Difference between revisions of "Part:BBa K523001"

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<partinfo>BBa_K523001 short</partinfo>
 
<partinfo>BBa_K523001 short</partinfo>
  
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This is the ''E. coli'' amylase gene ''malS''. The part contains the native Ribosome Binding Site.
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''E. coli'' periplasmic &alpha;-amylase gene ''malS'' (see [http://www.ncbi.nlm.nih.gov/nuccore/48994873?from=3735520&to=3737550&report=gbwithparts info from GenBank: U00096.2]).
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The native ribosome binding site is present.
  
 
The part was made using the strategy outlined in <partinfo>BBa_K523000</partinfo>, and therefore contains 4 extra bases at the 5' end which generate a BglII restriction site.
 
The part was made using the strategy outlined in <partinfo>BBa_K523000</partinfo>, and therefore contains 4 extra bases at the 5' end which generate a BglII restriction site.
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===Usage and Biology===
 
===Usage and Biology===
  
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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.
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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: [http://dx.doi.org/10.1128/JB.00767-08 Lengsfeld ''et al'' (2008)] state that "MalS produces preferentially maltohexaose from longer maltodextrins in the periplasm".
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Other interesting and useful facts about the MalS protein can be found in [http://dx.doi.org/10.1074/jbc.272.35.22125 Spiess ''et al'' (1997)].
  
 
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.
 
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.
  
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===Characterisation===
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<font color="red">Edinburgh 2011 carried out some experiments on this part under the control of the lac promoter - see details at part <partinfo>BBa_K523006</partinfo>.</font>
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====Iodine assay====
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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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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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<center>
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{| style="margin-top: 1em; margin-bottom: 1em;"
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|-
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| [[Image:523001-assay-pre2.jpg|200px]]
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| &nbsp; &nbsp;
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| [[Image:523001-assay-post.jpg|200px]]
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| &nbsp; &nbsp;
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| [[Image:523001-assay-post-post2.jpg|200px]]
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|-
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| width="200px" valign="top" | Before the assay. Colony 1 failed to grow. '''Control at top.'''
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| &nbsp; &nbsp;
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| width="200px" valign="top" | Immediately after iodine flooding.
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| &nbsp; &nbsp;
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| width="200px" valign="top" | 40 minutes after iodine flooding.
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|}
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</center>
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We made three observations:
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# The area under the negative control (top streak) turned black, while the areas under the ''malS'' streaks did not.
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# The iodine gradually evaporated, turning the plate clear again.
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# After 30 minutes, halos were seen around the two ''malS'' streaks.
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We can think of two ways to explain observation 1:
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* ''malS'' degraded starch, or:
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* 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.
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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.
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====Mucoid phenotype====
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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).
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<center>
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{| style="margin-top: 1em; margin-bottom: 1em;"
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|-
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|[[Image:523001-mucoid.jpg|200px]]
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|&nbsp; &nbsp;
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|[[Image:523001-mucoid-original.jpg|200px]]
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|-
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|width="200px" valign="top" | 3 days after iodine assay
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|&nbsp; &nbsp;
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|width="200px" valign="top" | (Different plate) 6 days after iodine assay
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|}
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</center>
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We have not yet confirmed whether such a phenotype arises without iodine treatment.
 
  
 
<span class='h3bb'>'''Sequence and Features'''</span>
 
<span class='h3bb'>'''Sequence and Features'''</span>

Latest revision as of 19:53, 18 September 2011

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

E. coli periplasmic α-amylase gene malS (see [http://www.ncbi.nlm.nih.gov/nuccore/48994873?from=3735520&to=3737550&report=gbwithparts info from GenBank: U00096.2]).

The native ribosome binding site is present.

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

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: [http://dx.doi.org/10.1128/JB.00767-08 Lengsfeld et al (2008)] state that "MalS produces preferentially maltohexaose from longer maltodextrins in the periplasm".

Other interesting and useful facts about the MalS protein can be found in [http://dx.doi.org/10.1074/jbc.272.35.22125 Spiess et al (1997)].

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.

Edinburgh 2011 carried out some experiments on this part under the control of the lac promoter - see details at part BBa_K523006.


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]