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:
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| Before the assay. Colony 1 failed to grow. Control at top. | Immediately after iodine flooding. | 40 minutes after iodine flooding. |
We made three observations:
- The area under the negative control (top streak) turned black, while the areas under the malS streaks did not.
- The iodine gradually evaporated, turning the plate clear again.
- After 40 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
The precise nature of the negative control above has been lost in the mists of time (it was JM109 E. coli with an unknown construct in pSB1C3).
We (Edinburgh 2011) 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.)
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| Control at top. Bottom two streaks are malS. | Same plate, time = 25 minutes. | Same plate, time = 70 minutes. While the iodine has faded from all the bacteria, halos are now visible around the malS streaks but not the control. |
DNS assay
We (Edinburgh 2011) made a cell extract using this part and compared it to a negative control (BBa_K523000) in the following way:
Cells were sonicated and fractionated to obtain cell lysate and cell debris. 0.2% starch solution and phosphate buffered saline (PBS) were mixed with cell extract and incubated at 37 C. Every 30 minutes, a sample was taken from the reaction and 3,5-dinitrosalicylic acid (DNS) was added. The sample was heated in boiling water for 10 minutes. The reaction was then stopped by addition of potassium tartrate. The sample was cooled to room temperature and OD575 was measured.
If the cell extract is capable of starch degradation, this will cause the liberated glucose to react with the DNS, increasing the OD575 reading.
Mucoid phenotype
Streaks of E. coli with this part, and grown on starch agar, eventually show a mucoid phenotype so pronounced they were described as "disgusting" by a hardened microbiologist. This phenotype is only visible after several days, and was first spotted on plates that had already been subjected to iodine treatment, so we (Edinburgh 2011) checked plates that were never subjected to iodine. These too show the effect:
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| Starch agar. Mucoid (slimy) phenotype. All colonies are malS. | Same plate, held up to the light. Control failed to grow because it wasn't chloramphenicol resistant. Oops. |
Future experiments
Final controls ought to be run testing:
- Whether this part displays the mucoid phenotype on normal agar.
- Whether normal E. coli displays a mucoid phenotype on starch agar (this is being done...)
Discussion
There is clear evidence that malS, when expressed from a high copy number plasmid, is capable of at least mild starch degradation.
The mucoid phenotype was unexpected. It is possible that starch degradation has led to a high supply of glucose, leading to extra exopolysaccharide being produced.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1841
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 607
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 977
Illegal AgeI site found at 2142 - 1000COMPATIBLE WITH RFC[1000]
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