Difference between revisions of "Part:BBa K1405004"

(Background)
(Background)
Line 12: Line 12:
  
  
<h2>Result</h2>
 
Figure 1 Expression and purification of ModA. Expression of ModA and its purification by His-chelating chromatography. Lane 1, molecular weight standards (kDa); lane 2, total bacterial proteins before IPTG induction; lane 3, total bacterial proteins after 0.5 mM IPTG induction; lane 4, total bacterial lysate after 0.5 mM IPTG induction; lane 5,the sediment after 16,000 rpm 30 min; lane 6 the flow through of column loading; lane 7,the flow of buffer B .lane 8 The purification of ModA by size-elution chromatography.
 
 
<br/>
 
<br/>
<a title="Figure 1 Expression and purification of ModA. Expression of ModA and its purification by His-chelating chromatography. Lane 1, molecular weight standards (kDa); lane 2, total bacterial proteins before IPTG induction; lane 3, total bacterial proteins after 0.5 mM IPTG induction; lane 4, total bacterial lysate after 0.5 mM IPTG induction; lane 5,the sediment after 16,000 rpm 30 min; lane 6 the flow through of column loading; lane 7,the flow of buffer B .lane 8 The purification of ModA by size-elution chromatography." href="https://static.igem.org/mediawiki/2014/c/c6/Bnu_moda3.png" rel="prettyPhoto"> <span class="overlay zoom" style="display: none;"></span><img width="60%" style="margin-left:180px" src="https://static.igem.org/mediawiki/2014/c/c6/Bnu_moda3.png"> </a>
+
<h2 id="results">Results</h2>
 
+
<p>插图 Expression and purification of ModA. Expression of ModA and its purification by His-chelating chromatography. Lane 1, molecular weight standards (kDa); lane 2, total bacterial proteins before IPTG induction; lane 3, total bacterial proteins after 0.5 mM IPTG induction; lane 4, total bacterial lysate after 0.5 mM IPTG induction; lane 5,the sediment after 16,000 rpm 30 min; lane 6 the flow through of column loading; lane 7,the flow of buffer B .lane 8 The purification of ModA by size-elution chromatography. 插图 Native-PAGE to provide the molybdate lane 1, the modA(acetic buffer PH 5.0); lane 2, the modA incubate with 20mM molybdate.</p>
<p class="fig">Figure 1 Expression and purification of ModA. Expression of ModA and its purification by His-chelating chromatography. Lane 1, molecular weight standards (kDa); lane 2, total bacterial proteins before IPTG induction; lane 3, total bacterial proteins after 0.5 mM IPTG induction; lane 4, total bacterial lysate after 0.5 mM IPTG induction; lane 5,the sediment after 16,000 rpm 30 min; lane 6 the flow through of column loading; lane 7,the flow of buffer B .lane 8 The purification of ModA by size-elution chromatography.</p>
+
 
<br/>
 
<br/>
 
+
<a title="Fig.1  Agar assay" href="https://static.igem.org/mediawiki/2014/7/70/Bnu_cb7.jpg" rel="prettyPhoto"> <span class="overlay zoom" style="display: none;"></span><img style="opacity: 1; width:40%; float: left; margin-left: 50px; border-radius: 0.5em 0.5em 0.5em 0.5em;"src="https://static.igem.org/mediawiki/2014/7/70/Bnu_cb7.jpg"></a>
<a title="Figure 2 Native-PAGE to provide the molybdate lane 1, the modA(acetic buffer PH 5.0); lane 2, the modA incubate with 20mM molybdate." href="https://static.igem.org/mediawiki/2014/9/90/Bnu_moda4.jpg" rel="prettyPhoto"> <span class="overlay zoom" style="display: none;"></span><img width="60%" style="margin-left:180px" src="https://static.igem.org/mediawiki/2014/e/e5/Bnu_moda4_01.jpg"> </a>
+
<br />
<p class="fig" style="width:85%; margin-left:50px">Figure 2 Native-PAGE to provide the molybdate lane 1, the modA(acetic buffer PH 5.0); lane 2, the modA incubate with 20mM molybdate.</p>
+
<div style="float:left; margin-left: 20px; margin-top: 40px; width: 40%; padding: 30px 20px 30px 30px;"><p style=" background-color: rgba(226,210,197,0.5);font-size:12px; padding: 8px 20px 8px 20px; border-radius: 0.5em 0.5em 0.5em 0.5em;  ">Fig.1 Agar assay: The three plates shows the chemotaxis results of 8F towards three kinds of attractants, malate, citrate and succinate from left to right. The filter paper of attractants is put on the left, the PBS is put on the right, bacteria is in the middle. Here, 8F swims fastest to malate. </p></div>
 +
<div style="width:100%; float: left;"><br /><br/></div>
 +
<a title="Fig.2 <i>E. coli</i>’s ability of chemotaxis towards different concentrations of succinate or malate." href="https://static.igem.org/mediawiki/2014/7/79/Bnu_delivery3.png" rel="prettyPhoto"> <span class="overlay zoom" style="display: none;"></span><img style="opacity: 1; width:80%; margin-left: 100px;" src="https://static.igem.org/mediawiki/2014/7/79/Bnu_delivery3.png"> </a>
 +
<br/><br/>
 +
<p style=" background-color: rgba(226,210,197,0.5);font-size:12px;padding: 8px 20px 8px 20px; border-radius: 0.5em 0.5em 0.5em 0.5em;  ">Fig.2 <i>E. coli</i>’s ability of chemotaxis towards different concentrations of succinate or malate.<br/>The cells were diluted 20000 times. 5A is a control which doesn’t have chemotaxis towards malate or succinate.<br/>
 +
<strong>Malate</strong>: 8F showed the strongest respond. The tendency of curve of 8F was biphasic with maximums at attractant concentration of about 10<sup>-2</sup> M and 10<sup>-5</sup> M, and reached minimum at attractant concentration of about 10<sup>-1</sup> M, which is similar to others’ work. With attractant concentration decreasing for 8F, the number of cells decreased slowly. McfR stayed stable at range of 10<sup>-7</sup> M to 10<sup>-3</sup> M, and had only one phase which reached its maximum at attractant concentration of 10<sup>-2</sup> M, then it went down sharply at 10<sup>-1</sup> M. There was a significant difference among 8F and 5A (p < 0.05), and quantity of cells of 8F was much more than that of 5A. So 8F has chemotaxis towards malate. On the other hand, McfR and 5A were not significantly different and quantity of cells of McfR was little less than it of 5A. So McfR shows no chemotaxis towards malate here.<br/>     
 +
<strong>Succinate</strong>: The tendencies of curves of 8F and McfR towards succinate are same. As the attractant concentration increased, the number of cells arose and reached the maximum at attractant concentration of about 10<sup>-2</sup> M and fell sharply with the minimum at attractant concentration of 10<sup>-1</sup> M. The quantities of cells of 8F and McfR were almost equivalent and did not have significant difference. But there were significant differences among 8F & 5A, and McfR & 5A: the number of cells of 5A are far less than 8F or McfR (p < 0.05), which demonstrated that 8F and McfR have chemotaxis towards succinate and the capacity of chemotaxis of 8F and McfR towards succinate are almost equal. Compared with chemotaxis towards malate, both of 8F and McfR show stronger chemotaxis towards succinate.
 +
</p>
 +
<br/><br/>
 +
</body>
 +
</html>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 14:07, 17 October 2014

McfR

Background

McfR is a TCA intermediates responsive chemoreceptor that originally found in ''Pseudomonas putida''. BBa_K1405004 contains McfR coding sequence, an insulator, RBS sequence, and the constitutive promoter BBa_J23100, which is similar to BBa_K515102 that can help us compare our work with iGEM11_Imperial_College _London had done. And we showed the results below. A 15 bp insulator sequence upstream of the RBS ensures tunability of expression through easy switching of promoters. In addition it allows the translation initiation rate (TIR) of the RBS to remain the same, when the promoter is replaced.This device is compatible with motile strains of E. coli. It has been transformed and tested in E. coli BL21 in the vector backbone pSB1C3. This device is used as an additional chemoreceptor for endogenous chemotaxis system in E. coli and responds to L(-) malic acid (HO2CCH2CHOHCO2H) and succinate (HOOC-(CH2)2-COOH).

Results

插图 Expression and purification of ModA. Expression of ModA and its purification by His-chelating chromatography. Lane 1, molecular weight standards (kDa); lane 2, total bacterial proteins before IPTG induction; lane 3, total bacterial proteins after 0.5 mM IPTG induction; lane 4, total bacterial lysate after 0.5 mM IPTG induction; lane 5,the sediment after 16,000 rpm 30 min; lane 6 the flow through of column loading; lane 7,the flow of buffer B .lane 8 The purification of ModA by size-elution chromatography. 插图 Native-PAGE to provide the molybdate lane 1, the modA(acetic buffer PH 5.0); lane 2, the modA incubate with 20mM molybdate.



Fig.1 Agar assay: The three plates shows the chemotaxis results of 8F towards three kinds of attractants, malate, citrate and succinate from left to right. The filter paper of attractants is put on the left, the PBS is put on the right, bacteria is in the middle. Here, 8F swims fastest to malate.





Fig.2 E. coli’s ability of chemotaxis towards different concentrations of succinate or malate.
The cells were diluted 20000 times. 5A is a control which doesn’t have chemotaxis towards malate or succinate.
Malate: 8F showed the strongest respond. The tendency of curve of 8F was biphasic with maximums at attractant concentration of about 10-2 M and 10-5 M, and reached minimum at attractant concentration of about 10-1 M, which is similar to others’ work. With attractant concentration decreasing for 8F, the number of cells decreased slowly. McfR stayed stable at range of 10-7 M to 10-3 M, and had only one phase which reached its maximum at attractant concentration of 10-2 M, then it went down sharply at 10-1 M. There was a significant difference among 8F and 5A (p < 0.05), and quantity of cells of 8F was much more than that of 5A. So 8F has chemotaxis towards malate. On the other hand, McfR and 5A were not significantly different and quantity of cells of McfR was little less than it of 5A. So McfR shows no chemotaxis towards malate here.
Succinate: The tendencies of curves of 8F and McfR towards succinate are same. As the attractant concentration increased, the number of cells arose and reached the maximum at attractant concentration of about 10-2 M and fell sharply with the minimum at attractant concentration of 10-1 M. The quantities of cells of 8F and McfR were almost equivalent and did not have significant difference. But there were significant differences among 8F & 5A, and McfR & 5A: the number of cells of 5A are far less than 8F or McfR (p < 0.05), which demonstrated that 8F and McfR have chemotaxis towards succinate and the capacity of chemotaxis of 8F and McfR towards succinate are almost equal. Compared with chemotaxis towards malate, both of 8F and McfR show stronger chemotaxis towards succinate.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1274
  • 1000
    COMPATIBLE WITH RFC[1000]