Part:BBa_K4286907
Suicide switch (Pcbh1-cbh1ls-mazEF-Tcbh1) for Trichoderma spp
The another suicide switch for Trichoderma atroviride is condition-triggered. cbh1 promoter, which was first discovered in Trichoderma reesei, is one of the strongest inducible promoters in the fungal kingdom. It is often used to construct expression vectors. The cbh1 promoter sequence contains cellulose and sophorose inducible binding sites and glucose feedback inhibitory binding sites. These sites are in different positions, which provide multiple possibilities for us to regulate the expression of genes downstream of the promoter. We selected a known 1360bp cbh1 promoter sequence from the cbh1 gene of Trichoderma koningii, retaining the inducible regulatory site of polysaccharide and glucose. We also retained the 17bp leader sequence between the cbh1 promoter and cbh1 gene. Correspondingly, we used a 790bp cbh1 terminator from Trichoderma reesei in the circuit of our Trichoderma suicide switch.
The Pcbh1-cbh1ls-mazEF-Tcbh1 circuit was integrated into pCAMBIA1302 vector by homologous recombination. We transformed the recombinant plasmid into Trichoderma atroviride by Agrobacterium-mediated transformation in order to achieve condition-triggered suicide of T. atroviride ultimately.
- In the lab and inside the wrapping materials, we cultured T. atroviride under glucose-rich conditions. Glucose within high concentration inhibited the cbh1 operon, rendering this suicide pathway unexpressed. - When T. atroviride are released to the field and glucose concentration gradually reduces in the the wrapping materials and surrounding environment, the inhibitory effect of glucose on the suicide switch reduces correspondingly. At the same time, the utilization of carbon sources by T. atroviride converts to many kinds of saccharides such as cellulose and sophorose. These saccharides induced the expression of cbh1 operon, leading to the suicide of T. atroviride.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1006
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2663
Illegal AgeI site found at 2007
Illegal AgeI site found at 2707 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2644
Illegal BsaI.rc site found at 2510
Illegal BsaI.rc site found at 2544
Illegal SapI site found at 2301
Plasmid production and preliminary validation
We constructed the recombinant vector [Pcbh1-cbh1ls-mazEF-Tcbh1]-pCAMBIA1302. In order to produce the recombinant vector in larger scale for subsequent experiments, we transferred the recombinant vector into E.coli DH5α. Transformants were clearly visible on the culture medium after 16 hours of incubation at 37℃.
We selected 8 single colonies on the culture medium and carried out colony PCR for plasmid amplification. The theoretical length of the amplified product was 4019bp. Electrophoresis was performed in a 1% agarose gel. The results showed that all the colonies were positive transformants, which indicated that the recombinant vector was successfully transformed.
We further cultured the transformants and extracted the plasmids from them. Restriction enzyme XhoI was used for single digestion. There are two restriction sites of XhoI on the plasmid [Pcbh1-cbh1ls-mazEF-Tcbh1]-pCAMBIA1302, the theoretical sizes of bands after digestion are 1094bp and 10268bp. Electrophoresis was performed in a 1% agarose gel. The results showed successful single-enzyme digestion and correct plasmid extraction. However, a band of 1094bp size was not shown because a few plasmids were simultaneously cleaved twice by the enzyme Xhol.
Genetic transformation of Trichoderma atroviride
To transfer recombinant plasmids into Trichoderma, we used Agrobacterium-mediated transformation (AMT) to integrate the circuit of Trichoderma suicide switch (on T-DNA fragment of plasmid pCAMBIA1302) into the genome of Trichoderma atroviride. Then we used positive Agrobacterium GV3101 to transform T.atroviride. We selected the recombinant T.atroviride by 50ug/ml Hygromycin B and PCR after extracting its genome. Unfortunately, due to lack of time, we did not carry out the subsequent characterization experiments.
Reference
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[5]Zhong Y, Liu X, Xiao P, Wei S, Wang T. Expression and secretion of the human erythropoietin using an optimized cbh1 promoter and the native CBH I signal sequence in the industrial fungus Trichoderma reesei. Appl Biochem Biotechnol. 2011 Nov;165(5-6):1169-77. doi: 10.1007/s12010-011-9334-8. Epub 2011 Aug 16.
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