Part:BBa_K523006

Plac + LacZ + malS

E. coli periplasmic α-amylase gene malS under control of lac promoter. LacZα is also present.

Usage and Biology

While an amylase ought to be capable of degrading starch, the product protein is believed to be periplasmic and thus ought to only degrade starch if it leaks from the periplasm in significant quantities. Its natural function in E. coli presumably involves degrading shorter glucose chains.

The SignalP program predicts that a 17 amino acid localisation signal (at the N terminal) is cleaved off before the protein reaches its mature form.

Iodine assay #1

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 (poorly characterised) 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:

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.

Iodine assay #2

We repeated the iodine assay with a known control, PlacLacZ-bglX (part BBa_K523014). This part is a good control since it has both Plac-LacZ (like this part) and a periplasmic protein (like this part), and is known to actually work.

(Another control, using only Plac-LacZ, failed to grow because the source cells had been in the cold room too long.)

More to follow.

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).

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]