Part:BBa_K4286203

PCaMV-prb1-6xHis-TNOS

The PRB1 gene encodes a serine protease which plays an important role in the destruction of the plant pathogens. It is widely found in various trichoderma species which attack a large variety of phytopathogenic fungi responsible for major crop diseases. During the interaction, Trichoderma penetrates into the host mycelium, by partial degradation of its cell wall. It appears that the main mechanism involved in the antagonism to pathogenic fungi by Trichoderma is the release of lytic enzymes including PRB1. PRB1 production in Trichoderma is controlled by two mechanisms: it is induced by the presence of a phytopathogenic fungus, or its cell walls, and repressed by glucose. Evidence suggests that over-expression of the proteinase-encoding gene prb1 improved biocontrol activity of Trichoderma. To prevent Rice Sheath Blight, we choose Trichoderma atroviride(T.atroviride) as our chassis to overexpress Prb 1. The gene circuit of PRB1 is consist of CaMV 35s promoter, prb1 gene, 6xHis affinity tag, NOS terminator.

Figure 1. The expression of Prb1 by engineered Trichoderma atroviride

Sequence and Features

Assembly Compatibility:

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

  Illegal XhoI site found at 1946
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1046
  Illegal NgoMIV site found at 1091
  Illegal NgoMIV site found at 1385
  Illegal NgoMIV site found at 1499
  Illegal AgeI site found at 2015
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 66
  Illegal BsaI site found at 1232
  Illegal BsaI.rc site found at 1109
  

Assembly

Plasmid construction

Through homologous recombination, the coding sequence of PRB1 gene was integrated into plasmid pCAMBIA1302, and the strong constitutive promoter CaMV 35S promoter and NOS terminator of pCAMBIA plasmid vector were used to express PRB1 gene. In addition, we ligated 6× His tag at the end of prb1 CDS to facilitate protein purification in subsequent experiments. The following figure shows the recombinant plasmid, [prb1]-pCAMBIA1302.

Figure 2. [prb1]-pCAMBIA1302

Overproduction of recombinant plasmids

Since we needed to transfer the plasmids into Trichoderma, which would require a large number of plasmids, we transferred pCAMBIA1302 recombinant plasmids with Epl 1, Prb 1, and Snakin 1 into E. coli DH5a, to amplify them in large quantities, thus obtaining a constant and large number of plasmids.

After transforming the recombinant pCAMBIA1302 plasmid into DH5a competent cells, the recombinants were screened by the kana resistance gene on the plasmid. Subsequently, we first performed colony PCR on the isolated colonies and selected the successfully transformed isolated colonies for simple amplification with the extracted plasmids. Then we verified them by PCR and double digestion. We designed three pairs of primers with theoretical PCR fragment sizes of Epl 1-565bp, Prb 1-1388bp, and Snakin 1-425bp, respectively. The PCR results of three plasmids are shown in Figure 3, and all the selected plasmids were in expected positions, consistent with the positions of the positive control.

Figure 3: 1% agarose gel stained with Epl 1、Prb 1、Snakin 1 intergration of pCAMCIA1302 in E.coli was checked by PCR. Agarose gel was used to validate the pcr results. This figure shows PCR results of Epl 1, Prb 1, and Snakin 1, respectively. E+, P+, S+ are positive controls for Epl 1, Prb 1, and Snakin 1(positive clone is the synthetic plasmid). The capitalized word with a number represents the sample we choose.

In the double digestion verification, we used EcoRI and Bgl II enzymes to cut the plasmid into two segments, the longer segment was 9729bp, and the shorter segments of Prb 1 was 2065bp. As shown in the electrophoresis diagram of Prb 1 plasmid in Figure 4, the lower plasmid is in the superhelical state, followed by a band in the target position, which is probably the linear band of the plasmid.

Figure 4: 1% agarose gel stained with epl 1、prb 1、snakin 1 intergration of pCAMCIA1302 in E.coli was checked by enzyme digestion. (A) The results of double digestion of Epl 1 plasmid. E5-0 and E6-0 are controls that were not treated with enzymes. E5-2 and E6-2 represent the results of double digestion of E5 and E6.(B) The results of double digestion of Prb 1 plasmid. P5-0 and P6-0 are controls that were not treated with enzymes. P5-2 and P6-2 represent the results of double digestion of P5 and P6.(C) The results of double digestion of Snakin 1 plasmid. S0 is control ,S1 is the result of single enzyme digestion and S2 is double digestion of Snakin 1.

These results show that the selected separated colonies are positive and we then amplified and cultured these bacteria, and then extracted the plasmids in bulk for subsequent transformation of Trichoderma.

Genetic transformation of Trichoderma

To transfer recombinant plasmids into Trichoderma, we first tried nanomaterials-mediated transformation as well as using cell penetrating peptides to transfer the plasmids, but neither of them succeeded. After that, we tried a more traditional way protoplasted-mediated transformation. However, this CaCl2-PEG induction method didn't work. All of these methods and tries can be viewed in Protocol and Notebook. Finally, we decided to use Agrobacterium-mediated transformation (AMT).

We first transferred the three recombinant plasmids into agrobacterium GV3101 and these were screened by Kanamycin and colony PCR.

Figure 5: Agarose gel stained with epl 1、prb 1、snakin 1 integration of pCAMCIA1302 in GV3101 was checked by colony PCR. (A)E1 and E2 represent the isolated Epl 1 transformed GV3101 colony. Their were in expected positions. (B)P1 and P2 represent the isolated Prb 1 transformed GV3101 colony. Their were in expected positions. (C)S1 and s2 represent the isolated Snakin 1 transformed GV3101 colony. Their were in expected positions.

These gel results showed that the recombinant plasmids had already been transformed into agrobacterium GV3101 correctly.

Then we used positive agrobacterium GV3101 to transform T.atroviride. After several attempts and having got advice from our PI, we finally obtained the transformed T.atroviride. We selected the recombinant T.atroviride by 50ug/ml Hygromycin-B and PCR after extracting its genome. Each potential transformant was selected by 50ug/ml Hygromycin-B 4 times in case of unstable genetic inheritance caused by gene fragment inserting in cytoplasmic genome.

Figure 6: Agarose gel stained with epl 1、prb 1、snakin 1 integration of T.atroviride genome was checked by PCR. (A) 1% agarose gel stained with Prb 1 integration PCR results. P+ is positive control which is Prb 1 plasmid checked by PCR and P1 and P2 represent recombinants we chose. It is shown on the picture that P2 is consist with P+, which means P2 is positive.(B) 2% agarose gel stained with Snakin 1 integration PCR results. S+ is positive control which is Snakin 1 plasmid checked by PCR and S1 represents recombinant we chose.

According to our PCR results, we can initially confirm that we have transformed Prb 1 and Snakin 1 into T.atroviride successfully. Epl 1 transformant failed to grow up in the second time of selecting.

SDS-PAGE

After verifying that our plasmid was successfully transferred into T. atroviride, we need to further verify its expression activity in T. atroviride. We first used PRB 1 transformants as an example, Prb-1 transformants were expanded in Mini medium, and R. solani fungus powder obtained by grinding in liquid nitrogen was used as an inducer to induce Prb-1 expression in T.atroviride. After two days of culture, we extracted the whole protein of T.atroviride using a fungal protein extraction kit and verified it by SDS-PAGE. As shown in Figure 5, lane 1 is the protein extract of the Prb 1 transformant that was successfully transferred into the plasmid, lane 3 is the protein extract of the wild-type T.atroviride, and the total protein concentration was adjusted to the same for both groups of samples during loading. It can be seen that they have a relatively obvious band at the position of about 42.3kD, and from the perspective of depth, the band corresponding to the Prb-1 transformant is darker than that of the wild type, that is, the content is higher.

Figure 7: Result of SDS-PAGE (The picture on the right is the result of grayscale processing of the picture on the left to facilitate observation and comparison)

Characterization

Enzyme activity test

Prb 1 protein is a kind of serine protease, so we test the enzyme activity of wild-type T.a(WT) and engineered T.a.

Figure 8: BSA standard curve.

Through this curve, we can figure out the protein concentration of the supernate. For wild-type supernate it is 1.694125326 mg/ml and it is 1.356005222 mg/ml for Prb 1 transformant.

Then test the enzyme activity of Prb 1 by AKP Activity Assay Kit. One unit of enzyme activity（1 U) is defined as catalyze to produce 1 umol of Tyrosine per minute per mg of Prb 1 at 40 °C. The enzyme activity of wild-type(control) T.a in supernate is 5.3319 U and for Prb 1 transformant the enzyme activity has been enhanced to 9.2195U.

Figure 9: Enzyme activity of Prb 1. Wild -type represents the Prb 1 enzyme activity of wild-type T.a and it is the control group. Prb 1+ represents the Prb 1 enzyme activity of Prb 1 integrated T.a. Prb 1 activity of wild-type is 5.3319U and 9.2195U for transformed T.a. After T test, the significance between wild-type T.a and Prb 1+ is confirmed.

Inhibition test

Otherwise, we compared the inhibition effect of wild-type Trichoderma and Prb 1-engineered Trichoderma. We conducted a three-day standoff experiment, and after using the algorithm to calculate the area of the R.soalni and calculating the area according to the following formula:

We got Figure 10, the comparison of inhibition rate between wild-type T.a and Prb 1 integrated T.a. According to the graph, the Prb 1 transformant has a higher inhibition rate than wild-type T.a, which means transformant has a better inhibition ability.

Figure 10: The inhibition test of wild-type T.a and Prb 1 integreatd T.a. Wild -type represents the inhibition rate of wild-type T.a and Prb 1+ refers to the inhibition rate of Prb 1integrated T.a. After T test, the significance between wild-type T.a and Prb 1+ is confirmed.

References

Flores A, Chet I, Herrera-Estrella A. Improved biocontrol activity of Trichoderma harzianum by over-expression of the proteinase-encoding gene prb1. Curr Genet. 1997 Jan;31(1):30-7. doi: 10.1007/s002940050173.