Composite

Part:BBa_K2999007

Designed by: Xin Ma   Group: iGEM19_YAU-China   (2019-10-16)


T7+malS

We (YAU-China) replaced Plac + lacZ in Plac+ lacZ + malS with T7 promoter to enhance the expression of starch hydrolase.


Usage and Biology

This year, we improved Plac + lacZ + malS (BBa_K523006), which can produce starch hydrolase that can degrade starch. We used T7 promoter to replace lac promoter sequence on the protoplast, hoping to enhance the expression of starch hydrolase.

We designed the following experiments: The first line and the second line: Replacing malS gene with non malS gene PA1908 (Plac +lacZ +PA1908)；The third line and the fourth line: Transform with the original plasmid and test its function (Plac + lacZ + malS)；The fifth line: replace lac promoter with T7 promoter (T7 + malS).

We (YAU-China) monitored these three experiments in the same starch medium and cultured the cells for two days.

It shows the growth of cells in starch medium after two days.
Figure 1.
It is the result of immediating the cells with iodine solution, which turns black immediately in case of starch.
Figure 2.
It shows the result of iodine volatilization for 60 min.
Figure 3.

Figure. 1 ：It shows the growth of cells in starch medium after two days； Figure. 2 ：It is the result of immediating the cells with iodine solution, which turns black immediately in case of starch； Figure. 3 ：It shows the result of iodine volatilization for 60 min.

We come to the following conclusion： 1. When the culture medium was immersed in iodine solution, the color of the first and second stripes was lighter than that of the third, fourth and fifth stripes (They have malS gene); 2. As time goes on, the medium becomes transparent after iodine volatilization; 3. After 60 minutes, halo appeared around 3, 4 and 5 stripes, and the halo size of 5 stripes was larger than that of 3 and 4 stripes.

Therefore, when the lac promoter was replaced by T7 promoter, the expression of starch hydrolase was enhanced.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 60
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1339
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 105
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 60
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 60
    Illegal AgeI site found at 475
    Illegal AgeI site found at 1640
  • 1000
    COMPATIBLE WITH RFC[1000]


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