Team:Goettingen/week21-2

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#2 Speed Improvement - 21st week

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V09_17

V09_17_1 Preparative double digestion of flhDC, FliC, 18K-RFP and pSB1C3 followed by ligation

Experiment:
In order to clone the flhDC, FliC and 18K-RFP constructs 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
Observation and Results:
Text

V09_18_2 Preparative double digestion of MotA, MotB, yhjH, 18C-RFP, 20E-RFP and 20I-RFP followed by ligation

Experiment:
TEXT

V09_18_3 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)

V09_19

V09_19_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
Observation and Results:
Text

V09_19_2 Chemical transformation

Experiment:
For the chemical retransformation 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
- pSB1C3-18K-RFP

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@@ Line 584: / Line 584: @@
 <tr bordercolor="black" valign="top">
 <td width="900" bordercolor="black" valign="top">
-<h2><b>V09_18 </b></h2><br>
+<h2><b>V09_19 </b></h2><br>
-<b> V09_18_1 Motility Assays</i></i></b><br>
+<b> V09_19_1 Motility Assays</i></i></b><br>
 <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>
@@ Line 592: / Line 592: @@
 </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> followed by ligation</i></b><br>
+<b>V09_19_2 Chemical transformation</b><br>
 <ul>
 <li>Experiment: <br>
-In order to test the influence of the different genes, we cloned the genes behind constitutive promoters with various promoter strength. <i> MotA, MotB</i> and <i>yhjH</i> were digested with XbaI and PstI. The psB1C3-Promoter backbones were digested with SpeI and PstI. Afterwards, the fragments of the desired size were cut out and purified via gel-extraction kit. Finally, over night ligation was started.<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>
+- pSB1C3-18C-<i>motA</i> (DH10B)<br>
+- pSB1C3-18C-<i>motB</i> (DH10B)<br>
+- pSB1C3-18C-<i>yhjH</i> (DH10B)<br>
+- pSB1C3-20E-<i>motA</i> (DH10B)<br>
+- pSB1C3-20E-<i>motA</i> (DH10B)<br>
+- pSB1C3-20E-<i>motA</i> (DH10B)<br>
+- pSB1C3-20I-<i>motA</i> (DH10B)<br>
+- pSB1C3-20I-<i>motB</i> (DH10B)<br>
+- pSB1C3-20I-<i>yhjH</i> (DH10B)<br>
+- pSB1C3-<i>RFP</i> (BL21)<br>
+<br>
 </ul>
 <br>
-<b>V09_18_3 Chemical transformation of different plasmid-gene constructs into <i>E. coli</i> (DH10B) or (MG1655)</b><br>
+<b>V09_19_3 Preparation of over night cultures</b><br>
 <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>
-- pSB1C3-<i>FliC</i> into <i>E. coli </i>(DH10B)<br>
+Over night cultures were prepared of three colonies of each constructs in order to isolate the plasmid and check the correct insert.<br>
-pSB1C3-<i>flhDC</i> into <i>E. coli </i>(DH10B)<br>
+- pSB1C3-<i>flhDC</i> <br>
-PSB1C3-<i>RFP</i> into <i>E. coli </i> (MG1655)<br>
+- pSB1C3-<i>fliC</i> <br>
+- pSB1C3-18K-<i>RFP</i> <br>
 <br>
 <br></td></tr>