Difference between revisions of "Team:NKU CHINA/Basic Part"

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<h1>Basic Parts</h1>
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.tuzhu {
A <b>basic part</b> is a functional unit of DNA that cannot be subdivided into smaller component parts. <a href="http://parts.igem.org/wiki/index.php/Part:BBa_R0051">BBa_R0051</a> is an example of a basic part, a promoter regulated by lambda cl.
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<p>Most genetically-encoded functions have not yet been converted to BioBrick parts. Thus, there are <b>many</b> opportunities to find new, cool, and important genetically encoded functions, and refine and convert the DNA encoding these functions into BioBrick standard biological parts. </p>
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<h3>Note</h3>
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<p>This page should list all the basic parts your team has made during your project. You must add all characterization information for your parts on the Registry. You should not put characterization information on this page. Remember judges will only look at the first part in the list for the Best Basic Part award, so put your best part first!</p>
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<h3>Best Basic Part Special Prize</h3>
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<p> To be eligible for this award, this part must adhere to <a href="http://parts.igem.org/DNA_Submission">Registry sample submission guidelines</a> and have been sent to the Registry of Standard Biological Parts. If you have a part you wish to nominate your team for this <a href="https://2018.igem.org/Judging/Awards">special prize</a>, make sure you add your part number to your <a href="https://2018.igem.org/Judging/Judging_Form">judging form</a> and delete the box at the top of this page.
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    <h1 class="text-center" style="font-size: 80px;font-weight: normal;color: white;padding-bottom: 0;margin-bottom: 20px; font-family: myTitle;margin-top: 30px;padding-top: 0;">Basic Parts</h1>
<b>Please note:</b> Judges will only look at the first part number you list, so please only enter ONE (1) part number in the judging form for this prize. </p>
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    <h3 class="basic_header_two">P<sub><i>gltAB</i></sub></h3>
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          <h2 class="basic_header">Design</h2>
 
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          <p class="basic_content">This sequence includes the promoter(forward) of <i>gltAB</i> (glutamate synthase) and the promoter (backward) of <i>gltC</i> (LysR family transcriptional regulator). GltC can bind specific DNA site on it. Thus the expression of downstream proteins is upregulated, which is repressed by high concentration glutamate.</p>
 +
          <p class="basic_content">Since P<sub><i>gltAB</i></sub> can sense intracellular glutamate level, we combined it with proteins such as GFP and LacI, so that these proteins can respond to intracellular glutamate concentration and carry out their own function.</p>
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          <h3 class="basic_header_two">Proof of Function</h3>
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          <h2 class="basic_header">Detection of <i>gltC</i> transcription level in LL3-P<sub><i>gltAB</i></sub>-GFP under different glutamate concentrations</h2>
 +
          <p class="basic_content">LL3-P<sub><i>gltAB</i></sub>-GFP was cultured in M9 medium with different extracellular glutamate concentrations. From the 6th hour, we extracted the total RNA of LL3-P<sub><i>gltAB</i></sub>-GFP every 3 hours and tested the transcription of <i>gltC</i> together with the respective intracellular glutamate concentrations. Transcription level of <i>gltC</i> in plateau phase is shown in <strong>Fig. 1</strong>. It could be indicated that the transcription of <i>gltC</i> was repressed with the increasing intracellular glutamate concentration. Primers used in the assay are listed in <strong>Table 1</strong>.</p>
 +
          <img src="https://static.igem.org/mediawiki/2018/f/fa/T--NKU_CHINA--gltC.png" class="img-responsive center-block" style="border-radius: 5px;">
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          <p class="tuzhu"> <strong>Fig. 1. The intracellular glutamate concentration and the relative expression level of <i>gltC</i> in LL3 with P<sub><i>gltAB</i></sub>-GFP in plateau stage.</strong> <strong>a. The intracellular glutamate concentration of LL3 with P<sub><i>gltAB</i></sub>-GFP in plateau stage.</strong> *Significantly different (P &#60; 0.05) by Student&#39;s t-test.<strong>b. The relative expression level of <i>gltC</i> in plateau stage. </strong>The value illustrates the effect of glutamate concentration on the transcription of <i>gltC</i>. ***Very very significantly different (P &#60; 0.005) by Student &#60;s t-test. The strains were cultured at 37 &#176;C in M9 medium with 5 &#181;g/mL chloromycetin under different extracellular glutamate concentration (0 g/L, 2.5 g/L, 5.0 g/L) for 24 hours. Data indicate mean values &#177; standard deviations from three independent experiments performed in triplicates.</p>
 +
          <img src="https://static.igem.org/mediawiki/2018/7/70/T--NKU_CHINA--basic_table.jpg" class="img-responsive center-block" style="border-radius: 5px;">
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          <h2 class="basic_header">GFP fluorescent intensity (FI) reports the P<sub><i>gltAB</i></sub> function</h2>
 +
          <p class="basic_content">P<sub><i>gltAB</i></sub>-GFP and P<sub>43</sub>-GFP were converted into both <i>B. amyloliquefaciens</i> LL3 &#916;<i>bam</i> and <i>B. amyloliquefaciens</i> LL3 &#916; <i>bam</i> -<i>icd</i> strain (with stronger promoter before <i>icd</i> gene), which were designated as LL3-P<sub><i>gltAB</i></sub>-GFP and LL3-<i>icd</i>-P<sub><i>gltAB</i></sub>-GFP respectively. The two mutants were cultured in M9 culture medium for 24 hours. If needed the medium was supplemented with antibiotics or glutamate at the following concentrations: 5 &#181;g/mL chloramphenicol, 0 g/L, 0.5 g/L, 1.0 g/L, 2.5 g/L or 5.0 g/L glutamate. During the fermentation, 1.5mL bacteria culture was taken out every 3 hours, of which 600&#181;L was for GFP FI measurement (395nm&#92;509nm) by microplate reader, and 900&#181;L for OD<sub>600</sub> measurement.</p>
 +
          <img src="https://static.igem.org/mediawiki/2018/7/78/T--NKU_CHINA--basic2.png" class="img-responsive center-block" style="border-radius: 5px;">
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          <p class="tuzhu"><strong>Fig.2 Principle for detecting the P<sub><i>gltAB</i></sub> function.</strong> Under high glutamate concentration, GltC level goes down, reducing the level of GFP.</p>
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          <p class="tuzhu"> <strong>Fig.3 FI of GFP in LL3-P<sub><i>gltAB</i></sub>-GFP and LL3–<i>icd</i>-P<sub><i>gltAB</i></sub>-GFP under different extracellular glutamate concentrations in plateau stage.</strong> <strong>a. The intracellular glutamate concentration under different extracellular glutamate concentrations in plateau stage.</strong> The value illustrates the relationship between glutamate concentration in medium and intracellular glutamate concentration. *Significantly different (P &#60; 0.05) by Student&#39;s t-test. <strong>b. FI of GFP in LL3-P<sub><i>gltAB</i></sub>-GFP under different extracellular glutamate concentrations in plateau stage.</strong> **Very significantly different (P &#60; 0.01) by Student&#39;s t-test. <strong>c. FI of GFP in LL3-<i>icd</i>-P<sub><i>gltAB</i></sub>-GFP under different extracellular glutamate concentrations in plateau stage.</strong> *** Very very significantly different (P &#60; 0.005) by Student&#39;s t-test. The strains were cultured at 37 &#176;C in M9 medium with 5 &#181;g/mL chloromycetin for 24 hours under different extracellular glutamate concentration (0 g/L, 0.5 g/L, 1.0 g/L, 2.5 g/L, 5.0 g/L). Intracellular glutamate concentration, fluorescence intensity of GFP and the OD<sub>600</sub> were measured. FI of GFP was normalized against OD<sub>600</sub>. Data indicate mean values &#177; standard deviations from three independent experiments performed in triplicates.</p>
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          <h2 class="basic_header">Reference</h2>
 +
          <p class="refer_content">Weitao G, Mingfeng C, Cunjiang S <i>et al</i>. Complete Genome Sequence of <i>Bacillus amyloliquefaciens</i> LL3, Which Exhibits Glutamic Acid-Independent Production of Poly-&#947;-Glutamic Acid. J Bacteriol. 2011 Jul; 193(13): 3393–3394.</p>
 +
          <p class="refer_content">Picossi S, Belitsky B R, Sonenshein A L. Molecular mechanism of the regulation of <i>Bacillus subtilis gltAB</i> expression by GltC[J]. J Mol Biol., 2007, 365(5):1298-1313.</p>
 +
          <p class="refer_content">Commichau FM, Herzberg C, Tripal P et al. A regulatory protein-protein interaction governs glutamate biosynthesis in <i>Bacillus subtilis</i>: the glutamate dehydrogenase RocG moonlights in controlling the transcription factor GltC. Mol Microbiol. 2007 Aug;65(3):642-654.</p>
 +
          <p class="refer_content">Bohannon D E and Sonenshein A L. Positive regulation of glutamate biosynthesis in <i>Bacillus subtilis</i>. J Bacteriol. 1989 Sep; 171(9): 4718–4727.</p>
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                    Nankai University</br>
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                    No.94 Weijin Road, Nankai District</br>
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                    Tianjin, P.R.China 300071</br>
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Revision as of 15:04, 17 October 2018

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Basic Parts

PgltAB

Design

This sequence includes the promoter(forward) of gltAB (glutamate synthase) and the promoter (backward) of gltC (LysR family transcriptional regulator). GltC can bind specific DNA site on it. Thus the expression of downstream proteins is upregulated, which is repressed by high concentration glutamate.

Since PgltAB can sense intracellular glutamate level, we combined it with proteins such as GFP and LacI, so that these proteins can respond to intracellular glutamate concentration and carry out their own function.

Proof of Function

Detection of gltC transcription level in LL3-PgltAB-GFP under different glutamate concentrations

LL3-PgltAB-GFP was cultured in M9 medium with different extracellular glutamate concentrations. From the 6th hour, we extracted the total RNA of LL3-PgltAB-GFP every 3 hours and tested the transcription of gltC together with the respective intracellular glutamate concentrations. Transcription level of gltC in plateau phase is shown in Fig. 1. It could be indicated that the transcription of gltC was repressed with the increasing intracellular glutamate concentration. Primers used in the assay are listed in Table 1.

Fig. 1. The intracellular glutamate concentration and the relative expression level of gltC in LL3 with PgltAB-GFP in plateau stage. a. The intracellular glutamate concentration of LL3 with PgltAB-GFP in plateau stage. *Significantly different (P < 0.05) by Student's t-test.b. The relative expression level of gltC in plateau stage. The value illustrates the effect of glutamate concentration on the transcription of gltC. ***Very very significantly different (P < 0.005) by Student <s t-test. The strains were cultured at 37 °C in M9 medium with 5 µg/mL chloromycetin under different extracellular glutamate concentration (0 g/L, 2.5 g/L, 5.0 g/L) for 24 hours. Data indicate mean values ± standard deviations from three independent experiments performed in triplicates.

GFP fluorescent intensity (FI) reports the PgltAB function

PgltAB-GFP and P43-GFP were converted into both B. amyloliquefaciens LL3 Δbam and B. amyloliquefaciens LL3 Δ bam -icd strain (with stronger promoter before icd gene), which were designated as LL3-PgltAB-GFP and LL3-icd-PgltAB-GFP respectively. The two mutants were cultured in M9 culture medium for 24 hours. If needed the medium was supplemented with antibiotics or glutamate at the following concentrations: 5 µg/mL chloramphenicol, 0 g/L, 0.5 g/L, 1.0 g/L, 2.5 g/L or 5.0 g/L glutamate. During the fermentation, 1.5mL bacteria culture was taken out every 3 hours, of which 600µL was for GFP FI measurement (395nm\509nm) by microplate reader, and 900µL for OD600 measurement.

Fig.2 Principle for detecting the PgltAB function. Under high glutamate concentration, GltC level goes down, reducing the level of GFP.

Fig.3 FI of GFP in LL3-PgltAB-GFP and LL3–icd-PgltAB-GFP under different extracellular glutamate concentrations in plateau stage. a. The intracellular glutamate concentration under different extracellular glutamate concentrations in plateau stage. The value illustrates the relationship between glutamate concentration in medium and intracellular glutamate concentration. *Significantly different (P < 0.05) by Student's t-test. b. FI of GFP in LL3-PgltAB-GFP under different extracellular glutamate concentrations in plateau stage. **Very significantly different (P < 0.01) by Student's t-test. c. FI of GFP in LL3-icd-PgltAB-GFP under different extracellular glutamate concentrations in plateau stage. *** Very very significantly different (P < 0.005) by Student's t-test. The strains were cultured at 37 °C in M9 medium with 5 µg/mL chloromycetin for 24 hours under different extracellular glutamate concentration (0 g/L, 0.5 g/L, 1.0 g/L, 2.5 g/L, 5.0 g/L). Intracellular glutamate concentration, fluorescence intensity of GFP and the OD600 were measured. FI of GFP was normalized against OD600. Data indicate mean values ± standard deviations from three independent experiments performed in triplicates.

Reference

Weitao G, Mingfeng C, Cunjiang S et al. Complete Genome Sequence of Bacillus amyloliquefaciens LL3, Which Exhibits Glutamic Acid-Independent Production of Poly-γ-Glutamic Acid. J Bacteriol. 2011 Jul; 193(13): 3393–3394.

Picossi S, Belitsky B R, Sonenshein A L. Molecular mechanism of the regulation of Bacillus subtilis gltAB expression by GltC[J]. J Mol Biol., 2007, 365(5):1298-1313.

Commichau FM, Herzberg C, Tripal P et al. A regulatory protein-protein interaction governs glutamate biosynthesis in Bacillus subtilis: the glutamate dehydrogenase RocG moonlights in controlling the transcription factor GltC. Mol Microbiol. 2007 Aug;65(3):642-654.

Bohannon D E and Sonenshein A L. Positive regulation of glutamate biosynthesis in Bacillus subtilis. J Bacteriol. 1989 Sep; 171(9): 4718–4727.

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