Difference between revisions of "Part:BBa K523006"

(Iodine assay)
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====Iodine assay====
 
====Iodine assay====
  
We (Edinburgh 2011) placed this part under the control of the lac promoter (creating <partinfo>BBa_K523006</partinfo>) and streaked colonies on a starch agar plate. A negative control without this construct was also streaked out. The cells were incubated for 3 days.
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We (Edinburgh 2011) placed <partinfo>BBa_K523001</partinfo> under the control of the lac promoter (creating <partinfo>BBa_K523006</partinfo>) and streaked colonies on a starch agar plate. A negative control without this construct was also streaked out. The cells were incubated for 3 days.
  
 
One colony failed to grow for some reason, but the others did. We flooded the plate with iodine, which turns black in the presence of starch:
 
One colony failed to grow for some reason, but the others did. We flooded the plate with iodine, which turns black in the presence of starch:

Revision as of 22:12, 12 September 2011

Plac + LacZ + malS

E. coli periplasmic alpha-amylase gene malS under control of lac promoter. LacZ is also present for blue/white screening.

Iodine assay

We (Edinburgh 2011) placed BBa_K523001 under the control of the lac promoter (creating BBa_K523006) and streaked colonies on a starch agar plate. A negative control without this construct was also streaked out. The cells were incubated for 3 days.

One colony failed to grow for some reason, but the others did. We flooded the plate with iodine, which turns black in the presence of starch:

523001-assay-pre2.jpg     523001-assay-post.jpg     523001-assay-post-post2.jpg
Before the assay. Colony 1 failed to grow. Control at top.     Immediately after iodine flooding.     40 minutes after iodine flooding.

We made three observations:

  1. The area under the negative control (top streak) turned black, while the areas under the malS streaks did not.
  2. The iodine gradually evaporated, turning the plate clear again.
  3. After 30 minutes, halos were seen around the two malS streaks.

We can think of two ways to explain observation 1:

  • malS degraded starch, or:
  • the control cells grew slower than the malS cells for some reason, and thus formed a thinner layer; iodine could pass through that layer to turn the starch underneath black, but could not pass through the thicker malS layer.

However, observation 3, the halos, seem to rule out this second explanation, and instead suggest actual diffusion of a starch-degrading enzyme (MalS) into the agar.

Mucoid phenotype

The plate was allowed to rest at room temperature (in other words: it was left on a table and forgotten about). Some days after the iodine assay, we noticed a slimy mucoid phenotype developing on the malS streaks, but not the control streak. This was also true of colonies on a similar plate that had also been assayed with iodine (this plate is not mentioned above due to the lack of a control).

523001-mucoid.jpg     523001-mucoid-original.jpg
3 days after iodine assay     (Different plate) 6 days after iodine assay

We have not yet confirmed whether such a phenotype arises without iodine treatment.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1841
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 607
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 977
    Illegal AgeI site found at 2142
  • 1000
    COMPATIBLE WITH RFC[1000]