also tested levan production at different temperatures. In all three systems, levan production was higher at around 30°C.

   

   Fig.7. pUCzli-SacB-PRFP, pUCzli-PlacsacBRFP, pUCzli-PUVsac-BRFP plasmids’ expression and functional identification.

Reference:

1. Liu L, et al. The potential use of Zymomonas mobilis for the food industry. Crit Rev Food Sci Nutr. 2022, 10.1080/10408398.2022.2139221
2. Dai Y, et al. Lowering whole cost for sugarcane based ethanol production by engineered Zymomonas mobilis. GCB Bioenergy, 2021, 13(12), 1894-1907.
3. Kondo Y, et al., Cloning and characterization of a pair of genes that stimulate the production and secretion of Zymomonas mobilis extracellular levansucrase and invertase. Bioscience, Biotechnology, and Biochemistry 1994, 58:3, 526-530

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 415
  Illegal XhoI site found at 5757
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 6172
  Illegal AgeI site found at 3526
  Illegal AgeI site found at 3638
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1751
  Illegal BsaI.rc site found at 5639
  Illegal SapI site found at 668