Part:BBa_K4286206

PCaMV-epl1-6xHis-TNOS

EPL1 is a kind of type II hydrophobin, which is mostly located on the outer surface of the cell wall and is involved in cell-cell or cell-surface contacts. EPL1 binds to various forms of chitin, but not to cellulose. Meanwhile, EPL1 belongs to the cerato-platanin protein family, which is only found in filamentous fungi and is involved in the induction of plant defense responses, but the molecular mechanisms involved are still largely unknown. Trichoderma atroviride has three genes that encode cerato-platanin proteins: EPL1, EPL2, and EPL3. The expression of epl1 gene mainly occurred in the process of mycelial growth and mycelial development, while the expression of epl2 occurred in the process of conidia, and epl3 was almost not expressed. Study shows that EPL1 can easily self-assemble and form a protein layer at the water interface, and the protein layer can be redissolved in water after stirring. The gene circuit of epl1 is consist of CamV 35s promoter, epl1 gene, 6xHis affinity tag, NOS terminator.

Figure 1. The expression of Epl1 by engineered Trichoderma atroviride

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 66
  

Assembly

Plasmid construction

Through homologous recombination, the coding sequence of EPL1 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 epl1 gene. In addition, we ligated 6× His tag at the end of epl1 CDS to facilitate protein purification in subsequent experiments. The following figure shows the recombinant plasmid, [epl1]-pCAMBIA1302.

Figure 2. [epl1]-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.

References

[1]Frischmann A, Neudl S, Gaderer R, Bonazza K, Zach S, Gruber S, Spadiut O, Friedbacher G, Grothe H, Seidl-Seiboth V. Self-assembly at air/water interfaces and carbohydrate binding properties of the small secreted protein EPL1 from the fungus Trichoderma atroviride. J Biol Chem. 2013 Feb 8;288(6):4278-87. doi: 10.1074/jbc.M112.427633. Epub 2012 Dec 17.

[2]Guzmán-Guzmán P, Alemán-Duarte MI, Delaye L, Herrera-Estrella A, Olmedo-Monfil V. Identification of effector-like proteins in Trichoderma spp. and role of a hydrophobin in the plant-fungus interaction and mycoparasitism. BMC Genet. 2017 Feb 15;18(1):16. doi: 10.1186/s12863-017-0481-y.

[3]Salas-Marina MA, Isordia-Jasso MI, Islas-Osuna MA, Delgado-Sánchez P, Jiménez-Bremont JF, Rodríguez-Kessler M, Rosales-Saavedra MT, Herrera-Estrella A, Casas-Flores S. The Epl1 and Sm1 proteins from Trichoderma atroviride and Trichoderma virens differentially modulate systemic disease resistance against different life style pathogens in Solanum lycopersicum. Front Plant Sci. 2015 Feb 23;6:77. doi: 10.3389/fpls.2015.00077.