Team:SDU-Denmark/project-r

From 2010.igem.org

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== Photosensor ==
== Photosensor ==
=== Biobrick assembly and sequence ===
=== Biobrick assembly and sequence ===
-
We succesfully assembled the biobrick K343007, which consists of the TetR repressible promoter R0040 followed by the ribosome binding site B0034, the coding sequence K343003 and the terminators B0010 and B0012.<br>
+
We succesfully assembled the biobrick [http://partsregistry.org/Part:BBa_K343007 K343007], which consists of the TetR repressible promoter R0040 followed by the ribosome binding site B0034, the coding sequence K343003 and the terminators B0010 and B0012.<br>
This was achieved through "standard" silver assembly and confirmed via sequencing with biobrick verification forward 2 (VF2) and verification reverse (VR) primers.<br>
This was achieved through "standard" silver assembly and confirmed via sequencing with biobrick verification forward 2 (VF2) and verification reverse (VR) primers.<br>
-
Sequencing results (as .ab1 files):<br>
 
<br>
<br>
-
[https://static.igem.org/mediawiki/2010/a/ab/Sequencing_K343007.ab1| Sequencing results K343007]
+
[http://partsregistry.org/cgi/sequencing/one_blast.cgi?id=8039| Sequencing results K343007]
 +
<br><br>
 +
The sequence obtained from sequencing is identical with the theoretical sequence of the part, except for a silent mutation in the coding sequence, which does not change the amino acid sequence.
 +
 
 +
=== Effect ===
 +
Our results from the experiments with semi-solid agar confirms that the bacteria carrying a plasmid with the biobrick have a altered motility, most likely by an alteration in tumbling frequency, when exposed to blue light compared to the wild type MG1655.<br>
<br>
<br>
 +
The videomicroscopy indicates that bacteria carrying a plasmid with the biobrick travel father and in a more straight path than the wild type MG1655 when exposed to blue light with a wavelength around 500nm, Thereby suggesting a lowered tubling frequency. <br>
<br>
<br>
-
=== Effect ===
+
Computeranalysis of videos recorded of swimming baceria containing this part, indicate that these bacteria have a tendency to swim towards the source of light, when exposed to a light gradient. A trackdiagram was constructed from the gathered data, and swimming bacteria tended to orientate their movement in the direction of the light source. This could mean that blue light acts as an attractant stimulus on the SopII-HtrII-Tar complex. <br><br>
-
 
+
The stability of pSB1C3-K343007 is most likely <20 generations, however the stability of pSB3T5-K343007 was not determined. <br>
-
 
+
Plasmids expressing K343007 does not seem to hinder the bacterial growth in any way.<br>
-
=== Stability assay ===
+
For further information, raw data and background of the assay see [https://2010.igem.org/Team:SDU-Denmark/K343007 characterization of K343007].
-
<html>
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  <meta content="text/html; charset=ISO-8859-1"
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  <title></title>
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</head>
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<body>
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<table x:str=""
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style="border-collapse: collapse; width: 428pt;" border="0"
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cellpadding="0" cellspacing="0" width="570">
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  <col style="width: 75pt; font-weight: bold;" width="100">
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  <col style="width: 79pt; font-weight: bold;" width="105">
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  <col style="width: 128pt; font-weight: bold;" width="170">
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  <col style="width: 53pt; font-weight: bold;" width="71">
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  <col style="width: 93pt; font-weight: bold;" width="124">
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  <tbody>
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    <tr style="height: 15pt;" height="20">
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      <td class="xl65"
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style="height: 15pt; width: 75pt; font-weight: bold;"
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height="20" width="100">Stability assay.</td>
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      <td class="xl65" style="width: 79pt;" width="105"></td>
+
-
      <td style="width: 128pt;" width="170"></td>
+
-
      <td style="width: 53pt;" width="71"></td>
+
-
      <td style="width: 93pt;" width="124"></td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20"></td>
+
-
      <td></td>
+
-
      <td></td>
+
-
      <td></td>
+
-
      <td></td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td class="xl65"
+
-
style="height: 15pt; text-decoration: underline;" height="20">0
+
-
generations</td>
+
-
      <td style="font-weight: bold;">Dilution number</td>
+
-
      <td style="font-weight: bold;">Colonies counted on
+
-
plate</td>
+
-
      <td style="font-weight: bold;">Cfu</td>
+
-
      <td style="font-weight: bold;">PS-chlor/PS+chlor</td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS1 +
+
-
chlor</td>
+
-
      <td x:num="" align="right">6</td>
+
-
      <td x:num="" align="right">168</td>
+
-
      <td x:num="8400000000" x:fmla="=(10^B4)*C4*50"
+
-
align="right">8.4E+09</td>
+
-
      <td x:num="" align="right">100</td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS1 -
+
-
chlor</td>
+
-
      <td x:num="" align="right">6</td>
+
-
      <td x:num="" align="right">157</td>
+
-
      <td x:num="7850000000" x:fmla="=(10^B5)*C5*50"
+
-
align="right">7.85E+09</td>
+
-
      <td></td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS2 +
+
-
chlor</td>
+
-
      <td x:num="" align="right">6</td>
+
-
      <td x:num="" align="right">192</td>
+
-
      <td x:num="9600000000" x:fmla="=(10^B6)*C6*50"
+
-
align="right">9.6E+09</td>
+
-
      <td x:num="" align="right">100</td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS2 -
+
-
chlor</td>
+
-
      <td x:num="" align="right">6</td>
+
-
      <td x:num="" align="right">216</td>
+
-
      <td x:num="10800000000" x:fmla="=(10^B7)*C7*50"
+
-
align="right">1.08E+10</td>
+
-
      <td></td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td class="xl65"
+
-
style="height: 15pt; text-decoration: underline;" height="20">20
+
-
generations</td>
+
-
      <td class="xl65"></td>
+
-
      <td></td>
+
-
      <td></td>
+
-
      <td></td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS1 +
+
-
chlor</td>
+
-
      <td x:num="" align="right">6</td>
+
-
      <td x:num="" align="right">157</td>
+
-
      <td x:num="7850000000" x:fmla="=(10^B9)*C9*50"
+
-
align="right">7.85E+09</td>
+
-
      <td x:num="59.245283018867923" x:fmla="=(D9/D10)*100"
+
-
align="right">59.24528302</td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS1 -
+
-
chlor</td>
+
-
      <td x:num="" align="right">6</td>
+
-
      <td x:num="" align="right">265</td>
+
-
      <td x:num="13250000000" x:fmla="=(10^B10)*C10*50"
+
-
align="right">1.325E+10</td>
+
-
      <td></td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS2 +
+
-
chlor</td>
+
-
      <td x:num="" align="right">6</td>
+
-
      <td x:num="" align="right">130</td>
+
-
      <td x:num="6500000000" x:fmla="=(10^B11)*C11*50"
+
-
align="right">6.5E+09</td>
+
-
      <td x:num="63.414634146341463" x:fmla="=(D11/D12)*100"
+
-
align="right">63.41463415</td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS2 -
+
-
chlor</td>
+
-
      <td x:num="" align="right">6</td>
+
-
      <td x:num="" align="right">205</td>
+
-
      <td x:num="10250000000" x:fmla="=(10^B12)*C12*50"
+
-
align="right">1.025E+10</td>
+
-
      <td></td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td class="xl65"
+
-
style="height: 15pt; text-decoration: underline;" height="20">40
+
-
generations</td>
+
-
      <td class="xl65"></td>
+
-
      <td></td>
+
-
      <td></td>
+
-
      <td></td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS1 +
+
-
chlor</td>
+
-
      <td x:num="" align="right">6</td>
+
-
      <td x:num="" align="right">143</td>
+
-
      <td x:num="7150000000" x:fmla="=(10^B14)*C14*50"
+
-
align="right">7.15E+09</td>
+
-
      <td x:num="95.973154362416096" x:fmla="=(D14/D15)*100"
+
-
align="right">95.97315436</td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS1 -
+
-
chlor</td>
+
-
      <td x:num="" align="right">6</td>
+
-
      <td x:num="" align="right">149</td>
+
-
      <td x:num="7450000000" x:fmla="=(10^B15)*C15*50"
+
-
align="right">7.45E+09</td>
+
-
      <td></td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS2 +
+
-
chlor</td>
+
-
      <td x:num="" align="right">6</td>
+
-
      <td x:num="" align="right">168</td>
+
-
      <td x:num="8400000000" x:fmla="=(10^B16)*C16*50"
+
-
align="right">8.4E+09</td>
+
-
      <td x:num="92.817679558011051" x:fmla="=(D16/D17)*100"
+
-
align="right">92.81767956</td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS2 -
+
-
chlor</td>
+
-
      <td x:num="" align="right">6</td>
+
-
      <td x:num="" align="right">181</td>
+
-
      <td x:num="9050000000" x:fmla="=(10^B17)*C17*50"
+
-
align="right">9.05E+09</td>
+
-
      <td></td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td class="xl65"
+
-
style="height: 15pt; text-decoration: underline;" height="20">60
+
-
generations</td>
+
-
      <td style="text-decoration: underline;" class="xl65"></td>
+
-
      <td style="text-decoration: underline;"></td>
+
-
      <td style="text-decoration: underline;"></td>
+
-
      <td style="text-decoration: underline;"></td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS1 +
+
-
chlor</td>
+
-
      <td x:num="" align="right">6</td>
+
-
      <td x:num="" align="right">147</td>
+
-
      <td x:num="7350000000" x:fmla="=(10^B19)*C19*50"
+
-
align="right">7.35E+09</td>
+
-
      <td x:num="89.634146341463421" x:fmla="=(D19/D20)*100"
+
-
align="right">89.63414634</td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS1 -
+
-
chlor</td>
+
-
      <td x:num="" align="right">6</td>
+
-
      <td x:num="" align="right">164</td>
+
-
      <td x:num="8200000000" x:fmla="=(10^B20)*C20*50"
+
-
align="right">8.2E+09</td>
+
-
      <td></td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS2 +
+
-
chlor</td>
+
-
      <td x:num="" align="right">6</td>
+
-
      <td x:num="" align="right">134</td>
+
-
      <td x:num="6700000000" x:fmla="=(10^B21)*C21*50"
+
-
align="right">6.7E+09</td>
+
-
      <td x:num="104.6875" x:fmla="=(D21/D22)*100"
+
-
align="right">104.6875</td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS2 -
+
-
chlor</td>
+
-
      <td x:num="" align="right">6</td>
+
-
      <td x:num="" align="right">128</td>
+
-
      <td x:num="6400000000" x:fmla="=(10^B22)*C22*50"
+
-
align="right">6.4E+09</td>
+
-
      <td></td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td class="xl65"
+
-
style="height: 15pt; text-decoration: underline;" height="20">80
+
-
generations</td>
+
-
      <td class="xl65"></td>
+
-
      <td></td>
+
-
      <td></td>
+
-
      <td></td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS1 +
+
-
chlor</td>
+
-
      <td x:num="" align="right">6</td>
+
-
      <td x:num="" align="right">127</td>
+
-
      <td x:num="6350000000" x:fmla="=(10^B24)*C24*50"
+
-
align="right">6.35E+09</td>
+
-
      <td x:num="136.55913978494624" x:fmla="=(D24/D25)*100"
+
-
align="right">136.5591398</td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS1 -
+
-
chlor</td>
+
-
      <td x:num="" align="right">6</td>
+
-
      <td x:num="" align="right">93</td>
+
-
      <td x:num="4650000000" x:fmla="=(10^B25)*C25*50"
+
-
align="right">4.65E+09</td>
+
-
      <td></td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS2 +
+
-
chlor</td>
+
-
      <td x:num="" align="right">6</td>
+
-
      <td x:num="" align="right">99</td>
+
-
      <td x:num="4950000000" x:fmla="=(10^B26)*C26*50"
+
-
align="right">4.95E+09</td>
+
-
      <td x:num="104.21052631578947" x:fmla="=(D26/D27)*100"
+
-
align="right">104.2105263</td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS2 -
+
-
chlor</td>
+
-
      <td x:num="" align="right">6</td>
+
-
      <td x:num="" align="right">95</td>
+
-
      <td x:num="4750000000" x:fmla="=(10^B27)*C27*50"
+
-
align="right">4.75E+09</td>
+
-
      <td></td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td class="xl65" colspan="2"
+
-
style="height: 15pt; text-decoration: underline;" height="20">100
+
-
generations</td>
+
-
      <td></td>
+
-
      <td></td>
+
-
      <td></td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS1 +
+
-
chlor</td>
+
-
      <td x:num="" align="right">5</td>
+
-
      <td x:num="" align="right">130</td>
+
-
      <td x:num="650000000" x:fmla="=(10^B29)*C29*50"
+
-
align="right">650000000</td>
+
-
      <td x:num="" x:fmla="=(D29/D30)*100"
+
-
align="right">125</td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS1 -
+
-
chlor</td>
+
-
      <td x:num="" align="right">5</td>
+
-
      <td x:num="" align="right">104</td>
+
-
      <td x:num="" x:fmla="=(10^B30)*C30*50"
+
-
align="right">520000000</td>
+
-
      <td></td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS2 +
+
-
chlor</td>
+
-
      <td x:num="" align="right">5</td>
+
-
      <td x:num="" align="right">124</td>
+
-
      <td x:num="620000000" x:fmla="=(10^B31)*C31*50"
+
-
align="right">620000000</td>
+
-
      <td x:num="118.0952380952381" x:fmla="=(D31/D32)*100"
+
-
align="right">118.0952381</td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt;" height="20">PS2 -
+
-
chlor</td>
+
-
      <td x:num="" align="right">5</td>
+
-
      <td x:num="" align="right">105</td>
+
-
      <td x:num="" x:fmla="=(10^B32)*C32*50"
+
-
align="right">525000000</td>
+
-
      <td></td>
+
-
    </tr>
+
-
  </tbody>
+
-
</table>
+
-
</body>
+
-
</html>
+
 +
==Flagella==
 +
=== Biobrick assembly and sequence ===
 +
We succesfully assembled the biobrick [http://partsregistry.org/Part:BBa_K343004 K343004], which consists of the TetR repressible promoter R0040 followed by the ribosome binding site B0034, the coding sequence K343000 and the terminators B0010 and B0012.<br>
 +
This was achieved through "standard" silver assembly and confirmed via sequencing with biobrick verification forward 2 (VF2) and verification reverse (VR) primers.<br>
 +
<br>
 +
[http://partsregistry.org/cgi/sequencing/one_blast.cgi?id=8031| Sequencing results for K343004]
<br><br>
<br><br>
 +
When comparing the theoretical sequence with the sequence done on the part K343004 the two sequences were identical.
-
<html>
+
=== Effect ===
-
<head>
+
Our results from the motility assay with semi-solid agar confirm that the biobrick does in fact increase the motility of the cells. We believe this is caused by hyperflagellation facilitated by the overexpression of the flhD/C operon. <br>
-
  <meta content="text/html; charset=ISO-8859-1"
+
The stability of pSB1C3-K343004 is most likely <20 generations, however the stability of pSB3T5-K343004 was not determined.<br>
-
http-equiv="content-type">
+
The growth assay showed no significant difference between the wild type and the cells containing either pSB1C3-K343004 or pSB3K3-K343004.<br>
-
  <title></title>
+
In a phase contrast microscope we observed that MG1655 containing pSB1C3-K343004 was longer that wild type. This support the theory that the cells are hyperflagellated and therefore stays longer in the G2-phase in the cell cycle.  
-
</head>
+
For further information, raw data and background of the assay see [https://2010.igem.org/Team:SDU-Denmark/K343004 characterization of K343004]. <br><br>
-
<body>
+
-
<table x:str=""
+
-
style="border-collapse: collapse; width: 507px;" border="0"
+
-
cellpadding="0" cellspacing="0" width="516">
+
-
  <col style="width: 110pt;" width="147"> <col
+
-
span="2" style="width: 48pt;" width="64"> <col
+
-
style="width: 85pt;" width="113"> <col
+
-
style="width: 96pt;" width="128"> <tbody>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt; font-weight: bold; width: 91px;"
+
-
height="20">Number of generations</td>
+
-
      <td style="font-weight: bold; width: 102px;">PS1</td>
+
-
      <td style="font-weight: bold; width: 102px;">PS2</td>
+
-
      <td style="font-weight: bold; width: 102px;">PS
+
-
(average)</td>
+
-
      <td style="font-weight: bold; width: 102px;">Standard
+
-
deviations</td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt; width: 91px;" x:num=""
+
-
align="right" height="20">0</td>
+
-
      <td style="width: 102px;" x:num="" align="right">100</td>
+
-
      <td style="width: 102px;" x:num="" align="right">100</td>
+
-
      <td style="width: 102px;" x:num=""
+
-
x:fmla="=(B2+C2)/2" align="right">100</td>
+
-
      <td style="width: 102px;" x:num=""
+
-
x:fmla="=STDEV(B2,C2)" align="right">0</td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt; width: 91px;" x:num=""
+
-
align="right" height="20">20</td>
+
-
      <td style="width: 102px;" x:num="59.245283018867923"
+
-
align="right">59.24528</td>
+
-
      <td style="width: 102px;" x:num="63.414634146341463"
+
-
align="right">63.41463</td>
+
-
      <td style="width: 102px;" x:num="61.329958582604689"
+
-
x:fmla="=(B3+C3)/2" align="right">61.32995858</td>
+
-
      <td style="width: 102px;" x:num="2.948176455384516"
+
-
x:fmla="=STDEV(B3,C3)" align="right">2.948176455</td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt; width: 91px;" x:num=""
+
-
align="right" height="20">40</td>
+
-
      <td style="width: 102px;" x:num="95.973154362416096"
+
-
align="right">95.97315</td>
+
-
      <td style="width: 102px;" x:num="92.817679558011051"
+
-
align="right">92.81768</td>
+
-
      <td style="width: 102px;" x:num="94.395416960213566"
+
-
x:fmla="=(B4+C4)/2" align="right">94.39541696</td>
+
-
      <td style="width: 102px;" x:num="2.2312576320582482"
+
-
x:fmla="=STDEV(B4,C4)" align="right">2.231257632</td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt; width: 91px;" x:num=""
+
-
align="right" height="20">60</td>
+
-
      <td style="width: 102px;" x:num="89.634146341463421"
+
-
align="right">89.63415</td>
+
-
      <td style="width: 102px;" x:num="104.6875"
+
-
align="right">104.6875</td>
+
-
      <td style="width: 102px;" x:num="97.160823170731703"
+
-
x:fmla="=(B5+C5)/2" align="right">97.16082317</td>
+
-
      <td style="width: 102px;" x:num="10.644328451550683"
+
-
x:fmla="=STDEV(B5,C5)" align="right">10.64432845</td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt; width: 91px;" x:num=""
+
-
align="right" height="20">80</td>
+
-
      <td style="width: 102px;" x:num="136.55913978494624"
+
-
align="right">136.5591</td>
+
-
      <td style="width: 102px;" x:num="104.21052631578947"
+
-
align="right">104.2105</td>
+
-
      <td style="width: 102px;" x:num="120.38483305036786"
+
-
x:fmla="=(B6+C6)/2" align="right">120.3848331</td>
+
-
      <td style="width: 102px;" x:num="22.873923946023144"
+
-
x:fmla="=STDEV(B6,C6)" align="right">22.87392395</td>
+
-
    </tr>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td style="height: 15pt; width: 91px;" x:num=""
+
-
align="right" height="20">100</td>
+
-
      <td style="width: 102px;" x:num="" align="right">125</td>
+
-
      <td style="width: 102px;" x:num="118.0952380952381"
+
-
align="right">118.0952</td>
+
-
      <td style="width: 102px;" x:num="121.54761904761905"
+
-
x:fmla="=(B7+C7)/2" align="right">121.547619</td>
+
-
      <td style="width: 102px;" x:num="4.8824039653356079"
+
-
x:fmla="=STDEV(B7,C7)" align="right">4.882403965</td>
+
-
    </tr>
+
-
  </tbody>
+
-
</table>
+
-
</body>
+
-
</html>
+
-
 
+
-
===Flagella===
+
-
-----
+
-
===Motility assay===
+
-
<br>Exp. 1<br>
+
-
 
+
-
The purpose of this experiment is to see if the cells containing pSB1C3-K343004 move farther than the wild type (MG1655) and the negative control (DH5alpha). This would be an indication of hyperflagellation. We also want to test if it makes a difference in the motility wether the bacteria contain low-medium- or high-copy plasmids. <br><br>
+
-
For the motility experiments we added 5ul of an ON culture to petridishes containing motility agar (LB media with 0.3% agar) instead of regular LA (Luria agar). This semi-solid media lets the bacteria swim more easily. <br>
+
-
The plates were incubated at 37 degrees celcius for 24 hours.<br><br>
+
-
[[Image:Team-SDU-Denmark-Flagellamotility-exp1-DH5a.JPG|250px|DH5alpha]]
+
-
[[Image:Team-SDU-Denmark-Flagellamotility-exp1-MG1655.JPG|250px|MG1655]]<br><br>
+
-
[[Image:Team-SDU-Denmark-Flagellamotility-exp1-FlhDCmutCP_i_pSB1C3_(LA+chlor).JPG|250px|pSB1C3-FlhDCmutCP(LA+chlor).]]
+
-
[[Image:Team-SDU-Denmark-Flagellamotility-exp1-FlhDCmutCP_i_pSB3K3_(LA+kan).JPG|250px|pSB3K3-FlhDCmutCP(LA+Kan)]]
+
-
<br><br>
+
-
The upper two plates did not contain antibiotics, and therefore contamination colonies are seen.<br>
+
-
The upper left picture is of ''E. coli'' strain '''DH5alpha''' that does not express flagella and therefor movement in the media should, as seen on the picture, be minimal. The upper right picture is of the wild type ''E. coli'' strain '''MG1655''', this strain has about 8-10 flagella per cell. These cells are, as seen, expected to move farther than the DH5alpha but not as far as the transformed cells. The lower left picture is of ''E. Coli'' strain '''MG1655 with pSB1C3-K343004''' this shows that these bacteria move farther than the wild type and the negative control. The lover right picture is of ''E. coli'' strain '''MG1655 with pSB3K3-K343004''' these bacteria move farther than the wild type, the negative control and MG1655 with pSB3K3-K343004.<br><br>
+
-
 
+
-
From the pictures above we can definately se that the bacteria containing our part is much more motile than the wild type. We assume this is caused by overexpression of the FlhDC master flagella operon which leads to hyperflagellation of the cells. <br> The two buttom pictures show that bacteria with pSB1C3-K343004 have not moved as far as the bacteria containing pSB3K3-K343004. pSB1C3 is a high copy plasmid while pSB3K3 is a low-medium copy plasmid. The promoters in K343004 is a constitutive promoter (tetR repressable promoter). Bacteria containing a high copy plasmid with a constitutive promoter are more metabolically challanged than bacteria containing a low- or medium-copy plasmid with a constitutive promoter because of the higher number of plasmids per the cell. Therefore the high copy plasmid bacteria are less motile than low- or medium-copy plasmid bacteria.<br>
+
== Retinal ==
== Retinal ==
-
 
+
=== Biobrick assembly and sequence ===
-
=== UV-vis determination of beta-carotene production ===  
+
We succesfully assembled the biobrick [http://partsregistry.org/Part:BBa_K343006 K343006], which consists of the TetR repressible promoter R0040 followed by the ribosome binding site B0034, the coding sequence K343002 and the terminators B0010 and B0012.<br>
-
<p style="text-align: justify;">
+
This was achieved through "standard" silver assembly and confirmed via sequencing with biobrick verification forward 2 (VF2) and verification reverse (VR) primers.<br>
 +
Sequencing results (as .ab1 files):<br>
<br>
<br>
-
In this experiment cells were prepared and harvested according to protocol [https://2010.igem.org/Team:SDU-Denmark/protocols#EX1.1]. This experiment was performed with four different strains of ''E. coli'': <br>
+
[http://partsregistry.org/cgi/sequencing/one_blast.cgi?id=8025| Sequencing results for K343006]
-
<br>
+
-
Wildtype Top10 <br>
+
-
Wildtype MG1655 <br>
+
-
Top10 containing PSB1A2 with constitutively active K274210 <br>
+
-
MG1655 containing PSB1A2 with  constitutively active K274210 <br> <br>
+
-
The measurements were performed on cells both in the exponential and stationary phase. The resulting graphs are presented below:<br>
+
-
<br>
+
-
[[Image:Team-SDU-denmarkBetacarotene samples with controles (exponential_phase).png |350px]]
+
-
[[Image:Team-SDU-denmarkBetacarotene samples with controles (stationary phase).png |350px]]
+
-
<br>
+
-
<br>
+
-
In order to assess the results, a series of standard dilutions of beta-carotene were made. The concentrations were 1mM, 100 µM, 50 µM, 25 µM, 10 µM, 5 µM, 1 µM, 100 nM and 10 nM. The standard dilutions were also measured on the spectrophotometer. The resulting graphs are presented low: <br>
+
-
<br>
+
-
[[Image:Team-SDU-denmarkStandart betacaroten curves.png |350px]]
+
-
<br><br>
+
-
When combining the graphs of the samples and the standard dilutions, it is clear that the spectres are uniform. This similarity of the spectres indicates that the cells actually do produce beta-carotene. The graphs also give a qualitative indication of the amount of beta-carotene produced by the MG1655 and the Top10 ''E. coli'' cells. The resulting graphs are presented below: <br><br>
+
-
[[Image:Image-Team-SDU-denmarkBetacarotene standarts and stationary samples.png |350px]]
+
<br><br>
<br><br>
-
</p>
+
When comparing the theoretical sequence with the sequence done on the part K343006 the two sequences was identical.
-
=== UV-Vis determination of beta-carotene and retinal production ===
+
=== Effect ===
-
<p style="text-align: justify;">In this experiment cells was prepare and harvested according to protocol [https://2010.igem.org/Team:SDU-Denmark/protocols#EX1.1]. This experiment was preformed with six different strain of E. Coli:
+
UV-Vis spectroskopy and HPLC analysis of organic extractions of the bacteria containing K343006 didn't give conclusive results conserning the Retinal production.<br>
-
wildtype Top10<br>
+
The stability of pSB1C3-K343006 is most likely <20 generations.<br>
-
wildtype MG1655<br>
+
The growth assay showed no significant difference between the wild type and the cells containing pSB1C3-K343006.<br>
-
Top10 containing PSB1A2 with K274210 constitutively active<br>
+
For further information, raw data and background of the assay see [https://2010.igem.org/Team:SDU-Denmark/K343006 characterization of K343006]
-
MG1655 containing PSB1A2 with K274210 constitutively active<br>
+
-
TOP10 containing PSB1A2 with K274210 plus PSB1C3 with K343005 both under a constitutively active promotor<br>
+
-
MG1655 containing PSB1A2 with K274210 plus PSB1C3 with K343005 both under a constitutively active promotor<br>
+
-
The measurements were preformed on cells after 20 hours of growth, because the first experiment showed none or little product produced in the exponential phase. The resulting graphs is presented beneath the text.<br>
+
-
[[Image: Team-SDU-denmark-Samples form top10 mg1655 and control.png |350px|]]
+
-
<br>
+
-
In the spectre a sudden drop at 320 nm occurs, this cannot be explained .<br>
+
-
The graph also shows that some cell material interferes with the UV-Vis measurement at the retinal has the strongest absorption, because of this some organic separation might be needed before the measurement is preformed.<br>
+
-
In order assess the results a standard dilution of beta-carotene and retinal was made and measured: 1mM, 100 µM, 50 µM, 25 µM, 10 µM, 5 µM, 1 µM, 100 nM and 10 nM. The standart dilutions was also measured on the spectrophotometer. The resulting graphs is presented beneath the text<br>
+
-
[[Image: Team-SDU-denmark-Beta-carotene standarts.png |350px|]]
+
-
[[Image: Team-SDU-denmark-Retinal standarts.png |350px|]]
+
-
<br>
+
-
The spectrum for the Beta-carotene looks normal and reliable, the standards for retinal however doesn’t look reliable. The spectrum for the three lowest concentrations appears to be give a wrong picture from the measurement.<br>  
+
-
The measurement jumps rapidly from nearly nothing to the maximum absorbance, this might be because the concentration of retinal is to low to be measured as a spectrum, but the concentration of the individual derivates of retinal might be high enough to be measured.<br>
+
-
It might also just be that the concentrations are to low and something ells are influencing the measurements.<br>
+
-
Because of the error in the UV-vis spectre concerning retinal detection in both the standards and samples the next characterization experiments is preformed on a HPLC.
+
-
<br><br>
+
-
</p>
+
-
 
-
 
-
== OD measurment of MG1655 ==
 
-
<br>
 
-
<html>
 
-
<head>
 
-
  <meta content="text/html; charset=ISO-8859-1"
 
-
http-equiv="content-type">
 
-
  <title></title>
 
-
</head>
 
-
<body>
 
-
<table x:str=""
 
-
style="border-collapse: collapse; background-color: white; width: 630px; height: 440px; text-align: left; margin-left: auto; margin-right: auto;"
 
-
border="1" cellpadding="2" cellspacing="2">
 
-
  <col style="width: 89pt;" width="119"> <col
 
-
span="2" style="width: 67pt;" width="89"> <col
 
-
style="width: 60pt;" width="80"> <col
 
-
style="width: 67pt;" width="89"> <col
 
-
style="width: 62pt;" width="82"> <col
 
-
style="width: 67pt;" width="89"> <col
 
-
style="width: 82pt;" width="109"> <col
 
-
style="width: 67pt;" width="89"> <col
 
-
style="width: 80pt;" width="107"> <col
 
-
style="width: 67pt;" width="89"> <col
 
-
style="width: 48pt;" width="64"> <col
 
-
style="width: 67pt;" width="89"> <tbody>
 
-
    <tr style="height: 15pt;" height="20">
 
-
      <td style="height: 15pt; width: 89pt;" height="20"
 
-
width="119"></td>
 
-
      <td style="width: 67pt; font-weight: bold;" width="89">Mg1655</td>
 
-
      <td style="width: 67pt; font-weight: bold;" width="89"></td>
 
-
      <td colspan="2" style="width: 127pt; font-weight: bold;"
 
-
width="169">PS in pSB3T5</td>
 
-
      <td colspan="2" style="width: 129pt; font-weight: bold;"
 
-
width="171">PS in pSB1C3</td>
 
-
      <td style="width: 82pt; font-weight: bold;"
 
-
x:str="flhDC in pSB3K3 " width="109">flhDC in pSB3K3<span
 
-
style="">&nbsp;</span></td>
 
-
      <td style="width: 67pt; font-weight: bold;" width="89"></td>
 
-
      <td style="width: 80pt; font-weight: bold;" width="107">flhDC
 
-
in pSB1C3</td>
 
-
      <td style="width: 67pt; font-weight: bold;" width="89"></td>
 
-
      <td colspan="2" style="width: 115pt; font-weight: bold;"
 
-
width="153">NinaB in pSB1C3</td>
 
-
    </tr>
 
-
    <tr style="height: 15pt;" height="20">
 
-
      <td style="height: 15pt; font-weight: bold;" height="20">Hours</td>
 
-
      <td x:num="" align="right">1</td>
 
-
      <td x:num="" align="right">2</td>
 
-
      <td x:num="" align="right">1</td>
 
-
      <td x:num="" align="right">2</td>
 
-
      <td x:num="" align="right">1</td>
 
-
      <td x:num="" align="right">2</td>
 
-
      <td x:num="" align="right">1</td>
 
-
      <td x:num="" align="right">2</td>
 
-
      <td x:num="" align="right">1</td>
 
-
      <td x:num="" align="right">2</td>
 
-
      <td x:num="" align="right">1</td>
 
-
      <td x:num="" align="right">2</td>
 
-
    </tr>
 
-
    <tr style="height: 15pt;" height="20">
 
-
      <td style="height: 15pt;" x:num="" align="right"
 
-
height="20">0</td>
 
-
      <td x:num="2.4E-2" align="right">0.024</td>
 
-
      <td x:num="2.5999999999999999E-2" align="right">0.026</td>
 
-
      <td x:num="2.8000000000000001E-2" align="right">0.028</td>
 
-
      <td x:num="" align="right">0.03</td>
 
-
      <td x:num="2.4E-2" align="right">0.024</td>
 
-
      <td x:num="2.4E-2" align="right">0.024</td>
 
-
      <td x:num="2.8000000000000001E-2" align="right">0.028</td>
 
-
      <td x:num="2.7E-2" align="right">0.027</td>
 
-
      <td x:num="2.8000000000000001E-2" align="right">0.028</td>
 
-
      <td x:num="2.7E-2" align="right">0.027</td>
 
-
      <td x:num="2.7E-2" align="right">0.027</td>
 
-
      <td x:num="2.7E-2" align="right">0.027</td>
 
-
    </tr>
 
-
    <tr style="height: 15pt;" height="20">
 
-
      <td style="height: 15pt;" x:num="" align="right"
 
-
height="20">1</td>
 
-
      <td class="xl66" x:num="8.2000000000000003E-2"
 
-
align="right">0.082</td>
 
-
      <td class="xl65" x:num="" align="right">0.03</td>
 
-
      <td x:num="5.8000000000000003E-2" align="right">0.058</td>
 
-
      <td x:num="" align="right">0.06</td>
 
-
      <td x:num="5.8999999999999997E-2" align="right">0.059</td>
 
-
      <td x:num="6.3E-2" align="right">0.063</td>
 
-
      <td x:num="6.9000000000000006E-2" align="right">0.069</td>
 
-
      <td x:num="7.6999999999999999E-2" align="right">0.077</td>
 
-
      <td x:num="5.1999999999999998E-2" align="right">0.052</td>
 
-
      <td x:num="5.0999999999999997E-2" align="right">0.051</td>
 
-
      <td x:num="8.4000000000000005E-2" align="right">0.084</td>
 
-
      <td x:num="7.1999999999999995E-2" align="right">0.072</td>
 
-
    </tr>
 
-
    <tr style="height: 15pt;" height="20">
 
-
      <td style="height: 15pt;" x:num="" align="right"
 
-
height="20">2.5</td>
 
-
      <td x:num="0.58" align="right">0.58</td>
 
-
      <td x:num="" align="right">0.65</td>
 
-
      <td x:num="" align="right">0.42</td>
 
-
      <td x:num="" align="right">0.45</td>
 
-
      <td x:num="" align="right">0.48</td>
 
-
      <td x:num="" align="right">0.44</td>
 
-
      <td x:num="" align="right">0.52</td>
 
-
      <td x:num="" align="right">0.6</td>
 
-
      <td x:num="" align="right">0.38</td>
 
-
      <td x:num="" align="right">0.34</td>
 
-
      <td x:num="" align="right">0.31</td>
 
-
      <td x:num="" align="right">0.54</td>
 
-
    </tr>
 
-
    <tr style="height: 15pt;" height="20">
 
-
      <td style="height: 15pt;" x:num="" align="right"
 
-
height="20">3</td>
 
-
      <td x:num="1.14" align="right">1.14</td>
 
-
      <td x:num="1.16" align="right">1.16</td>
 
-
      <td x:num="" align="right">0.99</td>
 
-
      <td x:num="" align="right">0.91</td>
 
-
      <td x:num="" align="right">0.94</td>
 
-
      <td x:num="" align="right">0.94</td>
 
-
      <td x:num="" align="right">1.06</td>
 
-
      <td x:num="1.16" align="right">1.16</td>
 
-
      <td x:num="" align="right">0.84</td>
 
-
      <td x:num="" align="right">0.78</td>
 
-
      <td x:num="" align="right">1.04</td>
 
-
      <td x:num="" align="right">1.03</td>
 
-
    </tr>
 
-
    <tr style="height: 15pt;" height="20">
 
-
      <td style="height: 15pt;" x:num="" align="right"
 
-
height="20">3.83</td>
 
-
      <td x:num="" align="right">1.54</td>
 
-
      <td x:num="" align="right">1.66</td>
 
-
      <td x:num="" align="right">1.52</td>
 
-
      <td x:num="" align="right">1.31</td>
 
-
      <td x:num="" align="right">1.38</td>
 
-
      <td x:num="" align="right">1.37</td>
 
-
      <td x:num="" align="right">1.54</td>
 
-
      <td x:num="" align="right">1.72</td>
 
-
      <td x:num="" align="right">1.33</td>
 
-
      <td x:num="" align="right">1.25</td>
 
-
      <td x:num="" align="right">1.91</td>
 
-
      <td x:num="" align="right">1.45</td>
 
-
    </tr>
 
-
    <tr style="height: 15pt;" height="20">
 
-
      <td style="height: 15pt;" x:num="" align="right"
 
-
height="20">5</td>
 
-
      <td x:num="4.4" align="right">4.4</td>
 
-
      <td x:num="" align="right">4.3</td>
 
-
      <td x:num="" align="right">3.2</td>
 
-
      <td x:num="" align="right">3.4</td>
 
-
      <td x:num="" align="right">4.2</td>
 
-
      <td x:num="" align="right">3.3</td>
 
-
      <td x:num="" align="right">4.2</td>
 
-
      <td class="xl65" x:num="2.2" align="right">2.2</td>
 
-
      <td x:num="" align="right">3.9</td>
 
-
      <td x:num="" align="right">4</td>
 
-
      <td x:num="" align="right">3.9</td>
 
-
      <td x:num="" align="right">3.9</td>
 
-
    </tr>
 
-
    <tr style="height: 15pt;" height="20">
 
-
      <td style="height: 15pt;" x:num="" align="right"
 
-
height="20">6</td>
 
-
      <td x:num="" align="right">4.7</td>
 
-
      <td x:num="" align="right">4.7</td>
 
-
      <td class="xl65" x:num="" align="right">2.8</td>
 
-
      <td x:num="" align="right">4.3</td>
 
-
      <td x:num="" align="right">4.2</td>
 
-
      <td x:num="" align="right">3.1</td>
 
-
      <td x:num="" align="right">4.7</td>
 
-
      <td x:num="4.4" align="right">4.4</td>
 
-
      <td x:num="" align="right">3.8</td>
 
-
      <td x:num="" align="right">5</td>
 
-
      <td x:num="" align="right">3.4</td>
 
-
      <td x:num="" align="right">4.7</td>
 
-
    </tr>
 
-
    <tr style="height: 15pt;" height="20">
 
-
      <td style="height: 15pt;" x:num="" align="right"
 
-
height="20">7</td>
 
-
      <td x:num="" align="right">4</td>
 
-
      <td x:num="" align="right">3.8</td>
 
-
      <td x:num="" align="right">3.3</td>
 
-
      <td x:num="" align="right">3.8</td>
 
-
      <td x:num="" align="right">4.2</td>
 
-
      <td x:num="" align="right">3.2</td>
 
-
      <td x:num="" align="right">4.3</td>
 
-
      <td x:num="" align="right">4.7</td>
 
-
      <td x:num="4.9" align="right">4.9</td>
 
-
      <td x:num="" align="right">4.5</td>
 
-
      <td x:num="" align="right">3.7</td>
 
-
      <td x:num="" align="right">3.9</td>
 
-
    </tr>
 
-
    <tr style="height: 15pt;" height="20">
 
-
      <td style="height: 15pt;" x:num="" align="right"
 
-
height="20">8</td>
 
-
      <td x:num="" align="right">5.5</td>
 
-
      <td x:num="" align="right">6.1</td>
 
-
      <td x:num="" align="right">3.9</td>
 
-
      <td x:num="" align="right">4</td>
 
-
      <td x:num="" align="right">3.5</td>
 
-
      <td class="xl65" x:num="" align="right">6.8</td>
 
-
      <td x:num="" align="right">4.2</td>
 
-
      <td x:num="" align="right">4.8</td>
 
-
      <td x:num="" align="right">5.4</td>
 
-
      <td x:num="" align="right">5.3</td>
 
-
      <td x:num="4.6" align="right">4.6</td>
 
-
      <td x:num="4.6" align="right">4.6</td>
 
-
    </tr>
 
-
    <tr style="height: 15pt;" height="20">
 
-
      <td style="height: 15pt;" x:num="" align="right"
 
-
height="20">9</td>
 
-
      <td x:num="4.1" align="right">4.1</td>
 
-
      <td x:num="4.1" align="right">4.1</td>
 
-
      <td x:num="" align="right">3.4</td>
 
-
      <td x:num="" align="right">2.6</td>
 
-
      <td x:num="" align="right">3.6</td>
 
-
      <td x:num="" align="right">4.7</td>
 
-
      <td class="xl65" x:num="" align="right">2.6</td>
 
-
      <td x:num="" align="right">3.1</td>
 
-
      <td x:num="" align="right">3.7</td>
 
-
      <td x:num="" align="right">3.1</td>
 
-
      <td x:num="" align="right">2.6</td>
 
-
      <td x:num="" align="right">3.6</td>
 
-
    </tr>
 
-
    <tr style="height: 15pt;" height="20">
 
-
      <td style="height: 15pt;" x:num="" align="right"
 
-
height="20">10</td>
 
-
      <td x:num="4.6" align="right">4.6</td>
 
-
      <td x:num="" align="right">6.1</td>
 
-
      <td x:num="" align="right">5</td>
 
-
      <td x:num="" align="right">4.8</td>
 
-
      <td x:num="" align="right">7.8</td>
 
-
      <td x:num="" align="right">4.5</td>
 
-
      <td x:num="" align="right">5</td>
 
-
      <td x:num="" align="right">6</td>
 
-
      <td x:num="" align="right">5.3</td>
 
-
      <td x:num="" align="right">5.3</td>
 
-
      <td x:num="4.4" align="right">4.4</td>
 
-
      <td x:num="" align="right">4.7</td>
 
-
    </tr>
 
-
    <tr style="height: 15pt;" height="20">
 
-
      <td style="height: 15pt;" x:num="" align="right"
 
-
height="20">11</td>
 
-
      <td x:num="5.1" align="right">5.1</td>
 
-
      <td x:num="" align="right">6.2</td>
 
-
      <td x:num="" align="right">4.7</td>
 
-
      <td x:num="" align="right">4</td>
 
-
      <td x:num="" align="right">6.1</td>
 
-
      <td x:num="" align="right">7.1</td>
 
-
      <td x:num="" align="right">6.6</td>
 
-
      <td class="xl65" x:num="8.3" align="right">8.3</td>
 
-
      <td x:num="" align="right">6.6</td>
 
-
      <td x:num="" align="right">6.4</td>
 
-
      <td class="xl65" x:num="" align="right">6.2</td>
 
-
      <td x:num="4.1" align="right">4.1</td>
 
-
    </tr>
 
-
    <tr style="height: 15pt;" height="20">
 
-
      <td style="height: 15pt;" x:num="" align="right"
 
-
height="20">12</td>
 
-
      <td x:num="5.1" align="right">5.1</td>
 
-
      <td x:num="" align="right">5</td>
 
-
      <td x:num="" align="right">3</td>
 
-
      <td x:num="4.4" align="right">4.4</td>
 
-
      <td x:num="4.4" align="right">4.4</td>
 
-
      <td x:num="" align="right">3.9</td>
 
-
      <td x:num="" align="right">4</td>
 
-
      <td x:num="" align="right">4.8</td>
 
-
      <td x:num="" align="right">5</td>
 
-
      <td x:num="" align="right">5.3</td>
 
-
      <td x:num="" align="right">4</td>
 
-
      <td x:num="" align="right">4.7</td>
 
-
    </tr>
 
-
    <tr style="height: 15pt;" height="20">
 
-
      <td style="height: 15pt;" x:num="" align="right"
 
-
height="20">24</td>
 
-
      <td x:num="" align="right">5.2</td>
 
-
      <td x:num="" align="right">5.6</td>
 
-
      <td x:num="" align="right">3.3</td>
 
-
      <td x:num="" align="right">5.4</td>
 
-
      <td x:num="" align="right">6.4</td>
 
-
      <td x:num="" align="right">5.8</td>
 
-
      <td x:num="4.6" align="right">4.6</td>
 
-
      <td class="xl65" x:num="" align="right">6.4</td>
 
-
      <td x:num="" align="right">7.1</td>
 
-
      <td x:num="" align="right">7.4</td>
 
-
      <td x:num="" align="right">5.6</td>
 
-
      <td x:num="" align="right">5.2</td>
 
-
    </tr>
 
-
  </tbody>
 
-
</table>
 
-
</body>
 
-
</html>
 
</div>
</div>

Latest revision as of 02:22, 28 October 2010

Retrieved from "http://2010.igem.org/Team:SDU-Denmark/project-r"