Difference between revisions of "Part:BBa K731040"
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A sfGFP tagged M256I CysE inducible by arabinose. This part was subcloned and characterized in pSB3C5.
 
A sfGFP tagged M256I CysE inducible by arabinose. This part was subcloned and characterized in pSB3C5.
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 +
This part is also available in pSB3C5. Please contact igemtrento[at]gmail.dot.com if you are interested in this part.
  
  
 
===Usage and Biology===
 
===Usage and Biology===
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CysE is a serine acetyltransferase that mediates the production of cysteine. More specifically, CysE catalyses the activation of L-serine by acetyl-CoA. Its product, 0-acetyl-L-serine (OAS), is then subsequently converted to L-cysteine by 0-acetyl-L-serine(thio1)lyase [1].
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Normally,the catalytic activity of CysE is sensitive to feedback inhibition by L-cysteine, but this is a mutated version (M256I) that have a 10-fold decrease in feedback inhibition by cysteine itself [2].
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<div style="text-align:center">[[Image:1040_1.jpg]]</div>
 
<div style="text-align:center">[[Image:1040_1.jpg]]</div>
 
<html><p style="width:600px; margin-left:150px; margin-bottom:60px;
 
<html><p style="width:600px; margin-left:150px; margin-bottom:60px;
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text-align:justify "><em><strong>FIGURE 1. CysE-sfGFP expression with different concentrations of arabinose, after 4 hours of induction.</strong><br/>Test was conducted in NEB10beta strain, with part BBa_K731240, but in pSB3C5. Cells were grown in LB brothuntil OD_600= 0.3 then centrifuged and resuspended in equal amount of minimal MOPS A medium (with 60mM glycerol as carbon source) and induced at OD_600 = 0.6 with 5mM arabinose concentration. Higher CysE-sfGFP expression is observed as the arabinose concentration increases. However, MOPS medium seem to negatively influence induction, delaying promoter response to higher arabinose concentration. No difference in fluorescence intensity between not induced and control NEB10beta cells was observed.</p></html>
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text-align:justify "><em><strong>FIGURE 1. CysE-sfGFP expression with different concentrations of arabinose, after 4 hours of induction.</strong><br/>NEB10beta cells expressing BBa_K731040 in pSB3C5 were grown in LB broth until OD_600= 0.3 then centrifuged and resuspended in equal amount of minimal MOPS A medium (with 60mM glycerol as carbon source) and induced at OD_600 = 0.6 with arabinose.</p></html>
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<div style="text-align:center">[[Image:1040_2.jpg]]</div>
 
<div style="text-align:center">[[Image:1040_2.jpg]]</div>
 
<html><p style="width:600px; margin-left:150px; margin-bottom:60px;
 
<html><p style="width:600px; margin-left:150px; margin-bottom:60px;
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text-align:justify "><em><strong>FIGURE 2. CysE-sfGFP expression with different concentrations of arabinose in presence or absence of glucose.</strong><br/>This chart shows the difference in expression levels when using glucose or glycerol as carbon source. Expression levels in MOPS 30mM glucose with 5mM arabinose are very similar to levels in MOPS 60mM glycerol and 0.1mM arabinose.</p></html>
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text-align:justify "><em><strong>FIGURE 2. CysE-sfGFP expression with different concentrations of arabinose in presence or absence of glucose.</strong><br/>For this test we used NEB10beta cells, expressing part BBa_K731040 in pSB3C5. The data shows the difference in expression levels when using glucose or glycerol as carbon source. MOPS A has 60mM glycerol as carbon source, MOPS B has 30mM glucose. Data relative to MOPS A are shown in orange, the ones relative MOPS B are in blue. Expression levels in MOPS B with 5mM arabinose are very similar to levels in MOPS A with 0.1mM arabinose.</p></html>
  
 
<div style="text-align:center">[[Image:1040_3.jpg]]</div>
 
<div style="text-align:center">[[Image:1040_3.jpg]]</div>
 
<html><p style="width:600px; margin-left:150px; margin-bottom:60px;
 
<html><p style="width:600px; margin-left:150px; margin-bottom:60px;
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text-align:justify "><em><strong>FIGURE 3. CysE-sfGFP expression over time, after induction.</strong><br/>Test was conducted in DH10beta strain, with part BBa_K731250, but in pSB3C5. Cells were grown in LB brothuntil OD_600= 0.3 then centrifuged and resuspended in equal amount of minimal MOPS medium and induced at OD_600 = 0.6 with 5mM arabinose concentration. MOPS A is 60mM glycerol as carbon source, MOPS B is 30mM glucose: in the chart, data relative to MOPS A are shown in orange, the ones relative MOPS B are in blue. In absence of glucose, increasing linear expression over time of GFP is observed, while in MOPS B, in presence of glucose, expression levels are much lower. </p></html>
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text-align:justify "><em><strong>FIGURE 3. CysE-sfGFP expression over time, after induction.</strong><br/>The test was conducted in NEB10beta strain, with part BBa_K731040 in pSB3C5. Cells were grown in LB broth until OD_600= 0.3 then centrifuged and resuspended in equal amount of minimal MOPS media (A or B, in orange and blue respictively) and induced at OD_600 = 0.6 with 5mM arabinose.In absence of glucose, increasing linear expression over time of GFP is observed, while in MOPS B, in presence of glucose, expression levels are much lower. </p></html>
  
 
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<partinfo>BBa_K731040 parameters</partinfo>
 
<partinfo>BBa_K731040 parameters</partinfo>
 
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===References===
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[1] Jones-Mortimer, M.C. (1968). The control of sulphate reduction in Escherichia coli by O-acetyl-l-serine. Biochem. J. 106, 33-36. <br>
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[2] Kredich & Tomkins, (1966). The enzymic synthesis of L-cysteine in Escherichia coli and Salmonella typhimurium. Biochem. J. 241, 4955-4965. <br>

Latest revision as of 21:22, 26 October 2012

Inducible araC-pBAD promoter regulating a sfGFP-tagged M256I CysE

A sfGFP tagged M256I CysE inducible by arabinose. This part was subcloned and characterized in pSB3C5.

This part is also available in pSB3C5. Please contact igemtrento[at]gmail.dot.com if you are interested in this part.


Usage and Biology

CysE is a serine acetyltransferase that mediates the production of cysteine. More specifically, CysE catalyses the activation of L-serine by acetyl-CoA. Its product, 0-acetyl-L-serine (OAS), is then subsequently converted to L-cysteine by 0-acetyl-L-serine(thio1)lyase [1]. Normally,the catalytic activity of CysE is sensitive to feedback inhibition by L-cysteine, but this is a mutated version (M256I) that have a 10-fold decrease in feedback inhibition by cysteine itself [2].


1040 1.jpg

FIGURE 1. CysE-sfGFP expression with different concentrations of arabinose, after 4 hours of induction.
NEB10beta cells expressing BBa_K731040 in pSB3C5 were grown in LB broth until OD_600= 0.3 then centrifuged and resuspended in equal amount of minimal MOPS A medium (with 60mM glycerol as carbon source) and induced at OD_600 = 0.6 with arabinose.


1040 2.jpg

FIGURE 2. CysE-sfGFP expression with different concentrations of arabinose in presence or absence of glucose.
For this test we used NEB10beta cells, expressing part BBa_K731040 in pSB3C5. The data shows the difference in expression levels when using glucose or glycerol as carbon source. MOPS A has 60mM glycerol as carbon source, MOPS B has 30mM glucose. Data relative to MOPS A are shown in orange, the ones relative MOPS B are in blue. Expression levels in MOPS B with 5mM arabinose are very similar to levels in MOPS A with 0.1mM arabinose.

1040 3.jpg

FIGURE 3. CysE-sfGFP expression over time, after induction.
The test was conducted in NEB10beta strain, with part BBa_K731040 in pSB3C5. Cells were grown in LB broth until OD_600= 0.3 then centrifuged and resuspended in equal amount of minimal MOPS media (A or B, in orange and blue respictively) and induced at OD_600 = 0.6 with 5mM arabinose.In absence of glucose, increasing linear expression over time of GFP is observed, while in MOPS B, in presence of glucose, expression levels are much lower.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
    Illegal BamHI site found at 2045
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
    Illegal AgeI site found at 1909
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961
    Illegal SapI.rc site found at 2075


References

[1] Jones-Mortimer, M.C. (1968). The control of sulphate reduction in Escherichia coli by O-acetyl-l-serine. Biochem. J. 106, 33-36.
[2] Kredich & Tomkins, (1966). The enzymic synthesis of L-cysteine in Escherichia coli and Salmonella typhimurium. Biochem. J. 241, 4955-4965.