Difference between revisions of "Part:BBa K523006"

Revision as of 10:42, 16 September 2011

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

Streaks of E. coli with this part show a mucoid phenotype. This was first spotted on plates that had been subjected to iodine treatment, so we made new plates that were never subjected to iodine. These too show the effect:

Mucoid phenotype.		Same plate, held up to the light.

(A negative control failed on this plate due to a lack of chloramphenicol resistance...)

Sequence and Features