Team:NEAU-Harbin/design.html

• Background
• Design
• Application
• Dry lab

1.Establishment of visual gene operation system

First, two reporter genes AmilCP (coding purple fluorescent protein) and cjBlue (coding blue fluorescent protein) 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 fluorescent protein genes will be inserted into the Gla site through homologous recombination. For the transgenic A. niger cell, both purple and blue fluorescence 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 with selective marker Hph-cjBlue deletion can be easily screened out due to the lack of blue fluorescence.

The purple fluorescence suggested that exogenous genes can be highly expressed and losing of blue fluorescence showed the selective marker gene can be deleted, which demonstrate that our visual gene operation system is effective.

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 purple fluorescence will disappear and blue fluorescence will be observed again. Then these target transformants will be easily picked out by the fluorescence of colonies without PCR or other molecular detection method. Based on the same mechanism, after several generations, transformants without the selective marker Hph-cjBlue will be accurately screened out by observing disappear of the blue fluorescence. These target gene transformants without any selective marker gene can also been used as an original strain for transformation of another target gene.

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). Based on these previous mechanisms, 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 AmyA 3. 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 blue fluorescence appearance and then disappearance. In a word, we can introduce more genes into more high expression sites in A. niger using our visual system, which make our system continuous.