Part:BBa_K4873055

pUCzli-SacB-PRFPT

Composite Part: BBa_K4873055 (pUCzli-SacB-PRFPT)

Construction Design

We planned to build 4 plasmids. At first, we only have an EV (empty vector) pUC19, so in order to construct a plasmid that can express itself by having fluorescence, we had 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 (BBa_K4873054), and finally plasmid 4 pUCzli-SacB-PRFPT (Part: BBa_K4873055). We expected to see the fluorescence of the red color from plasmid 4 (BBa_K4873055).

Fig.1 The map graph of pUCzli-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 (BBa_ K4873007) was added to the plasmid pUCzli-SacB-RFPT to allow RFP expression.

Experimental Approach

1. The fragment size of pzliE is approximately 300 bp. Figure 2 shows that the fragment lengths are consistent with the results.

   

   Fig.2 Identification of the promoter fragment after enzyme digestion

2. The sequencing results in Figure 3 showed no gene mutation. It indicates that our plasmid pUC-Zli-SacB-PRFPT has been successfully constructed.

   

   Fig 3. Sequencing results indicate the successful construction of pUC-Zli-SacB-PRFPT.

Characterization/Measurement

1. Determination of RFP fluorescence intensity

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

2. Determination of fructan content

   In order to verify whether the plasmid pUCzli-SacB-PRFP can decompose sucrose, we measured the yield of fructan at different temperatures. According to Figure 5, the yield of fructan increased first and then decreased with the increase of temperature, and the content of levoglucosan was the highest at 30 degrees. Thus, it can be proved that sacB has enzyme activity.

   

   Fig.5 The pUCzli-SacB-PRFP plasmids' expression and fructan determination

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 2045
  Illegal XhoI site found at 536
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 951
  Illegal AgeI site found at 6128
  Illegal AgeI site found at 6240
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 418
  Illegal BsaI.rc site found at 3381
  Illegal SapI site found at 2298