Part:BBa_K523001

RBS + malS (E. coli periplasmic α-amylase)

This is the E. coli amylase gene malS. The part contains the native Ribosome Binding Site.

The part was made using the strategy outlined in BBa_K523000, and therefore contains 4 extra bases at the 5' end which generate a BglII restriction site.

Usage and Biology

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.

Characterisation

Iodine assay

We (Edinburgh 2011) placed this part 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:

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).

3 days after iodine assay		(Different plate) 6 days after iodine assay

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1233
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 369
  Illegal AgeI site found at 1534
  
• 1000
  COMPATIBLE WITH RFC[1000]