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Team:NEAU-Harbin/design.html
From 2014.igem.org
1.Establishment of visual gene operation system
First, two reporter genes AmilCP (coding blue chromoprotein) and cjBlue (coding green chromoprotein) were introduced into the expression vector pSZH. AmilCP was driven by GlaA5 promoter and terminated by GlaA3, the fusion gene of Hph and cjBlue was driven by pgpdA promoter and terminated by GlaA3 (Figure 1). The resulted construct was transformed into A. niger cell then these two chromoprotein genes will be inserted into the Gla site through homologous recombination. For the transgenic A. niger cell, both blue and cyan color should be observed.
Because two GlaA3 sequences respectively located at flanking sites of Hph-cjBlue, there is the possibility to delete Hph-cjBlue through homologous recombination. The transformants without selective marker Hph-cjBlue can be easily screened out due to the lack of cyan color.
The blue color suggested that exogenous genes can be highly expressed and losing of cyan color showed the selective marker gene can be deleted, which demonstrate that our visual gene operation system is effective.
Figure 1
2.Single target gene
Based on the previous construct, a new construct with amilcp replaced by the target gene should be made (Figure 2). The cassette will include one target gene driven by GlaA5 and terminated by GlaA3, and Hph-cjBlue driven by pgpdA promoter and terminated by GlaA3. When the target gene is homologous recombined into the amilcp site, the blue color will disappear and cyan color will be observed. Then these target transformants will be easily picked out by the color of colonies without PCR or other molecular detection methods. Based on the same mechanism, after several generations, transformants without the selective marker Hph-cjBlue will be accurately screened out due to disappear of the cyan color. These target gene transformants without selective marker gene can also been used as an original strain for transformation of another target gene.
Figure 2
3.More genes, more sites
There are many other high expression sites can be used for homologous recombination in A. niger genome in addition to Gla site. Take Amy site for example, homology arm sequences of Amy can be designed as AmyA5 and AmyA3 (Figure 3). When we want to express another target gene into A. niger, similar construct can be made as follows: the second target gene was driven by AmyA5 and terminated by AmyA3, and selective marker gene Hph-cjBlue was driven by pgpdA promoter and terminated by AmyA3. The similar homologous recombination process will happen and result in the expression of the second target gene with selective marker gene deletion, which can be traced by cyan color disappearing. In a word, more genes can be introduced into A. niger using our visual system, which makes our system continuous.
Figure 3
