Difference between revisions of "Part:BBa K2999007"

(→‎Usage and Biology)
(→‎Usage and Biology)
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We designed the following experiments: 1, 2 to replace the mals gene with non malS gene A (Plac + LacZ + A)；3. 4. Transform with the original plasmid and test its function (Plac + lacZ + malS)；5. Replace lac promoter with T7 promoter (T7 + malS).
 
We designed the following experiments: 1, 2 to replace the mals gene with non malS gene A (Plac + LacZ + A)；3. 4. Transform with the original plasmid and test its function (Plac + lacZ + malS)；5. Replace lac promoter with T7 promoter (T7 + malS).
  
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We (YAY-China) monitored these three experiments in the same starch medium and cultured the cells for two days.
+
We (YAU-China) monitored these three experiments in the same starch medium and cultured the cells for two days.
 
[[File:T--YAU-China--iGEM2019-BBa -13.png|500px|thumb|none|alt=fwYellow spectum.|Figure 1. ]]
 
[[File:T--YAU-China--iGEM2019-BBa -13.png|500px|thumb|none|alt=fwYellow spectum.|Figure 1. ]]
 
[[File:T--YAU-China--iGEM2019-BBa -14.png|500px|thumb|none|alt=fwYellow spectum.|Figure 2. ]]
 
[[File:T--YAU-China--iGEM2019-BBa -14.png|500px|thumb|none|alt=fwYellow spectum.|Figure 2. ]]

Revision as of 17:17, 16 October 2019


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: 1, 2 to replace the mals gene with non malS gene A (Plac + LacZ + A)；3. 4. Transform with the original plasmid and test its function (Plac + lacZ + malS)；5. 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.

fwYellow spectum.
Figure 1.
fwYellow spectum.
Figure 2.
fwYellow spectum.
Figure 3.

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

We come to the following conclusion： 1. After immersion in iodine solution, the color of 1 and 2 stripes was not dark (3, 4, 5) with 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]