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.