Difference between revisions of "Part:BBa K2924021"
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[[File:PET21_ACC.png|500px|thumb|right|<i> <b>Fig. 1:</b> AccABCD in its order inside the pet21a backbone. Strong T7 Promotor and lac-O operon in front of it. The genes are followed by 6xHis-tag and the terminator. </i> ]]
 
[[File:PET21_ACC.png|500px|thumb|right|<i> <b>Fig. 1:</b> AccABCD in its order inside the pet21a backbone. Strong T7 Promotor and lac-O operon in front of it. The genes are followed by 6xHis-tag and the terminator. </i> ]]
 
[[File:ACCASE_Reaction.png|500px|thumb|right|<i><b>Fig. 2:</b> ACC-catalyzed formation of Malonyl-CoA from bicarbonate and Acetyl-CoA under usage of ATP</i>]]
 
[[File:ACCASE_Reaction.png|500px|thumb|right|<i><b>Fig. 2:</b> ACC-catalyzed formation of Malonyl-CoA from bicarbonate and Acetyl-CoA under usage of ATP</i>]]
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[[File:ACCase_pathway.png|500px|thumb|center|<i><b>Fig.3:</b> Bacterial fatty acid biosynthesis pathway in <i>E. coli</i>. AccABCD is highlighted and represents all parts involved in the ACC. Modified from Christoph Freiberg et al., 2014 Fig.1</i>]]
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ACC is allosterically inhibited by the accumulated fatty acyl-CoAs<sup>1</sup>. ACC catalyzes the first committed step in fatty acid biosynthesis - the ATP-dependent formation of malonyl-CoA from acetyl-CoA and bicarbonate (HCO<sub>3</sub><sup>-</sub>)<sup>2</sup> [Fig. 3].  
 
ACC is allosterically inhibited by the accumulated fatty acyl-CoAs<sup>1</sup>. ACC catalyzes the first committed step in fatty acid biosynthesis - the ATP-dependent formation of malonyl-CoA from acetyl-CoA and bicarbonate (HCO<sub>3</sub><sup>-</sub>)<sup>2</sup> [Fig. 3].  
 
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Malonyl-CoA further binds to the acyl carrier protein and forms Malonyl-ACP, an important precursor in the fatty acid synthesis. Fatty acids are carboxylic acids and have a chain length of 4 to 36 carbon atoms<sup>3</sup>. The genes were replicated and overexpressed in <i>E. coli</i> BL21 to increase carbon flux towards the precursor malonyl-CoA. An enhanced cellular concentration of malonyl-CoA contributes to increased production of malonyl-CoA and promotes the derived compounds, e.g. like fatty acids.  
 
Malonyl-CoA further binds to the acyl carrier protein and forms Malonyl-ACP, an important precursor in the fatty acid synthesis. Fatty acids are carboxylic acids and have a chain length of 4 to 36 carbon atoms<sup>3</sup>. The genes were replicated and overexpressed in <i>E. coli</i> BL21 to increase carbon flux towards the precursor malonyl-CoA. An enhanced cellular concentration of malonyl-CoA contributes to increased production of malonyl-CoA and promotes the derived compounds, e.g. like fatty acids.  
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[[File:ACCase_pathway.png|500px|thumb|center|<i><b>Fig.3:</b> Bacterial fatty acid biosynthesis pathway in <i>E. coli</i>. AccABCD is highlighted and represents all parts involved in the ACC. Modified from Christoph Freiberg et al., 2014 Fig.1</i>]]
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The <i>acc</i> genes were cloned in an operon-like manner to ensure transcription of all genes of the complex at the same level and from the same vector guided by the same conditions [Fig. 1].
 
The <i>acc</i> genes were cloned in an operon-like manner to ensure transcription of all genes of the complex at the same level and from the same vector guided by the same conditions [Fig. 1].
 
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[[File:Fatty_acid_profile_coli.png|400px|thumb|left|<i><b>Fig.4:</b> Gas chromatographic analysis of the fatty acid profile of modified E.coli strains</i>]]
 
[[File:Fatty_acid_profile_coli.png|400px|thumb|left|<i><b>Fig.4:</b> Gas chromatographic analysis of the fatty acid profile of modified E.coli strains</i>]]
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[[File:FA_quantification_coli.png|400px|thumb|right|<i><b>Fig.5:</b> Comparison of modified <i>E. coli</i> strains containing the fatty acid biosensor and analysed by C18:0 gas chromatography.</i>]]
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[[File:FA_quantification_coli.png|450px|thumb|right|<i><b>Fig.5:</b> Comparison of modified <i>E. coli</i> strains containing the fatty acid biosensor and analysed by C18:0 gas chromatography.</i>]]
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The rise in absolute fatty acid amount of ACC and tesA modified <i>E. coli</i> strains in comparison to Wild Type and HP(thioesterase of <i>Haematococcus pluvialis</i>) is not significant.
 
The rise in absolute fatty acid amount of ACC and tesA modified <i>E. coli</i> strains in comparison to Wild Type and HP(thioesterase of <i>Haematococcus pluvialis</i>) is not significant.
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Value of ACC with biosensor and gas chromatography for C18:0 do not vary significantly.
 
Value of ACC with biosensor and gas chromatography for C18:0 do not vary significantly.

Revision as of 21:58, 19 October 2019


accABCD

The composite part AccABCD is a protein complex of four subunits, coded in one transcription unit. It catalyzes the conversion of Acetyl-CoA to Malonyl-CoA.


Usage and Biology

The composite part AccABCD is used to overexpress the heteromeric protein complex of Acetyl-CoA carboxylase (ACC) from Escherichia coli (strain K12), involved in fatty acid biosynthesis. Cloned into an IPTG-inducible vector for expression (pET21a, Novagen), containing strong T7 promoter and carrying an ampicillin resistance and a C-terminal histidine tag.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 668
    Illegal AgeI site found at 1501
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 606
    Illegal SapI.rc site found at 803



Characterization

Fig. 1: AccABCD in its order inside the pet21a backbone. Strong T7 Promotor and lac-O operon in front of it. The genes are followed by 6xHis-tag and the terminator.
Fig. 2: ACC-catalyzed formation of Malonyl-CoA from bicarbonate and Acetyl-CoA under usage of ATP

ACC is allosterically inhibited by the accumulated fatty acyl-CoAs1. ACC catalyzes the first committed step in fatty acid biosynthesis - the ATP-dependent formation of malonyl-CoA from acetyl-CoA and bicarbonate (HCO3-)2 [Fig. 3].

Malonyl-CoA further binds to the acyl carrier protein and forms Malonyl-ACP, an important precursor in the fatty acid synthesis. Fatty acids are carboxylic acids and have a chain length of 4 to 36 carbon atoms3. The genes were replicated and overexpressed in E. coli BL21 to increase carbon flux towards the precursor malonyl-CoA. An enhanced cellular concentration of malonyl-CoA contributes to increased production of malonyl-CoA and promotes the derived compounds, e.g. like fatty acids.

Fig.3: Bacterial fatty acid biosynthesis pathway in <i>E. coli. AccABCD is highlighted and represents all parts involved in the ACC. Modified from Christoph Freiberg et al., 2014 Fig.1</i>

The acc genes were cloned in an operon-like manner to ensure transcription of all genes of the complex at the same level and from the same vector guided by the same conditions [Fig. 1].

Fig.4: Gas chromatographic analysis of the fatty acid profile of modified E.coli strains
Fig.5: Comparison of modified <i>E. coli strains containing the fatty acid biosensor and analysed by C18:0 gas chromatography.</i>



















The rise in absolute fatty acid amount of ACC and tesA modified E. coli strains in comparison to Wild Type and HP(thioesterase of Haematococcus pluvialis) is not significant.

Value of ACC with biosensor and gas chromatography for C18:0 do not vary significantly.