Part:BBa_K3838999

J-TES4

Butyric acid promotes the repair of intestinal epithelial cells, strengthens the intestinal mucosal barrier, and can inhibit multiple inflammatory pathways. Butyric acid is also the main energy source of colon epithelial mucosa. The proportion of patients with intestinal butyric acid bacteria decreased, and butyric acid can not be taken directly by mouth, only enema, etc., more painful. Engineered microorganisms enable sustained, long-term drug delivery. Solution: We use the fatty acid synthesis pathway of lactic acid bacteria and Escherichia coli and introduce exogenous Tes4 gene can achieve the production of butyric acid with glucose as substrate. Tes4 enzyme has some specificity for four-carbon fatty acids. When the fatty acid is synthesized to tetracarbon, the enzyme will release it from ACP to obtain butyric acid. In our team project,the related part is composed as follows: J23100 promoter+RBS+tes4+6*his tag+T7Te terminator+rrnB T1 terminator.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 322
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal PstI site found at 322
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 351
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 322
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 322
  
• 1000
  COMPATIBLE WITH RFC[1000]

Data:SZU-China 2021 TEAM

1. The DNA level The plasmid PJT was selected for validation and transformed into DH5α (DJT) and Nissle 1917(NJT). Plasmids were transformed into DH5a for plasmid running gel validation. As can be seen from figure 1, the sample band was about 5000 bp, basically conforming to the target band of 4994 bp, which proved that our plasmid transformation was successful. As the plasmid concentration extracted by Nissle 1917 was too low, PCR validation was carried out on the plasmids in NJT, as shown in figure 1. The target band size was 945 bp, and the actual electrophoresis results met the target band size.

Fig.1 A Plasmid was extracted from transformed DH5α and gel electrophoresis was performed. The target band size was 5477bp. B PCR was performed on plasmids extracted from transformed Nissle 1917.

2. Protein level We then verified the engineering bacteria at the protein level. Intracellular protein was extracted from the cultured bacteria and purified by affinity with 6x His tag. The target protein size was 19 kDa. Purification results are shown in figure 2. In the purification, Tes4 protein was not fully combined, and the presence of hybrid proteins with the same size of Tes4 protein led to the existence of bands with the same size in the eluent, which was expected by us. However, due to time constraints, we did not have time to explore the details of elution conditions. Therefore, due to problems in elution gradient, concentration control and sample grafting in the experimental process, there were other hybrid proteins in the eluent, but the purified band of Tes4 protein was clearly visible, which effectively indicated that the engineered bacteria successfully expressed BSH protein. For Nissle 1917, SDS-Page also shows clearly visible bands of expression, as shown in red box in figure 2.

Fig.2 A SDS-PAGE of affinity purification of transformed DH5α intracellular protein. B SDS-PAGE electrophoretic diagram of Nissle 1917 intracellular protein.

3. Functional representation We will convert the pJTes4 plasmid DH5a and Nissle 1917 cells broken overnight after the training, the supernatant were collected for detection of butyric acid. In order to be more efficient to test whether our engineering bacteria produced butyric acid, the samples were collected for the derivatization of short-chain fatty acids, as the following figure. The benzene ring is introduced, in order to increase its color, which is easier to be detected at 248nm.

Fig.3 Derivatization equation.

We diluted the sample 800 times and used Kinetex C18 2.6um 100×2.1mm; Mobile phase: A:0.1% phosphoric acid-water, B:0.1% phosphoric acid-acetonitrile; Flow rate: 0.5 mL/min; Injection volume: 1μL; Column temperature: 35℃; The mobile phase gradient was carried out in the following table.

Fig.4 Mobile phase gradient.

As shown in the figure, we selected butyric acid standard as the positive control, LB medium as the negative control, WT as wild-type strain Nissle 1917 blank control. Our engineered bacteria samples D (DH5α -JTES4) and NJT(Nissle 1917-JTES4) showed the same retention time as the butyric acid sample. Compared with the negative control and blank control, it can be considered that our engineered bacteria successfully expressed the target protein and produced butyric acid.

Fig.5 Derivatization equation.

However, the results of wild-type Nissle 1917 chromatogram in the above method showed the shortcomings of this method, which could not completely separate different substances. We hoped to further optimize this method to completely separate substances, but due to time constraints, we failed to optimize the chromatographic conditions, and the chromatogram produced was not ideal.Therefore, we searched for more literature materials to try, and finally we used the following method to separate different short-chain fatty acids, so as to better detect butyric acid. In the following equation, o-benzyl hydroxylamine was replaced with 3-nitrophenylhydrazine for derivatization, so as to detect the derivatized substance at 355nm.

Fig.6 Derivatization reaction equation.

We mixed 0.4 mL of standard solution/sample with 0.2 mL of 200 mM 3-NPH-HCl solution and 0.2 mL of 120 mM EDC-HCl-6% pyridine solution. The mixture was reacted for 45 min at 40 ℃ and cooled for 1 min after reaction. 14.2 mL water was added to the mixture to obtain 15.0 mL sample solution. Mobile phase: A:water, B:ACN Flow rate:1.0 mL/min; Injection volume: 20 μL; Column temperature: 25℃; The mobile phase gradient was carried out in the following table.

Fig.7 Derivatization equation.

As shown in the figure, we compared the mixed acid (butyric acid + isobutyric acid) 、butyric acid 、Nissle 1917、NJT(Nissle 1917-JTES4) and DJT(DH5α -Jtes4).The retention time of butyric acid and butyric acid in the mixed acid is exactly the same, which proves that this chromatographic condition has a good separation effect. At the same time, there are peaks similar to butyric acid in our sample.Although both NJT(Nissle 1917-JTES4) and Nissle1917 detected only trace amounts of the substance.

Fig.8 Comparison of chromatograms of different samples.

To further confirm the presence of butyric acid in our sample, we mixed DJT(DH5α -Jtes4) with butyric acid sample and detected whether their peaks overlapped with each other. The results are as follows.The retention time of DJT(DH5α -Jtes4) mixed with butyric acid is the same, and the peak area increases, indicating that there is a certain amount of butyric acid in our sample, but the yield is still low.

Fig.9 Mixed sample comparison.