Team:Uppsala University/Backbones

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Revision as of 18:45, 23 September 2012

Team Uppsala University – iGEM 2012

Standard low-copy backbones

Registry ID	Name	Ori	Resistance	Insert	Size	Status
	pSB4A15	pSC101	Amp	pUCori-red		Finished
	pSB4C15	pSC101	Cm	pUCori-red		Finished
	pSB4K15	pSC101	Kan	pUCori-red		Finished
	pSB4S15	pSC101	Spec	pUCori-red		Finished
	pSB4A15(Frt)	pSC101	Amp	pUCori-red		Finished
	pSB4C15(Frt)	pSC101	Cm	pUCori-red		Finished
	pSB4K15(Frt)	pSC101	Kan	pUCori-red		Finished
	pSB4S15(Frt)	pSC101	Spec	pUCori-red		Finished

LacIq backbones

For expression of toxic genes, or simply genes where you want to be able to tune the expression level, we constructed a series of lacIq bacbones. Including the lacIq casette on the plasmid ensures that the copy number of the repression always follows that of your inserted genes, providing guranteed strong repression without inducing unneccessary metabolic load.

Registry ID	Name	Ori	Resistance	Insert	Size	Status
	pSB4A15Iq	pSC101	Amp	pUCori-redq		Finished
	pSB4C15Iq	pSC101	Cm	pUCori-redq		Finished
	pSB4K15Iq	pSC101	Kan	pUCori-redq		Finished
	pSB4S15Iq	pSC101	Spec	pUCori-redq		Finished
	pSB4A15Iq(Frt)	pSC101	Amp	pUCori-redq		Planning
	pSB4C15Iq(Frt)	pSC101	Cm	pUCori-redq		Finished
	pSB4K15Iq(Frt)	pSC101	Kan	pUCori-redq		Finished
	pSB4S15Iq(Frt)	pSC101	Spec	pUCori-redq		Planning

Thermosensitive backbones

The pSB8 backbones contain the pSC101ts ori, which does not replicate at 42°C. Strains carrying pSB8x15 plasmids can be grown stably at 30°C, but the plasmid will be lost at 42°C. For removing the plasmid, the strain can be streaken on a antibiotic-free LB agar plate and grown at 42° C overnight. Plasmid loss can be confirmed by streaking the new colonies on a plate with the relevant antibiotic (See figure).
All backbones are available with a pUC ori and a constitutive RFP casette in the BioBrick site. This allows rapid growth at 37° C, high plasmid yields and faster red color expression. Temporary exposure to higher temperatures does not affect plasmid maintenance noticeably, and transformation recovery can be done at 37°.

Registry ID	Name	Ori	Resistance	Insert	Size	Status
	pSB4A15	pSC101ts	Amp	pUCori-redq		Finished
	pSB4C15	pSC101ts	Cm	pUCori-redq		Finished
	pSB4K15	pSC101ts	Kan	pUCori-redq		Finished
	pSB4S15	pSC101ts	Spec	pUCori-redq		Finished
	pSB4A15(Frt)	pSC101ts	Amp	pUCori-redq		Planning
	pSB4C15(Frt)	pSC101ts	Cm	pUCori-redq		Planning
	pSB4K15(Frt)	pSC101ts	Kan	pUCori-redq		Finished
	pSB4S15(Frt)	pSC101ts	Spec	pUCori-redq		Planning

@@ Line 41: / Line 41: @@
 <td></td><td>pSB4C15</td> <td>pSC101</td><td>Cm</td><td>pUCori-red</td><td></td><td>Finished</td>
 </tr><tr>
-<td></td><td>pSB4K15</td> <td>pSC101</td><td>Kan</td><td>pUCori-red</td><td></td><td>Planning</td>
+<td></td><td>pSB4K15</td> <td>pSC101</td><td>Kan</td><td>pUCori-red</td><td></td><td>Finished</td>
 </tr><tr>
 <td></td><td>pSB4S15</td> <td>pSC101</td><td>Spec</td><td>pUCori-red</td><td></td><td>Finished</td>
 </tr><tr>
-<td></td><td>pSB4T15</td> <td>pSC101</td><td>Tet</td><td>pUCori-red</td><td></td><td>Finished</td>
+<td></td><td>pSB4A15(Frt)</td> <td>pSC101</td><td>Amp</td><td>pUCori-red</td><td></td><td>Finished</td>
-</tr>
+</tr><tr>
+<td></td><td>pSB4C15(Frt)</td> <td>pSC101</td><td>Cm</td><td>pUCori-red</td><td></td><td>Finished</td>
+</tr><tr>
+<td></td><td>pSB4K15(Frt)</td> <td>pSC101</td><td>Kan</td><td>pUCori-red</td><td></td><td>Finished</td>
+</tr><tr>
+<td></td><td>pSB4S15(Frt)</td> <td>pSC101</td><td>Spec</td><td>pUCori-red</td><td></td><td>Finished</td>
+</tr></td>
 </table>
@@ Line 76: / Line 82: @@
 <td width="100"><b>Registry ID</b></td><td width="100"><b>Name</b></td> <td width="70"><b>Ori<b> </td> <td width="80"><b>Resistance</b></td> <td width="100"><b>Insert</b><td width="70"><b>Size</b></td></td> <td width="100"><b>Status</b></td>
 </tr><tr>
-<td></td><td>pSB4A15Iq</td> <td>pSC101</td><td>Amp</td><td>pUCori-redq</td><td></td><td>Planning</td>
+<td></td><td>pSB4A15Iq</td> <td>pSC101</td><td>Amp</td><td>pUCori-redq</td><td></td><td>Finished</td>
 </tr><tr>
 <td></td><td>pSB4C15Iq</td> <td>pSC101</td><td>Cm</td><td>pUCori-redq</td><td></td><td>Finished</td>
 </tr><tr>
-<td></td><td>pSB4K15Iq</td> <td>pSC101</td><td>Kan</td><td>pUCori-redq</td><td></td><td>Planning</td>
+<td></td><td>pSB4K15Iq</td> <td>pSC101</td><td>Kan</td><td>pUCori-redq</td><td></td><td>Finished</td>
 </tr><tr>
-<td></td><td>pSB4S15Iq</td> <td>pSC101</td><td>Spec</td><td>pUCori-redq</td><td></td><td>Planning</td>
+<td></td><td>pSB4S15Iq</td> <td>pSC101</td><td>Spec</td><td>pUCori-redq</td><td></td><td>Finished</td>
 </tr><tr>
-<td></td><td>pSB4T15Iq</td> <td>pSC101</td><td>Tet</td><td>pUCori-redq</td><td></td><td>Finished</td>
+<td></td><td>pSB4A15Iq(Frt)</td> <td>pSC101</td><td>Amp</td><td>pUCori-redq</td><td></td><td>Planning</td>
+</tr><tr>
+<td></td><td>pSB4C15Iq(Frt)</td> <td>pSC101</td><td>Cm</td><td>pUCori-redq</td><td></td><td>Finished</td>
+</tr><tr>
+<td></td><td>pSB4K15Iq(Frt)</td> <td>pSC101</td><td>Kan</td><td>pUCori-redq</td><td></td><td>Finished</td>
+</tr><tr>
+<td></td><td>pSB4S15Iq(Frt)</td> <td>pSC101</td><td>Spec</td><td>pUCori-redq</td><td></td><td>Planning</td>
 </tr>
 </table>
@@ Line 102: / Line 114: @@
 <tr>
-<td class="subtext"><h2>Chromosomal intergration backbones</h2></td>
+<td class="subtext"><h2>Thermosensitive backbones</h2></td>
 <td valign="bottom"><a id="top" href="#top">Back to top</a></td></tr>
 </table>
@@ Line 111: / Line 123: @@
 <td valign="top">
 <p>
-We have made special low copy backbones for recombination onto the bacterial chromosome. The antibiotic resistance casette is flanked by Flp recombinase recognition target sites (FRT sites), meaning that the casette can be flipped out of the chromosome after integration and selection. Since low copy plasmids still have a higher copy number than one, it can be also useful to to repress toxic genes during cloning work with the lacIq system.
+The pSB8 backbones contain the pSC101ts ori, which does not replicate at 42°C. Strains carrying pSB8x15 plasmids can be grown stably at 30°C, but the plasmid will be lost at 42°C. For removing the plasmid, the strain can be streaken on a antibiotic-free LB agar plate and grown at 42° C overnight. Plasmid loss can be confirmed by streaking the new colonies on a plate with the relevant antibiotic <i>(See figure)</i>. <br>
+All backbones are available with a pUC ori  and a constitutive RFP casette in the BioBrick site. This allows rapid growth at 37° C, high plasmid yields and faster red color expression. Temporary exposure to higher temperatures does not affect plasmid maintenance noticeably, and transformation recovery can be done at 37°.
 <table>
 <tr>
 <td width="100"><b>Registry ID</b></td><td width="100"><b>Name</b></td> <td width="70"><b>Ori<b> </td> <td width="80"><b>Resistance</b></td> <td width="100"><b>Insert</b><td width="70"><b>Size</b></td></td> <td width="100"><b>Status</b></td>
+</tr>><tr>
+<td></td><td>pSB4A15</td> <td>pSC101ts</td><td>Amp</td><td>pUCori-redq</td><td></td><td>Finished</td>
 </tr><tr>
-<td></td><td>pSB4A15Flp</td> <td>pSC101</td><td>Amp</td><td>pUCori-red</td><td></td><td>Finished</td>
+<td></td><td>pSB4C15</td> <td>pSC101ts</td><td>Cm</td><td>pUCori-redq</td><td></td><td>Finished</td>
 </tr><tr>
-<td></td><td>pSB4C15Flp</td> <td>pSC101</td><td>Cm</td><td>pUCori-red</td><td></td><td>Finished</td>
+<td></td><td>pSB4K15</td> <td>pSC101ts</td><td>Kan</td><td>pUCori-redq</td><td></td><td>Finished</td>
 </tr><tr>
-<td></td><td>pSB4K15Flp</td> <td>pSC101</td><td>Kan</td><td>pUCori-red</td><td></td><td>Finished</td>
+<td></td><td>pSB4S15</td> <td>pSC101ts</td><td>Spec</td><td>pUCori-redq</td><td></td><td>Finished</td>
 </tr><tr>
-<td></td><td>pSB4S15Flp</td> <td>pSC101</td><td>Spec</td><td>pUCori-red</td><td></td><td>Finished</td>
+<td></td><td>pSB4A15(Frt)</td> <td>pSC101ts</td><td>Amp</td><td>pUCori-redq</td><td></td><td>Planning</td>
 </tr><tr>
-<td></td><td>pSB4T15Flp</td> <td>pSC101</td><td>Tet</td><td>pUCori-red</td><td></td><td>Finished</td>
+<td></td><td>pSB4C15(Frt)</td> <td>pSC101ts</td><td>Cm</td><td>pUCori-redq</td><td></td><td>Planning</td>
 </tr><tr>
-<td></td><td>pSB4A15FlpIq</td> <td>pSC101</td><td>Amp</td><td>pUCori-redq</td><td></td><td>Planning</td>
+<td></td><td>pSB4K15(Frt)</td> <td>pSC101ts</td><td>Kan</td><td>pUCori-redq</td><td></td><td>Finished</td>
 </tr><tr>
-<td></td><td>pSB4C15FlpIq</td> <td>pSC101</td><td>Cm</td><td>pUCori-redq</td><td></td><td>Finished</td>
+<td></td><td>pSB4S15(Frt)</td> <td>pSC101ts</td><td>Spec</td><td>pUCori-redq</td><td></td><td>Planning</td>
-</tr><tr>
+</tr
-<td></td><td>pSB4K15FlpIq</td> <td>pSC101</td><td>Kan</td><td>pUCori-redq</td><td></td><td>Finished</td>
-</tr><tr>
-<td></td><td>pSB4S15FlpIq</td> <td>pSC101</td><td>Spec</td><td>pUCori-redq</td><td></td><td>Planning</td>
-</tr><tr>
-<td></td><td>pSB4T15FlpIq</td> <td>pSC101</td><td>Tet</td><td>pUCori-redq</td><td></td><td>Planning</td>
-</tr>
 </table>