Difference between revisions of "Part:BBa K4989000"

Revision as of 19:13, 4 October 2023

==Acetyl-CoA C-acetyltransferase (thl)== It is an improved basic part of <a href="https://parts.igem.org/Part:BBa_K1618040">BBa_K1618040</a> of iGEM 2015 NRP-UEA-Norwich.

==Alternative names of the enzyme==

Other names that the acetyl-CoA C-acetyl transferase enzyme could be found in the bibliography are:

Systematic name: acetyl-CoA:acetyl-CoA C-acetyltransferase

Else:3-ketoacyl CoA thiolase, 3-ketoacyl coenzyme A thiolase,

3-ketoacyl thiolase, 3-ketoacyl-CoA thiolase, 3-ketothiolase,

3-oxoacyl-CoA thiolase, 3-oxoacyl-coenzyme A thiolase,

6-oxoacyl-CoA thiolase, β-ketoacyl coenzyme A thiolase,

β-ketoacyl-CoA thiolase, β-ketoadipyl coenzyme A thiolase,

β-ketoadipyl-CoA thiolase, β-ketothiolase KAT, acetoacetyl-CoA β-ketothiolase,

acetyl-CoA acyltransferase, ketoacyl-CoA acyltransferase,

ketoacyl-coenzyme A thiolase, long-chain 3-oxoacyl-CoA thiolase,

oxoacyl-coenzyme A thiolase, pro-3-ketoacyl-CoA thiolase,

thiolase I, 2-methylacetoacetyl-CoA thiolase.

==Application in the field of biology==

The thl gene codes for an enzyme named acetyl-CoA C-acetyltransferase which belongs to the family of thiolases. This is the key enzyme for the condensation of two molecules of acetyl-CoA, which are produced from the glycolysis path, and the acetoacetyl-CoA production [a]. This is a reaction that thermodynamically itself is not favored. It starts with the acylation of a nucleophilic cysteine at the active site by acetyl-CoA (or a different acyl group) with the release of the CoA group. Then the acyl group is transferred to an acetyl-CoA molecule.

The chemical reactions that take place are shown below:

(1) acyl-CoA + acetyl-CoA = CoA + 3-oxoacyl-CoA
(1a) acetyl-CoA C-acyltransferase-S-acyl-L-cyteine + acetyl-CoA = 3-oxoacyl-CoA + acetyl-CoA C-acyltransferase-L-cyteine
(1b) acyl-CoA + acetyl-CoA C-acyltransferase-L-cyteine = acetyl-CoA C-acyltransferase-S-acyl-L-cyteine + CoA

This enzyme can catalyze the reaction of the condensation of the two molecules of acetyl-CoA to acetoacetyl-CoA for both the reactions of acetic acid and butyric acid production.

[a]Genome.jp, 2023, www.genome.jp/Fig/reaction/R08095.gif. Accessed 2 Oct.2023.

It is reported that the enzyme has a molecular mass of 44kDa and it is composed of 4 identical subunits. This is not a standard number since different bacterial genera (such as Clostridium, Firmicutes, etc.) have various differences in the sequences and the molecular masses of the enzyme. Nonetheless, they catalyze the same reaction. Comparing the reaction of the butyrate and the acetate synthesis, which are both produced from the acetyl-CoA molecule, the latter produces a yield of ATP that is twice as much as that of the former[1].

Below there is a crystal structure of the enzyme acetyl-CoA C-acetyltransferase from the bacterium Clostridium acetobutylicum. It is worth noting that the enzyme's crystal structure is different from organism to organism.

The enzyme is found both in the eukaryotes and the prokaryotes. Studies (primarily in a Clostridium strain) show that the change in the pH of the environment does not affect the regulation of the enzyme. It is inhibited by micromolar levels of CoA ,ATP, and butanoyl-CoA. Also, the concentration of CoASH(the Coenzyme A synthetase) plays an important role in the net rate of the condensation of the two acetyl-CoA molecules [1,2].

==Configuration of the new part==

The previous part <a data-fr-linked="true" href="https://parts.igem.org/Part:BBa_K1618040">BBa_K1618040</a> originated from Coprococcus sp. L2-50 DSM. There were some issues in the sequence and the origin of it that we addressed and solved as follows:

1. The sequence did contain a start and a stop codon but due to the results of the iGEM 2015 NRP-UEA-Norwich team, we decided to change them according to a research paper that provides all the sequences of the enzyme’s genes for the butyrate-producing pathway from the same strain Coprococcus sp.L2-50 [3,4].

2. We optimized our sequence in order to be expressed in both Lactobacillus species and E.coli.
3. We excluded all the restriction sites of the endonucleases that we used for cloning. We used the GenSmart Optimization Tool to optimize our sequences and exclude the formation of the restriction sites of the enzymes that we used for cloning.

==The obtainment of the sequence and its difficulties==

We obtained our sequence from iGEM's 2023 sponsor Twist Bioscience, via synthesis. Due to the large size of the part we faced a small difficulty in synthesizing this part along with others of the butyrate-producing pathway as a whole and proceeded with gene cloning. However, we were able, through gBlocks, to synthesize the part.

==Biosafety==

Our part is safe to be synthesized and utilized on an open bench. Also, the product of the gene does not include any biohazard and does not pose any threat even if by chance there is a leak.

==Characterization== The previous team, iGEM 2015 NRP-UEA-Norwich, did not perform any characterization. Our team, chose for the gold medal to create a new improved part and characterize it as well as possible for the competition standards and the other IGEM teams. So we did not characterize this specific part, as something that is crucial to be conducted in the future.

==References==

[1]Wiesenborn, Dennis P., et al. “Thiolase from Clostridium Acetobutylicum ATCC 824 and Its Role in the Synthesis of Acids and Solvents.” Applied and Environmental Microbiology, vol. 54, no. 11, 1988, pp. 2717–22,<a href="https://doi.org/10.1128/aem.54.11.2717-2722.1988"> https://doi.org/10.1128/aem.54.11.2717-2722.1988</a>. Accessed 21 Apr. 2021.
[2]Stim-Herndon, Kathleen P., et al. “Characterization of an Acetyl-CoA C Acetyltransferase (Thiolase) Gene from Clostridium Acetobutylicum ATCC 824.”Gene, vol. 154, no. 1, Feb. 1995, pp. 81–85,<a href="https://doi.org/10.1016/03781119(94)00838-j">https://doi.org/10.1016/03781119(94)00838-j. </a> Accessed on 20 Feb 2021

[3]Petra Louis, Sheila I. McCrae, Cédric Charrier, Harry J. Flint, Organization of butyrate synthetic genes in human colonic bacteria: phylogenetic conservation and horizontal gene transfer, FEMS Microbiology Letters, Volume 269, Issue 2, April 2007, Pages 240–247, <a href="https://doi.org/10.1111/j.1574-6968.2006.00629.x" style="text-align: start;color: rgb(0, 111, 183);background-color: rgb(255, 255, 255);font-size: 15px;">https://doi.org/10.1111/j.1574-6968.2006.00629.x</a>
[4]“Butyrate-Producing Bacterium L2-50 Putative Fe-S Oxidoreductase Gene, Partial Cds; Thiolase, Crotonase, Beta Hydroxybutyryl-CoA Dehydrogenase, Butyryl-CoA Dehydrogenase, Electron Transfer Flavoprotein Beta-Subunit, and Electron Transfer Flavoprotein Alpha-Subunit Genes, Complete Cds; and Putative Multidrug Efflux Pump Gene, Partial Cds.” NCBI Nucleotide, July 2016,<a href="www.ncbi.nlm.nih.gov/nuccore/DQ987697.1/">www.ncbi.nlm.nih.gov/nuccore/DQ987697.1/</a>

==Toolbox's links==

<a href="https://www.genscript.com/tools/gensmart-codon-optimization">Genscript's GenSmart Optimization Tool</a>

Other tools for optimizing a sequence: <a href="https://eu.idtdna.com/CodonOpt"> IDT's Optimization tool</a>

@@ Line 1: / Line 1: @@
-==<strong>Acetyl-CoA C-acetyltransferase <i>(thl)</i></strong>==
+<p><strong>==Acetyl-CoA C-acetyltransferase <em>(thl)</em></strong>== It is an improved basic part of <a href="https://parts.igem.org/Part:BBa_K1618040">BBa_K1618040</a> of iGEM 2015 NRP-UEA-Norwich.&nbsp;</p>
-It is an improved basic part of <a href="https://parts.igem.org/Part:BBa_K1618040">BBa_K1618040</a> of iGEM 2015 NRP-UEA-Norwich.
+<p><br></p>
+<p><br></p>
+<p>==<strong>Alternative names of the enzyme</strong>==&nbsp;</p>
-==<strong>Alternative names of the enzyme</strong>==
+<p>Other names that the acetyl-CoA C-acetyl transferase enzyme could be found in the bibliography are:</p>
-Other names that the acetyl-CoA C-acetyl transferase enzyme could be found in the bibliography are:
+<p>Systematic name: acetyl-CoA:acetyl-CoA C-acetyltransferase</p>
-<br>
+<p>Else:3-ketoacyl CoA thiolase, 3-ketoacyl coenzyme A thiolase,&nbsp;</p>
-Systematic name: acetyl-CoA:acetyl-CoA C-acetyltransferase
+<p>3-ketoacyl thiolase, 3-ketoacyl-CoA thiolase, 3-ketothiolase,</p>
-<br>
+<p>3-oxoacyl-CoA thiolase, 3-oxoacyl-coenzyme A thiolase,&nbsp;</p>
-Else:3-ketoacyl CoA thiolase,<br>3-ketoacyl coenzyme A thiolase,<br>3-ketoacyl thiolase, 3-ketoacyl-CoA thiolase,
+<p>6-oxoacyl-CoA thiolase, &beta;-ketoacyl coenzyme A thiolase,</p>
--ketothiolase,<br>3-oxoacyl-CoA thiolase,<br>3-oxoacyl-coenzyme A thiolase,<br>6-oxoacyl-CoA thiolase,<br>β-ketoacyl coenzyme A thiolase,<br>β-ketoacyl-CoA thiolase,<br>β-ketoadipyl coenzyme A thiolase,<br>β-ketoadipyl-CoA thiolase,<br>β-ketothiolase
+<p>&beta;-ketoacyl-CoA thiolase, &beta;-ketoadipyl coenzyme A thiolase,&nbsp;</p>
-KAT,<br>acetoacetyl-CoA β-ketothiolase,<br>acetyl-CoA acyltransferase,<br>ketoacyl-CoA acyltransferase,<br>ketoacyl-coenzyme A thiolase,<br>long-chain 3-oxoacyl-CoA thiolase,<br>oxoacyl-coenzyme A thiolase,<br>pro-3-ketoacyl-CoA thiolase,<br>thiolase I, 2-methylacetoacetyl-CoA thiolase.
+<p>&beta;-ketoadipyl-CoA thiolase, &beta;-ketothiolase KAT, acetoacetyl-CoA &beta;-ketothiolase,</p>
+<p>acetyl-CoA acyltransferase, ketoacyl-CoA acyltransferase,&nbsp;</p>
+<p>ketoacyl-coenzyme A thiolase, long-chain 3-oxoacyl-CoA thiolase,</p>
+<p>oxoacyl-coenzyme A thiolase, pro-3-ketoacyl-CoA thiolase,</p>
-==<strong>Application in the field of biology</strong>==
+<p>thiolase I, 2-methylacetoacetyl-CoA thiolase.&nbsp;</p>
-The <i>thl</i> gene codes for an enzyme named <em><b>acetyl-CoA C-acetyltransferase</em></b> which belongs to the family of <i>thiolases</i>. This is the key enzyme for the condensation of two molecules of acetyl-CoA, which are produced from the glycolysis path, and the acetoacetyl-CoA production [a]. This is a reaction that thermodynamically itself is not favored. It starts with the acylation of a nucleophilic cysteine at the active site by acetyl-CoA (or a different acyl group) with the release of the CoA group. Then the acyl group is transferred to an acetyl-CoA molecule.
+<p><br></p>
+<p><br></p>
-<p>The chemical reactions that take place are shown below:
+<p>==<strong>Application in the field of biology</strong>==&nbsp;</p>
-<br>
+<p>The <em>thl</em> gene codes for an enzyme named <em><strong>acetyl-CoA C-acetyltransferase</strong></em> which belongs to the family of <em>thiolases</em>. This is the key enzyme for the condensation of two molecules of acetyl-CoA, which are produced from the glycolysis path, and the acetoacetyl-CoA production [a]. This is a reaction that thermodynamically itself is not favored. It starts with the acylation of a nucleophilic cysteine at the active site by acetyl-CoA (or a different acyl group) with the release of the CoA group. Then the acyl group is transferred to an acetyl-CoA molecule.</p>
-(1) acyl-CoA + acetyl-CoA = CoA + 3-oxoacyl-CoA
+<p>The chemical reactions that take place are shown below:</p>
-<br>
+<p>(1) acyl-CoA + acetyl-CoA = CoA + 3-oxoacyl-CoA<br>(1a) acetyl-CoA C-acyltransferase-S-acyl-L-cyteine + acetyl-CoA = 3-oxoacyl-CoA + acetyl-CoA C-acyltransferase-L-cyteine<br>(1b) acyl-CoA + acetyl-CoA C-acyltransferase-L-cyteine = acetyl-CoA C-acyltransferase-S-acyl-L-cyteine + CoA</p>
-(1a) acetyl-CoA C-acyltransferase-S-acyl-L-cyteine + acetyl-CoA = 3-oxoacyl-CoA + acetyl-CoA C-acyltransferase-L-cyteine
+<p>This enzyme can catalyze the reaction of the condensation of the two molecules of acetyl-CoA to acetoacetyl-CoA for both the reactions of acetic acid and butyric acid production.</p>
-<br>
+<p><img 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bLx5D8b9Q1TQtNv/ABF4nklfQLuW7lXYP7Pihj8zbgLiVGPRuMHnpWtdapqln8ZPC3hddevL23uIP7N1jQlfattCLBtskcewb0aT5jcbsqQExzigDV8I/FrQPEHxdPiOy07xK2j6/ZW2g6frcmlY0y5eCW5lV0lDl9rmZlVmjVDsGGORXvFeCfDPw98W/Cej+DfAbWOkaboPht4rW58Tx3azNqWnwKVjijtimYpXURh2ZsLhipORj3ugAr5i+LXjDwfa/ETxXFe+K4bV3l8Ni7kTT7qe305rHUHu3W5uI42iiLpIoXew2nlsDmvp2vmVtL8beAfD/wASvAln4BvvEk3iXUtSvNJ1uGa3+wSJfFmP2xncPGYi5UgI25UXbnOAAfS8MyXESSxOskbqGV0OQwPIIPcU+sD4f+GW8E+A/Dnh57j7W+k6bbWDXBz+8MUSpu/Hbn8a36ACiiigArx/47azovhvxL8PtZ13xFo/h+wsL29aRtVvVgaTzLGaFRGp5chpFzjoMntXsFeDeM7sfDv9oiXxj4g0PVNW8Pah4ch0yx1LTdMl1A6dcRzzPLE0cKu6CZZI8OFwTFtJHFAHonwRtF0/4N+BbJbyz1D7JodlatdadcLPbyNHAiM0ci8OuVOGHWu1ryT9mnw1qHh/wXrdze6VN4ft9a8QX+r2GjToI5LG1mkzHG0Y4jY4MhQfdMhHUGvW6ACiiigAqrqel2etafcWGo2kF/Y3CGOa2uolkikU9VZWBBHsatUUAYGieAPDHhqyt7PSPDmk6XaW832mGGzsookjl2lfMUKow+0kbuuCRWtb6baWt1c3UNrDDc3RUzzRxhXlKjapdgMtgcDPQVZooAKKKKACiiigAooooAKKKKACiiigAooooAKKKKACiiigAooooAKKKKACiiigAooooAKKKKACiiigAooooAKKKKACiiigAooooAKKKKACiiigAooooAKKKKACiivn24+LXjBPA+ufEhdU0aLRbWy1SWHwncWbC5ElqJFTNwJMk5j3Ovl8BsDpkgH0FXl0f7NfgWPXjqK2V8LX7f8A2r/Yf9pT/wBlfbN2/wA/7Jv8rfv+f7uN3zYzzXB638VvHWk6jeeE11fTJdY06+vUk1s6acXEUGm2t8F+z+ZhS32ryywY4CA4y3Frw38b/FPiDxBod+q6eukahqNpon9kCI7vOm0RNU88XG7jDP5WzZjb82c8UAen3fwd8J33hXX/AA5NpjPo+vX0upahb/aJB51xJKJXfduyuXUHAIHGMYrtK8L074leNtc8H+HraWfS9H8T6z4t1Lw9JeW1s9xb2iWzXzhkjdlMh22aoSSv3mbAwFr0b4R+Mbn4hfDPw14kvIYre61OxjuZYoCSisw5C55xQB11FFFABRRRQAUUUUAFFFFABRRRQAUUUUAFFFFABRRRQAUUUUAFFFFABRRRQAUUUUAFFFFABRRRQAUUUUAFFFFABRRRQAUUUUAFFFFABRRRQAUUUUAFFFFABRRRQAUUUUAFFFFABRRRQAUUUUAFFFFABRRRQAUUUUAFFFFABRRRQAVzyfD3wxH4gv8AXF8P6b/bF/EYLu9+yoZZ4yACrtjkEAA56gV0NFAHHH4O+B28N2/h8+E9IOiW8/2mKxNohiSUnJcDH3vetCX4e+GZ/EB12TQNPbWTbfYvtxtl83ycY2bsZxjj6cdK6GigDhrH4F/D3TfD99oVp4M0W30e+lWe5sY7NFilkU5DsuOWyTz15NdnZ2cGn2sNtawx29tCoSOGJQqIoGAABwAKmooAKKKKACiiigAooooAKKKKACiiigAooooAKKKKACiiigAooooAKKKKACiiigAooooAKKKKACiiigAooooAKKKKACiiigAooooA/9k=" width="554" height="249"></p>
-(1b) acyl-CoA + acetyl-CoA C-acyltransferase-L-cyteine = acetyl-CoA C-acyltransferase-S-acyl-L-cyteine + CoA
+<p>[a]<span style='font-size:15px;font-family:"Calibri",sans-serif;'><em>Genome.jp</em>, 2023, www.genome.jp/Fig/reaction/R08095.gif. Accessed 2 Oct.2023.</span></p>
-</p> This enzyme can catalyze the reaction of the condensation of the two molecules of acetyl-CoA to acetoacetyl-CoA for both the reactions of acetic acid and butyric acid production.
+<p><br></p>
-<br>
+<p>It is reported that the enzyme has a molecular mass of 44kDa and it is composed of 4 identical subunits. This is not a standard number since different bacterial genera (such as <em>Clostridium</em>, <em>Firmicutes</em>, etc.) have various differences in the sequences and the molecular masses of the enzyme. Nonetheless, they catalyze the same reaction. Comparing the reaction of the butyrate and the acetate synthesis, which are both produced from the acetyl-CoA molecule, the latter produces a yield of ATP that is twice as much as that of the former[1].</p>
+<p>Below there is a crystal structure of the enzyme acetyl-CoA C-acetyltransferase <strong>from the bacterium&nbsp;</strong><em><strong>Clostridium acetobutylicum</strong></em>. It is worth noting that the enzyme&apos;s crystal structure is different from organism to organism.</p>
-[[File:reaction.jpg|center|400px]]
+<p><br></p>
-<br>
+<p><br></p>
+<p>The enzyme is found both in the eukaryotes and the prokaryotes. Studies (primarily in a <em>Clostridium</em> strain) show that the change in the pH of the environment does not affect the regulation of the enzyme. It is inhibited by micromolar levels of <strong>CoA</strong> ,<strong>ATP</strong>, and <strong>butanoyl-CoA</strong>. Also, the concentration of <strong>CoASH</strong>(the Coenzyme A synthetase) plays an important role in the net rate of the condensation of the two acetyl-CoA molecules [1,2].&nbsp;</p>
-It is reported that the enzyme has a molecular mass of 44kDa and it is composed of 4 identical subunits. This is not a standard number since different bacterial genera (such as <I>Clostridium</i>, Firmicutes, etc.) have various differences in the sequences and the molecular masses of the enzyme. Nonetheless, they catalyze the same reaction. Comparing the reaction of the butyrate and the acetate synthesis, which are both produced from the acetyl-CoA molecule, the latter produces a yield of ATP that is twice as much as that of the former[1].
+<p><br></p>
-<br>
+<p><br></p>
-Below there is a crystal structure of the enzyme acetyl-CoA C-acetyltransferase from the bacterium <i>Clostridium acetobutylicum</i>. It is worth noting that the crystal structure of the enzyme is different from organism to organism.
+<p>==<strong>Configuration of the new part</strong>==&nbsp;</p>
-<video width="1258" height="1048" controls>
+<p>The previous part <strong><em><a data-fr-linked="true" href="https://parts.igem.org/Part:BBa_K1618040">BBa_K1618040</a></em></strong><em>&nbsp;originated from <strong><em>Coprococcus sp. L2-50 DSM</em></strong>.&nbsp;</em>There were some issues in the sequence and the origin of it that we addressed and solved as follows:&nbsp;</p>
-<source src="4WYR_camera-spin" type="video/mp4">
+<p><br>1. The sequence did contain a start and a stop codon but due to the results of the iGEM 2015 NRP-UEA-Norwich team, we decided to change them according to a research paper that provides all the sequences of the enzyme&rsquo;s genes for the butyrate-producing pathway from the same strain <em>Coprococcus sp.</em>L2-50 [3,4].&nbsp;</p>
-</video>
+<p>2. We optimized our sequence in order to be expressed in both <em>Lactobacillus</em> species and<em>&nbsp;E.coli</em>.<em>&nbsp;</em><br>3. We excluded all the restriction sites of the endonucleases that we used for cloning. We used the GenSmart Optimization Tool to optimize our sequences and exclude the formation of the restriction sites of the enzymes that we used for cloning.</p>
+<p><br></p>
-<br>
+<p><br></p>
-The enzyme is found both in the eukaryotes and the prokaryotes. Studies (primarily in a <i>Clostridium</i> strain) show that the change in the pH of the environment does not affect the regulation of the enzyme. It is inhibited by micromolar levels of <b>CoA</b> and <b>ATP</b>, as well as <b>butanoyl-CoA</b>. Also, the concentration of <b>CoASH</b>(the Coenzyme A synthetase) plays an important role in the net rate of the condensation of the two acetyl-CoA molecules [1,2].
+<p>&nbsp;==<strong>The obtainment of the sequence and its difficulties</strong>==&nbsp;</p>
+<p>We obtained our sequence from iGEM&apos;s 2023 sponsor Twist Bioscience, via synthesis. Due to the large size of the part we faced a small difficulty in synthesizing this part along with others of the butyrate-producing pathway as a whole and proceeded with gene cloning. However, we were able, through gBlocks, to synthesize the part.&nbsp;</p>
+<p><br></p>
+<p><br></p>
+<p>==<strong>Biosafety</strong>==&nbsp;</p>
-==<strong>Configuration of the new part</strong>==
+<p>Our part is safe to be synthesized and utilized on an open bench. Also, the product of the gene does not include any biohazard and does not pose any threat even if by chance there is a leak.</p>
-The previous part <b><i>BBa_K1618040</b><i/> was originated from <strong><i>Coprococcus sp.L2-50 DSM</strong></i>.There were some issues in the sequence and the origin of it that we addressed and solved as follows:
+<p><br></p>
-<br>
+<p><br></p>
-. The sequence did contain a start and a stop codon but due to the results of the iGEM 2015 NRP-UEA-Norwich team, we decided to change them according to a research paper that provides all the sequences of the enzyme’s genes for the butyrate-producing pathway from the same strain <i>Coprococcus sp.L2-50</i>.
+<p>&nbsp;==<strong>Characterization</strong>== The previous team, iGEM 2015 NRP-UEA-Norwich, did not perform any characterization. Our team, chose for the gold medal to create a new improved part and characterize it as well as possible for the competition standards and the other IGEM teams. So we did not characterize this specific part, as something that is crucial to be conducted in the future.&nbsp;</p>
-. We optimized our sequence in order to be expressed in both <i>Lactobacillus</i> species and <i>E.coli</i>.
+<p><br></p>
-<br>
+<p><br></p>
-. We excluded all the restriction sites of the endonucleases that we used for cloning.
+<p>==<strong>References</strong>==&nbsp;</p>
+<p>[1]Wiesenborn, Dennis P., et al. &ldquo;Thiolase from Clostridium Acetobutylicum ATCC 824 and Its Role in the Synthesis of Acids and Solvents.&rdquo; Applied and Environmental Microbiology, vol. 54, no. 11, 1988, pp. 2717&ndash;22,<a href="https://doi.org/10.1128/aem.54.11.2717-2722.1988">&nbsp;https://doi.org/10.1128/aem.54.11.2717-2722.1988</a>. Accessed 21 Apr. 2021.&nbsp;<br> [2]Stim-Herndon, Kathleen P., et al. &ldquo;Characterization of an Acetyl-CoA C Acetyltransferase (Thiolase) Gene from Clostridium Acetobutylicum ATCC 824.&rdquo;Gene, vol. 154, no. 1, Feb. 1995, pp. 81&ndash;85,<a href="https://doi.org/10.1016/03781119(94)00838-j"><span style='font-size:15px;font-family:"Calibri",sans-serif;'>https://doi.org/10.1016/03781119(94)00838-j.&nbsp;</span></a> Accessed on 20 Feb 2021</p>
-We used the GenSmart Optimization Tool for the optimization of our sequences and the exclusion of the formation of the restriction sites of the enzymes that we used for cloning.
+<p>[3]<span style="text-align: start;color: rgb(42, 42, 42);background-color: rgb(255, 255, 255);font-size: 15px;">Petra Louis, Sheila I. McCrae, C&eacute;dric Charrier, Harry J. Flint, Organization of butyrate synthetic genes in human colonic bacteria: phylogenetic conservation and horizontal gene transfer,&nbsp;</span>FEMS Microbiology Letters<span style="text-align: start;color: rgb(42, 42, 42);background-color: rgb(255, 255, 255);font-size: 15px;">, Volume 269, Issue 2, April 2007, Pages 240&ndash;247,&nbsp;</span><a href="https://doi.org/10.1111/j.1574-6968.2006.00629.x" style="text-align: start;color: rgb(0, 111, 183);background-color: rgb(255, 255, 255);font-size: 15px;">https://doi.org/10.1111/j.1574-6968.2006.00629.x</a><br> [4]&ldquo;Butyrate-Producing Bacterium L2-50 Putative Fe-S Oxidoreductase Gene, Partial Cds; Thiolase, Crotonase, Beta Hydroxybutyryl-CoA Dehydrogenase, Butyryl-CoA Dehydrogenase, Electron Transfer Flavoprotein Beta-Subunit, and Electron Transfer Flavoprotein Alpha-Subunit Genes, Complete Cds; and Putative Multidrug Efflux Pump Gene, Partial Cds.&rdquo; NCBI Nucleotide, July 2016,<a href="www.ncbi.nlm.nih.gov/nuccore/DQ987697.1/">www.ncbi.nlm.nih.gov/nuccore/DQ987697.1/</a>&nbsp;</p>
+<p>==<strong>Toolbox&apos;s links</strong>==&nbsp;</p>
+<p><a href="https://www.genscript.com/tools/gensmart-codon-optimization">Genscript&apos;s GenSmart Optimization Tool</a>&nbsp;</p>
+<p>Other tools for optimizing a sequence: <a href="https://eu.idtdna.com/CodonOpt">&nbsp;IDT&apos;s Optimization tool</a> </p>
-==<strong>The obtainment of the sequence and its difficulties</strong>==
+<p><br></p>
-We obtained our sequence from iGEM's 2023 sponsor Twist Bioscience, via synthesis. Due to the large size of the part we faced a small difficulty in synthesizing this part along with others of the butyrate-producing pathway as a whole and proceeded with gene cloning. However, we were able, through gBlocks, to synthesize the part.
-==<strong>Biosafety</strong>==
-Our part is safe to be synthesized and utilized on an open bench. Also, the product of the gene does not include any biohazard and does not pose any threat even if by chance there is a leak.
-==<strong>Characterization</strong>==
-The previous team, iGEM 2015 NRP-UEA-Norwich, did not perform any characterization. Our team, chose for the gold medal to create a new improved part and characterize it as well as possible for the competition standards and the other IGEM teams. So we did not characterize this specific part, as something that is crucial to be conducted in the future.
-==<strong>References</strong>==
-[1]Wiesenborn, Dennis P., et al. “Thiolase from Clostridium Acetobutylicum ATCC 824 and Its Role in the Synthesis of Acids and Solvents.” Applied and Environmental Microbiology, vol. 54, no. 11, 1988, pp. 2717–22,<a href="https://doi.org/10.1128/aem.54.11.2717-2722.1988"> https://doi.org/10.1128/aem.54.11.2717-2722.1988</a>. Accessed 21 Apr. 2021.
-<br>
-[2]Stim-Herndon, Kathleen P., et al. “Characterization of an Acetyl-CoA C Acetyltransferase (Thiolase) Gene from Clostridium Acetobutylicum ATCC 824.”Gene, vol. 154, no. 1, Feb. 1995, pp. 81–85,<a href="https://doi.org/10.1016/03781119(94)00838-j"https://doi.org/10.1016/03781119(94)00838-j</a>. Accessed 20 Feb. 2021.
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-[4]Petra Louis, Sheila I. McCrae, Cédric Charrier, Harry J. Flint, Organization of butyrate synthetic genes in human colonic bacteria: phylogenetic conservation and horizontal gene transfer, FEMS Microbiology Letters, Volume 269, Issue 2, April 2007, Pages 240–247,<a href="https://doi.org/10.1111/j.1574-6968.2006.00629.x">https://doi.org/10.1111/j.1574-6968.2006.00629.x</a>
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-[5]“Butyrate-Producing Bacterium L2-50 Putative Fe-S Oxidoreductase Gene, Partial Cds; Thiolase, Crotonase, Beta Hydroxybutyryl-CoA Dehydrogenase, Butyryl-CoA Dehydrogenase, Electron Transfer Flavoprotein Beta-Subunit, and Electron Transfer Flavoprotein Alpha-Subunit Genes, Complete Cds; and Putative Multidrug Efflux Pump Gene, Partial Cds.” NCBI Nucleotide, July 2016,<a href="www.ncbi.nlm.nih.gov/nuccore/DQ987697.1/">www.ncbi.nlm.nih.gov/nuccore/DQ987697.1/</a>
-==<strong>Toolbox's links</strong>==
-<a href="https://www.genscript.com/tools/gensmart-codon-optimization">Genscript's GenSmart Optimization Tool</a>
-<p>
-Other tools for optimizing a sequence:
-<a href="https://eu.idtdna.com/CodonOpt"> IDT's Optimization tool</a>