Team:Goettingen/week21-2

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<td width="900" bordercolor="black" valign="top">
<td width="900" bordercolor="black" valign="top">
<h2><b>V09_17 </b></h2><br>
<h2><b>V09_17 </b></h2><br>
-
<b>V09_17_1 Preparative double digestion of <i>flhDC</i>, <i>FliC</i>, 18K-<i>RFP</i> and pSB1C3 followed by ligation</i></b><br>
+
<b>V09_17_1 Preparative double digestion of <i>flhDC</i>, <i>FliC</i> and pSB1C3 followed by ligation</i></b><br>
<ul>
<ul>
<li>Experiment: <br>  
<li>Experiment: <br>  
-
In order to clone the <i>flhDC</i>, <i>FliC</i> and 18K-<i>RFP</i> constructs into pSB1C3,  all components were digested with EcoRI and PstI according to the <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. The restriction products were subsequently separated on a gel in order to verify the digestions success and purify the desired fragments. After measuring the concentration of the purified fragments, an overnight ligation was initiated.<br>
+
In order to clone the <i>flhDC</i> and <i>FliC</i> into pSB1C3,  all components were digested with <i>EcoRI</i> and <i>PstI</i> according to the <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. The restriction products were subsequently separated on a gel in order to verify the digestions success and purify the desired fragments. After measuring the concentration of the purified fragments, an overnight ligation was initiated.<br>
</ul>
</ul>
<br>
<br>
<b>V09_17_2 Preparation of over night cultures</b><br>
<b>V09_17_2 Preparation of over night cultures</b><br>
         <ul>
         <ul>
-
<li>Experiment: We prepared overnight cultures of different promoter-gene constructs in two different E. coli strains (MG1655 & BL21) in order to test wether the motility is influenced. The following constructs were used: <br>
+
<li>Experiment:<br>
 +
We prepared overnight cultures of different promoter-gene constructs in two different E. coli strains (MG1655 & BL21) in order to test wether the motility is influenced. The following constructs were used: <br>
<br>
<br>
- 20E_<i>flhDC</i> <br>
- 20E_<i>flhDC</i> <br>
Line 51: Line 52:
<ul>
<ul>
<li>Experiment: For the motility assay the over night cultures, with different constructs, were spun down and dropped onto M9- or 1% trypton-agar. The exact protocol you can find <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">HERE</a> <br>
<li>Experiment: For the motility assay the over night cultures, with different constructs, were spun down and dropped onto M9- or 1% trypton-agar. The exact protocol you can find <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">HERE</a> <br>
 +
</ul>
 +
<br>
 +
<b>V09_18_2 Preparative double digestion of <i>MotA</i>, <i>MotB</i>, <i>yhjH</i>, 18C-<i>RFP</i>, 20E-<i>RFP</i> and 20I-<i>RFP</i></b><br>
 +
<ul>
 +
<li>Experiment: <br>
 +
The genes of interest, motA, motB and yhjH were digested with <i>XbaI</i> and <i>PstI</i>. The plasmids with the different promoters were treated with <i>SpeI</i> and <i>PstI</i>. The digestion was then controlled via gel-electrophoresis.<br>
<li> Observation and Results:<br>
<li> Observation and Results:<br>
-
Text<br>
+
Since for all clones band of the expected size could be obtained these were isolated from the gel and purified using PeqGOLD Gelextraction Kit (Peqlab) according to the manual.<br>
</ul>
</ul>
<br>
<br>
-
<b>V09_18_2 Preparative double digestion of <i>MotA</i>, <i>MotB</i>, <i>yhjH</i>, 18C-<i>RFP</i>, 20E-<i>RFP</i> and 20I-<i>RFP</i> followed by ligation</i></b><br>
+
<b>V09_18_3 Ligation of <i>MotA</i>, <i>MotB</i> and <i>yhjH</i> into 18C-<i>RFP</i>, 20E-<i>RFP</i> and 20I-<i>RFP</i></b><br>
<ul>
<ul>
<li>Experiment: <br>
<li>Experiment: <br>
-
TEXT<br>
+
<i>motA</i>, <i>motB</i> and <i>yhjH</i> were ligated into the plasmid pSB1C3 with different promoters (20E, 20I and 18C) according to the <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>.<br>
</ul>
</ul>
<br>
<br>
-
<b>V09_18_3 Chemical transformation of different plasmid-gene constructs into <i>E. coli</i> (DH10B) or (MG1655)</b><br>
+
<b>V09_18_4 Chemical transformation of different plasmid-gene constructs into <i>E. coli</i> (DH10B) or (MG1655)</b><br>
<ul>
<ul>
<li>Experiment:<br> Chemical transformation was done according to the standard <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. The following constructs were used:<br>
<li>Experiment:<br> Chemical transformation was done according to the standard <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. The following constructs were used:<br>
Line 68: Line 75:
- pSB1C3-<i>flhDC</i> into <i>E. coli </i>(DH10B)<br>
- pSB1C3-<i>flhDC</i> into <i>E. coli </i>(DH10B)<br>
- PSB1C3-<i>RFP</i> into <i>E. coli </i> (MG1655)<br>
- PSB1C3-<i>RFP</i> into <i>E. coli </i> (MG1655)<br>
 +
<br>
 +
<li>Observations & Results:<br>
 +
On all plates only a few colonies were observed.
 +
</ul>
<br>
<br>
<br></td></tr>
<br></td></tr>
</table>
</table>
 +
 +
 +
<table cellpadding="20 px" border="1" bordercolor="black" valign="top">
<table cellpadding="20 px" border="1" bordercolor="black" valign="top">
<tr bordercolor="black" valign="top">
<tr bordercolor="black" valign="top">
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<b> V09_19_1 Motility Assays</i></i></b><br>
<b> V09_19_1 Motility Assays</i></i></b><br>
<ul>
<ul>
-
<li>Experiment: For the motility assay the over night cultures, with different constructs, were spun down and dropped onto M9- or 1% trypton-agar. The exact protocol you can find <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">HERE</a> <br>
+
<li>Experiment: For the motility assay the cultures with different constructs were grown for 6 hours. After that they were spun down and dropped onto M9- or 1% trypton-agar. The exact protocol you can find <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">HERE</a><br>
 +
The Following constructs in the <i>E. coli</>strain MG1655 were used:<br>
 +
<br>
 +
-pSB1C3-20I-<i>flhDC</i><br>
 +
-pSB1C3-18C-<i>flhDC</i><br>
 +
-pSB1C3-20E-<i>flhDC</i><br>
 +
-pSB1C3-<i>RFP</i><br>
 +
<br>
<li> Observation and Results:<br>
<li> Observation and Results:<br>
-
Text<br>
+
At the following day (20th of september) the colonies still looked exactly the same. Neither chemotaxis nor swimming could be observed.<br>
</ul>
</ul>
<br>
<br>
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<ul>
<ul>
<li>Experiment: <br>
<li>Experiment: <br>
-
For the chemical retransformation the standard <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a> was followed. The following constructs were transformed:<br>
+
For the chemical transformation the standard <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a> was followed. The following constructs were transformed:<br>
 +
<br>
- pSB1C3-18C-<i>motA</i> (DH10B)<br>
- pSB1C3-18C-<i>motA</i> (DH10B)<br>
- pSB1C3-18C-<i>motB</i> (DH10B)<br>
- pSB1C3-18C-<i>motB</i> (DH10B)<br>
Line 105: Line 127:
- pSB1C3-<i>flhDC</i> <br>
- pSB1C3-<i>flhDC</i> <br>
- pSB1C3-<i>fliC</i> <br>
- pSB1C3-<i>fliC</i> <br>
-
- pSB1C3-18K-<i>RFP</i> <br>
 
<br>
<br>
<br></td></tr>
<br></td></tr>
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<ul>
<ul>
<li>Experiment: <br>
<li>Experiment: <br>
-
Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.All constructs were digested with XbaI and SpeI conducted as described in the <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol. </a> <br>
+
Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual. All constructs were digested with XbaI and SpeI conducted as described in the <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol. </a> <br>
<br>
<br>
- pSB1C3-<i>flhDC</i> <br>
- pSB1C3-<i>flhDC</i> <br>
- pSB1C3-<i>fliC</i> <br>
- pSB1C3-<i>fliC</i> <br>
-
- pSB1C3-18K-<i>RFP</i> <br>
 
<br>
<br>
<li> Observation and Results:<br>
<li> Observation and Results:<br>
-
All three the pSB1C3-18K-<i>RFP</i> clones host the correctly inserted gene in the plasmid. The pSB1C3-<i>flhDC / fliC</i> clones do not host the correctly inserted gene.<br>
+
The pSB1C3-<i>flhDC / fliC</i> clones do not host the correctly inserted gene.<br>
</ul>
</ul>
<br>
<br>
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<ul>
<ul>
<li>Experiment: <br>
<li>Experiment: <br>
-
In order to get more <i>fliC</i>, the already quikchanged fliC was amplified via PCR. The PCR product was analyzed via gel-electrophoresis and subsequently purified using peqGOLD Gel Extraction Kit (Peqlab) according to the manual.<br>
+
In order to get more <i>fliC</i>, the already quikchanged <i>fliC</i> was amplified via PCR. The PCR product was analyzed via gel-electrophoresis and subsequently purified using peqGOLD Gel Extraction Kit (Peqlab) according to the manual.<br>
 +
<li> Observation and Results:<br>
 +
The PCR was not succesful, we could not observe a band at the expected size.<br>
</ul>
</ul>
<br>
<br>
-
<b>V09_20_3 Repetition of preparative double digestion of <i>flhDC, FliC</i> and pSB1C3</b><br>
+
<b>V09_20_3 Repetition of preparative double digestion of <i>flhDC and pSB1C3</b><br>
<ul>
<ul>
-
<li>Experiment:<br> In order to clone the flhDC, FliC constructs into pSB1C3, all components were digested with EcoRI and PstI according to the protocol.<br>
+
<li>Experiment:<br> In order to clone the <i>flhDC</i> into pSB1C3, all components were digested with <i>EcoRI</i> and </i>PstI according to the <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. The prodcuts were seperated via gel electrophoresis and purified using PeqGOLD Gelextraction Kit (Peqlab).<br>
</ul>
</ul>
<br>
<br>
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<td width="900" bordercolor="black" valign="top">
<td width="900" bordercolor="black" valign="top">
<h2><b>V09_21 </b></h2><br>
<h2><b>V09_21 </b></h2><br>
-
<b> V09_21_1 Ligation and chemical Transformation of <i>flhDC, FliC</i> and <i> pSB1C3</i></b><br>
+
<b> V09_21_1 Ligation and chemical Transformation of pSB1C3-<i>flhDC</i></b><br>
<ul>
<ul>
<li>Experiment: <br>
<li>Experiment: <br>
Line 170: Line 192:
</ul>
</ul>
<br>
<br>
-
<b>V09_21_2 Mini Prep and test digestion of pSB1C3-gene constructs</b><br>
+
<b>V09_21_2 Mini Prep and test digestion of pSB1C3-promoter-gene constructs</b><br>
<ul>
<ul>
<li>Experiment: <br>
<li>Experiment: <br>
-
Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.All constructs were digested with XbaI and SpeI conducted as described in the <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>.<br>
+
Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual. All constructs were digested with <i>XbaI</i> and <i>SpeI</i> conducted as described in the <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>.<br>
<br>
<br>
- pSB1C3-18C-<i>motA</i> (DH10B)<br>
- pSB1C3-18C-<i>motA</i> (DH10B)<br>
Line 187: Line 209:
<br>
<br>
<li> Observation and Results:<br>
<li> Observation and Results:<br>
-
All three the pSB1C3-18K-<i>RFP</i> clones host the correctly inserted gene in the plasmid. The pSB1C3-<i>flhDC / fliC</i> clones do not host the correctly inserted gene.<br>
+
All clones host the correctly inserted gene in the plasmid.<br>
</ul>
</ul>
-
<br>
 
-
<b>V09_21_3 TITEL</b><br>
 
-
<ul>
 
-
<li>Experiment:<br>
 
-
TEXT
 
<br>
<br>
<br></td></tr>
<br></td></tr>

Latest revision as of 23:27, 26 September 2012

Deutsch  / English 

#2 Speed Improvement - 21st week

Back to overview

V09_17


V09_17_1 Preparative double digestion of flhDC, FliC and pSB1C3 followed by ligation
  • Experiment:
    In order to clone the flhDC and FliC into pSB1C3, all components were digested with EcoRI and PstI according to the protocol. The restriction products were subsequently separated on a gel in order to verify the digestions success and purify the desired fragments. After measuring the concentration of the purified fragments, an overnight ligation was initiated.

V09_17_2 Preparation of over night cultures
  • Experiment:
    We prepared overnight cultures of different promoter-gene constructs in two different E. coli strains (MG1655 & BL21) in order to test wether the motility is influenced. The following constructs were used:

    - 20E_flhDC
    - 18C_flhDC
    - 20I_flhdc
    - 20E_motA
    - 18C_motA
    - 20E_motB
    - 18C_motB


V09_18


V09_18_1 Motility Assays
  • Experiment: For the motility assay the over night cultures, with different constructs, were spun down and dropped onto M9- or 1% trypton-agar. The exact protocol you can find HERE

V09_18_2 Preparative double digestion of MotA, MotB, yhjH, 18C-RFP, 20E-RFP and 20I-RFP
  • Experiment:
    The genes of interest, motA, motB and yhjH were digested with XbaI and PstI. The plasmids with the different promoters were treated with SpeI and PstI. The digestion was then controlled via gel-electrophoresis.
  • Observation and Results:
    Since for all clones band of the expected size could be obtained these were isolated from the gel and purified using PeqGOLD Gelextraction Kit (Peqlab) according to the manual.

V09_18_3 Ligation of MotA, MotB and yhjH into 18C-RFP, 20E-RFP and 20I-RFP
  • Experiment:
    motA, motB and yhjH were ligated into the plasmid pSB1C3 with different promoters (20E, 20I and 18C) according to the protocol.

V09_18_4 Chemical transformation of different plasmid-gene constructs into E. coli (DH10B) or (MG1655)
  • Experiment:
    Chemical transformation was done according to the standard protocol. The following constructs were used:

    - pSB1C3-FliC into E. coli (DH10B)
    - pSB1C3-flhDC into E. coli (DH10B)
    - PSB1C3-RFP into E. coli (MG1655)

  • Observations & Results:
    On all plates only a few colonies were observed.


V09_19


V09_19_1 Motility Assays
  • Experiment: For the motility assay the cultures with different constructs were grown for 6 hours. After that they were spun down and dropped onto M9- or 1% trypton-agar. The exact protocol you can find HERE
    The Following constructs in the E. colistrain MG1655 were used:

    -pSB1C3-20I-flhDC
    -pSB1C3-18C-flhDC
    -pSB1C3-20E-flhDC
    -pSB1C3-RFP

  • Observation and Results:
    At the following day (20th of september) the colonies still looked exactly the same. Neither chemotaxis nor swimming could be observed.

V09_19_2 Chemical transformation
  • Experiment:
    For the chemical transformation the standard protocol was followed. The following constructs were transformed:

    - pSB1C3-18C-motA (DH10B)
    - pSB1C3-18C-motB (DH10B)
    - pSB1C3-18C-yhjH (DH10B)
    - pSB1C3-20E-motA (DH10B)
    - pSB1C3-20E-motA (DH10B)
    - pSB1C3-20E-motA (DH10B)
    - pSB1C3-20I-motA (DH10B)
    - pSB1C3-20I-motB (DH10B)
    - pSB1C3-20I-yhjH (DH10B)
    - pSB1C3-RFP (BL21)


V09_19_3 Preparation of over night cultures
  • Experiment:
    Over night cultures were prepared of three colonies of each constructs in order to isolate the plasmid and check the correct insert.
    - pSB1C3-flhDC
    - pSB1C3-fliC


V09_20


V09_20_1 Mini Prep and test digestion of pSB1C3-gene constructs
  • Experiment:
    Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual. All constructs were digested with XbaI and SpeI conducted as described in the protocol.

    - pSB1C3-flhDC
    - pSB1C3-fliC

  • Observation and Results:
    The pSB1C3-flhDC / fliC clones do not host the correctly inserted gene.

V09_20_2 PCR of fliC
  • Experiment:
    In order to get more fliC, the already quikchanged fliC was amplified via PCR. The PCR product was analyzed via gel-electrophoresis and subsequently purified using peqGOLD Gel Extraction Kit (Peqlab) according to the manual.
  • Observation and Results:
    The PCR was not succesful, we could not observe a band at the expected size.

V09_20_3 Repetition of preparative double digestion of flhDC and pSB1C3
  • Experiment:
    In order to clone the flhDC into pSB1C3, all components were digested with EcoRI and     PstI according to the protocol. The prodcuts were seperated via gel electrophoresis and purified using PeqGOLD Gelextraction Kit (Peqlab).

V09_20_4 Preparation of over night cultures
  • Experiment:
    Over night cultures were prepared of three colonies of each constructs in order to isolate the plasmid and check the correct insert.

    - pSB1C3-18C-motA (DH10B)
    - pSB1C3-18C-motB (DH10B)
    - pSB1C3-18C-yhjH (DH10B)
    - pSB1C3-20E-motA (DH10B)
    - pSB1C3-20E-motA (DH10B)
    - pSB1C3-20E-motA (DH10B)
    - pSB1C3-20I-motA (DH10B)
    - pSB1C3-20I-motB (DH10B)
    - pSB1C3-20I-yhjH (DH10B)
    - pSB1C3-RFP (BL21)

V09_21


V09_21_1 Ligation and chemical Transformation of pSB1C3-flhDC
  • Experiment:
    The restriction products were separated on a gel in order to verify the digestions success and purify the desired fragments. After ligation of the genes into pSB1C3, we transformed the ligated plasmids into E. coli DH10B according to the protocol.


V09_21_2 Mini Prep and test digestion of pSB1C3-promoter-gene constructs
  • Experiment:
    Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual. All constructs were digested with XbaI and SpeI conducted as described in the protocol.

    - pSB1C3-18C-motA (DH10B)
    - pSB1C3-18C-motB (DH10B)
    - pSB1C3-18C-yhjH (DH10B)
    - pSB1C3-20E-motA (DH10B)
    - pSB1C3-20E-motA (DH10B)
    - pSB1C3-20E-motA (DH10B)
    - pSB1C3-20I-motA (DH10B)
    - pSB1C3-20I-motB (DH10B)
    - pSB1C3-20I-yhjH (DH10B)
    - pSB1C3-RFP (BL21)

  • Observation and Results:
    All clones host the correctly inserted gene in the plasmid.



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