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>
<br><br>
-
When comparing the sequence that came back from sequencing with the theoretical sequence the part should have it is identical, except for a silent mutation in the coding sequence, which does not change the amino acid sequence.
+
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 ===
=== Effect ===
-
Our results from the experiments with semi-solid agar confirm that the biobrick does indeed couple itself to the bacterial chemotaxis pathway and modify the bacterial motility pattern by modifying the tumbling frequency. For further information on this conclusion see [https://2010.igem.org/Team:SDU-Denmark/project-p#K343007| Characterization - K343007 - 1. Growth of bacterial culture on semi-solid agar plates (Experiment 1 and 2)]<br>
+
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 blue light with a wavelength around 500nm leads to CheA's autophosphorylation being downregulated. This means that the bacterial tumbling frequency gets reduced and the bacteria will spend an increased time in the "run" mode of propulsion. This means that the bacteria will travel further in a certain amount of time, compared to wildtype bacteria. More details can be found here [https://2010.igem.org/Team:SDU-Denmark/project-p#K343007| Characterization - K343007 - 2. Videomicroscopy and computer analysis of bacterial motility]<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>
 +
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>
 +
For further information, raw data and background of the assay see [https://2010.igem.org/Team:SDU-Denmark/K343007 characterization of K343007].
-
=== Stability assay ===
+
==Flagella==
-
<html>
+
=== Biobrick assembly and sequence ===
-
<head>
+
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>
-
  <meta content="text/html; charset=ISO-8859-1"
+
This was achieved through "standard" silver assembly and confirmed via sequencing with biobrick verification forward 2 (VF2) and verification reverse (VR) primers.<br>
-
http-equiv="content-type">
+
<br>
-
  <title></title>
+
[http://partsregistry.org/cgi/sequencing/one_blast.cgi?id=8031| Sequencing results for K343004]
-
</head>
+
-
<body>
+
-
<table x:str=""
+
-
style="border-collapse: collapse; width: 428pt;" border="0"
+
-
cellpadding="0" cellspacing="0" width="570">
+
-
  <col style="width: 75pt; font-weight: bold;" width="100">
+
-
  <col style="width: 79pt; font-weight: bold;" width="105">
+
-
  <col style="width: 128pt; font-weight: bold;" width="170">
+
-
  <col style="width: 53pt; font-weight: bold;" width="71">
+
-
  <col style="width: 93pt; font-weight: bold;" width="124">
+
-
  <tbody>
+
-
    <tr style="height: 15pt;" height="20">
+
-
      <td class="xl65"
+
-
style="height: 15pt; width: 75pt; font-weight: bold;"
+
-
height="20" width="100">Stability assay.</td>
+
-
      <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>
+
-
 
+
<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>
+
== Retinal ==
-
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>
+
=== Biobrick assembly and sequence ===
-
The plates were incubated at 37 degrees celcius for 24 hours.<br><br>
+
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>
-
[[Image:Team-SDU-Denmark-Flagellamotility-exp1-DH5a.JPG|250px|DH5alpha]]
+
This was achieved through "standard" silver assembly and confirmed via sequencing with biobrick verification forward 2 (VF2) and verification reverse (VR) primers.<br>
-
[[Image:Team-SDU-Denmark-Flagellamotility-exp1-MG1655.JPG|250px|MG1655]]<br><br>
+
Sequencing results (as .ab1 files):<br>
-
[[Image:Team-SDU-Denmark-Flagellamotility-exp1-FlhDCmutCP_i_pSB1C3_(LA+chlor).JPG|250px|pSB1C3-FlhDCmutCP(LA+chlor).]]
+
<br>
-
[[Image:Team-SDU-Denmark-Flagellamotility-exp1-FlhDCmutCP_i_pSB3K3_(LA+kan).JPG|250px|pSB3K3-FlhDCmutCP(LA+Kan)]]
+
[http://partsregistry.org/cgi/sequencing/one_blast.cgi?id=8025| Sequencing results for K343006]
<br><br>
<br><br>
-
The upper two plates did not contain antibiotics, and therefore contamination colonies are seen.<br>
+
When comparing the theoretical sequence with the sequence done on the part K343006 the two sequences was identical.
-
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>
+
=== Effect ===
 +
UV-Vis spectroskopy and HPLC analysis of organic extractions of the bacteria containing K343006 didn't give conclusive results conserning the Retinal production.<br>
 +
The stability of pSB1C3-K343006 is most likely <20 generations.<br>
 +
The growth assay showed no significant difference between the wild type and the cells containing pSB1C3-K343006.<br>
 +
For further information, raw data and background of the assay see [https://2010.igem.org/Team:SDU-Denmark/K343006 characterization of K343006]
-
== 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"