Template:Team:Edinburgh/Notebook/Week04

From 2012.igem.org

(Difference between revisions)
m
m
 
(6 intermediate revisions not shown)
Line 1: Line 1:
 +
{{:Team:Edinburgh/header}}
{{Team:Edinburgh/css/wiki-edit-structure.css}}
{{Team:Edinburgh/css/wiki-edit-structure.css}}
-
{{Team:Edinburgh/css/notebook-week-style.css}}
+
{{Team:Edinburgh/css/notebook-style.css}}
 +
{{Team:Edinburgh/css/article.css}}
 +
 
 +
{{Team:Edinburgh/notebook-navigation}}
<html>
<html>
-
<div id="week04">
+
<style type="text/css">
-
<div class="edi-lab-book-entry" id="w04-16-07">
+
#week04 a,
-
<div class="entry-title">
+
#week04 a:hover,
-
<p><a name="week04-16-07">Week 4, 16.07.2012, Monday</a></p>
+
#week04 a:visited,
-
</div>
+
#week04 span{
-
<div class="entry-details">
+
color: #7f7f7f;
-
<p>Posted on 16/07/2012</p>
+
font: bold 14px/18px tahoma, calibri, sans-serif;
-
</div>
+
}
-
<div class="entry-text">
+
#week04 .navigation-description{
-
<p>
+
color: #ccc;
-
<b>1) The M9 agar plates were inoculated with C.freundii SBS 197, C.freundii NCIMB, E.coli J109, E.coli + sucrose hydrolase. The plates were left o/n 37ºC. </b> <br/>
+
font: normal 12px/14px tahoma, calibri, sans-serif;
 +
}
 +
</style>
 +
<div id="page-content">
 +
<div class="article" id="w04-16-07">
 +
<div class="article-title">
 +
<p><a name="week04-16-07">Week 4, 16.07.2012, Monday</a></p>
 +
</div>
 +
<div class="article-details">
 +
<p>Posted on 16/07/2012</p>
 +
</div>
 +
<div class="article-text">
 +
<p>
 +
<b>1) The M9 agar plates were inoculated with C.freundii SBS 197, C.freundii NCIMB, E.coli J109, E.coli + sucrose hydrolase. The plates were left o/n 37ºC. </b> <br/>
 +
 
 +
<b> 2) LB plates with 200 µl carbinicillin were prepared: </b> <br/>
 +
2* 50 µl MTZ (out of 50 mg/ml stock) 100 µg/ml+ 25 µg/ml IPTG <br/>
 +
2*45 µl MTZ (out of 50 mg/ml stock) 90 µg/ml+ 25 µg/ml IPTG <br/>
 +
5 µl NFT (out of 5 mg/ml stock) 1 µg/ml +25 µg/ml IPTG <br/>
 +
1 µl NFT (out of 5 mg/ml stock) 0.2 µg/ml +25 µg/ml IPTG <br/>
 +
50 µl DNBA (out of 50 mg/ml stock) 100 µg/ml +25 µg/ml IPTG <br/>
 +
75 µl DNBA (out of 50 mg/ml stock) 150 µg/ml +25 µg/ml IPTG <br/>
 +
Control +25 µg/ml IPTG <br/>
 +
Control – IPTG  <br/>
 +
The MTZ plates were streaked with either BS control or BS nitred. All other plates were streaked with the four nitroreductase strains. <br/><br/>
 +
<b> Protein assay of cytochrome expression cell extracts</b><br/>
 +
Protein assay was performed for 4 samples of cytochrome expression cell extracts (<a href="http://openwetware.org/wiki/Cfrench:ProteinAssay"> protocol</a>). Results:<br/>
 +
Anaerobic culture with nitrate: 0,094 mg protein/ml<br/>
 +
Aerobic culture with nitrate: 0,113 mg protein/ml <br/>
 +
Anaerobic culture with TMAO: 0,134 mg protein/ml <br/>
 +
Aerobic culture with TMAO: 0,725 mg protein/ml <br/><br/>
 +
<b> minipreps of ccm plasmids </b><br/>
 +
Minipreps were prepared for ccm plasmids (<a href="http://openwetware.org/wiki/Cfrench:minipreps1">protocol</a>).
 +
</p>
 +
</div><!-- /article-text -->
 +
</div><!-- /article -->
 +
<div class="article" id="w04-17-07">
 +
<div class="article-title">
 +
<p><a name="week04-17-07">Week 4, 17.07.2012, Tuesday</a></p>
 +
</div>
 +
<div class="article-details">
 +
<p>Posted on 17/07/2012</p>
 +
</div>
 +
<div class="article-text">
 +
<p>
 +
<b> 1)The M9 agar plates were examined. </b> All strains grew on all plates even the no sugar ones. <br/>
 +
The M9 agar plates were repeated by inoculating the remaining plates with the 4 strains present on the glucose plates (from 16th July 2012). <br/>  
-
<b> 2) LB plates with 200 µl carbinicillin were prepared: </b> <br/>
+
<b> 2)LB plates were examined: </b> <br/>
-
2* 50 µl MTZ (out of 50 mg/ml stock) 100 µg/ml+ 25 µg/ml IPTG <br/>
+
Both BS control and BS nitred grew ( BS-contol much better) on the MTZ plates.  <br/>
-
2*45 µl MTZ (out of 50 mg/ml stock) 90 µg/ml+ 25 µg/ml IPTG <br/>
+
No difference between the strains was seen in the NFT, DNBA and control plates. <br/>
-
5 µl NFT (out of 5 mg/ml stock) 1 µg/ml +25 µg/ml IPTG <br/>
+
-
1 µl NFT (out of 5 mg/ml stock) 0.2 µg/ml +25 µg/ml IPTG <br/>
+
-
50 µl DNBA (out of 50 mg/ml stock) 100 µg/ml +25 µg/ml IPTG <br/>
+
-
75 µl DNBA (out of 50 mg/ml stock) 150 µg/ml +25 µg/ml IPTG <br/>
+
-
Control +25 µg/ml IPTG <br/>
+
-
Control – IPTG  <br/>
+
-
The MTZ plates were streaked with either BS control or BS nitred. All other plates were streaked with the four nitroreductase strains. <br/>
+
-
</p>
+
-
</div>
+
-
</div>
+
-
<div class="edi-lab-book-entry" id="w04-17-07">
+
<b> 3)LB plates were prepared  </b>(200 µl carbinicillin) <br/>
-
<div class="entry-title">
+
2* 100 µg/ml MTZ + 25 µg/ml IPTG+80 µg/ml Xgal  <br/>
-
<p><a name="week04-17-07">Week 4, 17.07.2012, Tuesday</a></p>
+
110 µg/ml MTZ + 25 µg/ml IPTG+ 80 µg/ml Xgal <br/>
-
</div>
+
2* 120 µg/ml MTZ + 25 µg/ml IPTG+ 80 µg/ml Xgal <br/>
-
<div class="entry-details">
+
2* 130 µg/ml MTZ + 25 µg/ml IPTG+ 80 µg/ml Xgal <br/>
-
<p>Posted on 17/07/2012</p>
+
2* control + 25 µg/ml IPTG + 80 µg/ml Xgal <br/>
-
</div>
+
0.5  µg/ml NFT + 25 µg/ml IPTG+ 40 µg/ml Xgal <br/>
-
<div class="entry-text">
+
1 µg/ml NFT + 25 µg/ml IPTG+ 40 µg/ml Xgal <br/>
-
<p>
+
100 µg/ml DNBA + 25 µg/ml IPTG+ 40 µg/ml Xgal <br/>
-
<b> 1)The M9 agar plates were examined. </b> All strains grew on all plates even the no sugar ones. <br/>  
+
150 µg/ml DNBA + 25 µg/ml IPTG+ 40 µg/ml Xgal <br/>
-
The M9 agar plates were repeated by inoculating the remaining plates with the 4 strains present on the glucose plates (from 16th July 2012). <br/>  
+
200 µg/ml DNBA + 25 µg/ml IPTG+ 40 µg/ml Xgal <br/>
-
<b> 2)LB plates were examined: </b> <br/>
+
<b> 4)Liquid cultures of BS-control, BS-nitred, nitred-6 and CFX-nitred were prepared. </b> <br/>
-
Both BS control and BS nitred grew ( BS-contol much better) on the MTZ plates. <br/>
+
Carbinicillin (5  µl) was added to LB (5 ml) and the bottles were inoculated with the four strains. These were left o/n at 37ºC with agitation. <br/><br/>
-
No difference between the strains was seen in the NFT, DNBA and control plates. <br/>
+
<b> Ccm miniprep analysis </b><br/>
 +
ccm minipreprs were digested with EcoRI (<a href="http://openwetware.org/wiki/Cfrench:restriction1"> protocol </a>)<br/>
 +
Following the digestion, samples were analysed by gel electrophoresis (<a href="http://openwetware.org/wiki/Cfrench:AGE> protocol</a>)<br/><br/>
 +
<b> SDS PAGE of ccm cell extracts </b><br/>
 +
cell extracts (25 ul) were mixed with SDS loading buffer (25 ul) and boiled for a short duration<br/>
 +
Pre-cast gel was used;<br/>
 +
haem staining and commasie staining were peformed for the gels <br/>
 +
</p>
 +
</div><!-- /article-text -->
 +
</div><!-- /article -->
 +
<div class="article" id="w04-18-07">
 +
<div class="article-title">
 +
<p><a name="week04-18-07">Week 4, 18.07.2012, Wednesday</a></p>
 +
</div>
 +
<div class="article-details">
 +
<p>Posted on 18/07/2012</p>
 +
</div>
 +
<div class="article-text">
 +
<p>
 +
<b>1)The M9 agar plates were examined.</b> Again, all strains grew on all plates. A fresh batch of M9 agar (without a carbon source) was also prepared and will be inoculated tomorrow. <br/>
-
<b> 3)LB plates were prepared  </b>(200 µl carbinicillin) <br/>
+
<b> 2)The OD600 of the 4 nitroreductase liquid cultures was measured with LB blank: </b> <br/>
-
2* 100 µg/ml MTZ + 25 µg/ml IPTG+80 µg/ml Xgal  <br/>
+
BS-nitred: 1.246 <br/>
-
110 µg/ml MTZ + 25 µg/ml IPTG+ 80 µg/ml Xgal <br/>
+
BS-control: 1.305 <br/>
-
2* 120 µg/ml MTZ + 25 µg/ml IPTG+ 80 µg/ml Xgal <br/>
+
Nitred-6: 1.765 <br/>
-
2* 130 µg/ml MTZ + 25 µg/ml IPTG+ 80 µg/ml Xgal <br/>
+
CFX-nitred: 1.294 <br/>
-
2* control + 25 µg/ml IPTG + 80 µg/ml Xgal <br/>
+
-
0.5  µg/ml NFT + 25 µg/ml IPTG+ 40 µg/ml Xgal <br/>
+
-
1 µg/ml NFT + 25 µg/ml IPTG+ 40 µg/ml Xgal <br/>
+
-
100 µg/ml DNBA + 25 µg/ml IPTG+ 40 µg/ml Xgal <br/>
+
-
150 µg/ml DNBA + 25 µg/ml IPTG+ 40 µg/ml Xgal <br/>
+
-
200 µg/ml DNBA + 25 µg/ml IPTG+ 40 µg/ml Xgal <br/>
+
-
<b> 4)Liquid cultures of BS-control, BS-nitred, nitred-6 and CFX-nitred were prepared. </b> <br/>
+
The MTZ plates and controls were spread with BS-nitred (105 µl) or BS-control (100 µl) liquid culture.
-
Carbinicillin (5  µl) was added to LB (5 ml) and the bottles were inoculated with the four strains. These were left o/n at 37ºC with agitation. <br/>
+
The DNBA/NFT plates were streaked with BS-nitred (26 µl), BS-control (24 µl), nitred-6 (18 µl) and CFX-nitred (25 µl) liquid culture. <br/>
-
</p>
+
The plates were left o/n at 37ºC <br/>
-
</div>
+
-
</div>
+
-
<div class="edi-lab-book-entry" id="w04-18-07">
+
-
<div class="entry-title">
+
-
<p><a name="week04-18-07">Week 4, 18.07.2012, Wednesday</a></p>
+
-
</div>
+
-
<div class="entry-details">
+
-
<p>Posted on 18/07/2012</p>
+
-
</div>
+
-
<div class="entry-text">
+
-
<p>
+
-
<b>1)The M9 agar plates were examined.</b> Again, all strains grew on all plates. A fresh batch of M9 agar (without a carbon source) was also prepared and will be inoculated tomorrow. <br/>
+
-
<b> 2)The OD600 of the 4 nitroreductase liquid cultures was measured with LB blank: </b> <br/>
+
<b>3) Lac promoter characterization – results</b><br/>
-
BS-nitred: 1.246 <br/>
+
Three different sets of E. coli and Cf were grown overnight in LB, their OD was measured in order to normalize the number of cells and the normalized dilutions were used to inoculate 2.5ml M9 media that contained either no IPTG or 1, 2, 3, 4 or 5 μl IPTG. These bottles were then incubated at 37C for 24 hours. <br/> The fluorescence of the cultures was measured just after inoculation and it was fairly even within the two species, averaging 1669.88 FSU for E. coli and 1235.91 FSU for E. coli. <br/> Their fluorescence and OD was again measured after 24 hours in order to quantify RFP expression. These readings were normalized by dividing the fluorescence with the OD and the averages of the three sets were calculated. The graphs showing these averages can be seen below. <br/>
-
BS-control: 1.305 <br/>
+
<img style="width:612px;" src="https://static.igem.org/mediawiki/2012/3/36/E._coli_IPTG.PNG" /><br/>
-
Nitred-6: 1.765 <br/>
+
<img style="width:612px;" src="https://static.igem.org/mediawiki/2012/4/42/Cf_IPTG.PNG" /><br/>
-
CFX-nitred: 1.294 <br/>
+
The peaks that can be seen in Cf at 2 and 4 μl IPTG are due very high fluorescence readings in one of the sets, which have skewed these averages somewhat. <br/> These results do, however, show that there is a significant difference in RFP expression in E. coli with and without IPTG, while no significant difference in the levels of RFP expression is observable in Cf. This suggests that E. coli has got a native LacI gene that represses the Lac promoter on the plasmid, while Cf lacks a native LacI gene, which results in the RFP gene being constitutively expressed.</br><br/><br/>
-
The MTZ plates and controls were spread with BS-nitred (105 µl) or BS-control (100 µl) liquid culture.
+
<b>4) Gel analysis of ccm minipreps </b><br/>
-
The DNBA/NFT plates were streaked with BS-nitred (26 µl), BS-control (24 µl), nitred-6 (18 µl) and CFX-nitred (25 µl) liquid culture. <br/>
+
<img style="width:612px;" src="http://i1056.photobucket.com/albums/t366/edigem12/SkitchImage0.png" />
-
The plates were left o/n at 37ºC <br/>
+
-
<b>Lac promoter characterization – results</b><br/>
+
</p>
-
Three different sets of E. coli and Cf were grown overnight in LB, their OD was measured in order to normalize the number of cells and the normalized dilutions were used to inoculate 2.5ml M9 media that contained either no IPTG or 1, 2, 3, 4 or 5 μl IPTG. These bottles were then incubated at 37C for 24 hours. <br/> The fluorescence of the cultures was measured just after inoculation and it was fairly even within the two species, averaging 1669.88 FSU for E. coli and 1235.91 FSU for E. coli. <br/> Their fluorescence and OD was again measured after 24 hours in order to quantify RFP expression. These readings were normalized by dividing the fluorescence with the OD and the averages of the three sets were calculated. The graphs showing these averages can be seen below. <br/>
+
</div><!-- /article-text -->
-
<img style="width:612px;" src="https://static.igem.org/mediawiki/2012/3/36/E._coli_IPTG.PNG" /><br/>
+
</div><!-- /article -->
-
<img style="width:612px;" src="https://static.igem.org/mediawiki/2012/4/42/Cf_IPTG.PNG" />
+
<div class="article" id="w04-19-07">
-
The peaks that can be seen in Cf at 2 and 4 μl IPTG are due very high fluorescence readings in one of the sets, which have skewed these averages somewhat. <br/> These results do, however, show that there is a significant difference in RFP expression in E. coli with and without IPTG, while no significant difference in the levels of RFP expression is observable in Cf. This suggests that E. coli has got a native LacI gene that represses the Lac promoter on the plasmid, while Cf lacks a native LacI gene, which results in the RFP gene being constitutively expressed.
+
<div class="article-title">
 +
<p><a name="week04-19-07">Week 4, 19.07.2012, Thursday</a></p>
 +
</div>
 +
<div class="article-details">
 +
<p><Posted on 19/07/2012></p>
 +
</div>
 +
<div class="entry-text">
 +
<p>
 +
<b>1) ccm PCR product re-digestion</b><br/>
 +
PCR product for ccm was digested with EcoRI and PstI using buffer 4 and buffer EcoRI, following the digestion samples were ligated overnight in 16 C waterbath.<br/> <br/>
 +
<b>2) Glycerol stock preparation</b>
 +
Glycerol stocks were made of E. coli JM109 and Cf NCIMB and SBS197 strains by mixing 700 μl of culture with 300 μl 50% glycerol. They are stored in the -80C freezer.<br/>
-
</p>
+
</p>
-
</div>
+
</div><!-- /article-text -->
-
</div>
+
</div><!-- /article -->
-
</div>
+
<div class="article" id="w04-20-07">
 +
<div class="article-title">
 +
<p><a name="week04-20-07">Week 4, 20.07.2012, Friday</a></p>
 +
</div>
 +
<div class="article-details">
 +
<p><Posted on 1/8/2012></p>
 +
</div>
 +
<div class="entry-text">
 +
<p>
 +
<b>1) Gel analysis of ligation sample </b><br/>
 +
<img style="width:612px;" src="http://i1056.photobucket.com/albums/t366/edigem12/SkitchImage0-1.png" /><br/><br/>
 +
<b>2) Multi-host vector characterization in Cf – results II</b><br/>
 +
The Cf + ptg262 plasmid grew on all plates to some extent whereas the no plasmid control grew only below 8 μl chloramphenicol and there were fewer colonies on the plates compared to the Cf + ptg262. This suggests that the plasmid must work, but very weakly.<br/><br/>
 +
</p>
 +
</div><!-- /article-text -->
 +
</div><!-- /article -->
 +
</div><!-- /page-content -->
 +
</div><!-- /page-middle -->
</html>
</html>

Latest revision as of 15:41, 1 August 2012

Week 4, 16.07.2012, Monday

Posted on 16/07/2012

1) The M9 agar plates were inoculated with C.freundii SBS 197, C.freundii NCIMB, E.coli J109, E.coli + sucrose hydrolase. The plates were left o/n 37ºC.
2) LB plates with 200 µl carbinicillin were prepared:
2* 50 µl MTZ (out of 50 mg/ml stock) 100 µg/ml+ 25 µg/ml IPTG
2*45 µl MTZ (out of 50 mg/ml stock) 90 µg/ml+ 25 µg/ml IPTG
5 µl NFT (out of 5 mg/ml stock) 1 µg/ml +25 µg/ml IPTG
1 µl NFT (out of 5 mg/ml stock) 0.2 µg/ml +25 µg/ml IPTG
50 µl DNBA (out of 50 mg/ml stock) 100 µg/ml +25 µg/ml IPTG
75 µl DNBA (out of 50 mg/ml stock) 150 µg/ml +25 µg/ml IPTG
Control +25 µg/ml IPTG
Control – IPTG
The MTZ plates were streaked with either BS control or BS nitred. All other plates were streaked with the four nitroreductase strains.

Protein assay of cytochrome expression cell extracts
Protein assay was performed for 4 samples of cytochrome expression cell extracts ( protocol). Results:
Anaerobic culture with nitrate: 0,094 mg protein/ml
Aerobic culture with nitrate: 0,113 mg protein/ml
Anaerobic culture with TMAO: 0,134 mg protein/ml
Aerobic culture with TMAO: 0,725 mg protein/ml

minipreps of ccm plasmids
Minipreps were prepared for ccm plasmids (protocol).

Week 4, 17.07.2012, Tuesday

Posted on 17/07/2012

1)The M9 agar plates were examined. All strains grew on all plates even the no sugar ones.
The M9 agar plates were repeated by inoculating the remaining plates with the 4 strains present on the glucose plates (from 16th July 2012).
2)LB plates were examined:
Both BS control and BS nitred grew ( BS-contol much better) on the MTZ plates.
No difference between the strains was seen in the NFT, DNBA and control plates.
3)LB plates were prepared (200 µl carbinicillin)
2* 100 µg/ml MTZ + 25 µg/ml IPTG+80 µg/ml Xgal
110 µg/ml MTZ + 25 µg/ml IPTG+ 80 µg/ml Xgal
2* 120 µg/ml MTZ + 25 µg/ml IPTG+ 80 µg/ml Xgal
2* 130 µg/ml MTZ + 25 µg/ml IPTG+ 80 µg/ml Xgal
2* control + 25 µg/ml IPTG + 80 µg/ml Xgal
0.5 µg/ml NFT + 25 µg/ml IPTG+ 40 µg/ml Xgal
1 µg/ml NFT + 25 µg/ml IPTG+ 40 µg/ml Xgal
100 µg/ml DNBA + 25 µg/ml IPTG+ 40 µg/ml Xgal
150 µg/ml DNBA + 25 µg/ml IPTG+ 40 µg/ml Xgal
200 µg/ml DNBA + 25 µg/ml IPTG+ 40 µg/ml Xgal
4)Liquid cultures of BS-control, BS-nitred, nitred-6 and CFX-nitred were prepared.
Carbinicillin (5 µl) was added to LB (5 ml) and the bottles were inoculated with the four strains. These were left o/n at 37ºC with agitation.

Ccm miniprep analysis
ccm minipreprs were digested with EcoRI ( protocol )
Following the digestion, samples were analysed by gel electrophoresis (

Week 4, 18.07.2012, Wednesday

Posted on 18/07/2012

1)The M9 agar plates were examined. Again, all strains grew on all plates. A fresh batch of M9 agar (without a carbon source) was also prepared and will be inoculated tomorrow.
2)The OD600 of the 4 nitroreductase liquid cultures was measured with LB blank:
BS-nitred: 1.246
BS-control: 1.305
Nitred-6: 1.765
CFX-nitred: 1.294
The MTZ plates and controls were spread with BS-nitred (105 µl) or BS-control (100 µl) liquid culture. The DNBA/NFT plates were streaked with BS-nitred (26 µl), BS-control (24 µl), nitred-6 (18 µl) and CFX-nitred (25 µl) liquid culture.
The plates were left o/n at 37ºC
3) Lac promoter characterization – results
Three different sets of E. coli and Cf were grown overnight in LB, their OD was measured in order to normalize the number of cells and the normalized dilutions were used to inoculate 2.5ml M9 media that contained either no IPTG or 1, 2, 3, 4 or 5 μl IPTG. These bottles were then incubated at 37C for 24 hours.
The fluorescence of the cultures was measured just after inoculation and it was fairly even within the two species, averaging 1669.88 FSU for E. coli and 1235.91 FSU for E. coli.
Their fluorescence and OD was again measured after 24 hours in order to quantify RFP expression. These readings were normalized by dividing the fluorescence with the OD and the averages of the three sets were calculated. The graphs showing these averages can be seen below.


The peaks that can be seen in Cf at 2 and 4 μl IPTG are due very high fluorescence readings in one of the sets, which have skewed these averages somewhat.
These results do, however, show that there is a significant difference in RFP expression in E. coli with and without IPTG, while no significant difference in the levels of RFP expression is observable in Cf. This suggests that E. coli has got a native LacI gene that represses the Lac promoter on the plasmid, while Cf lacks a native LacI gene, which results in the RFP gene being constitutively expressed.


4) Gel analysis of ccm minipreps

Week 4, 19.07.2012, Thursday

1) ccm PCR product re-digestion
PCR product for ccm was digested with EcoRI and PstI using buffer 4 and buffer EcoRI, following the digestion samples were ligated overnight in 16 C waterbath.

2) Glycerol stock preparation Glycerol stocks were made of E. coli JM109 and Cf NCIMB and SBS197 strains by mixing 700 μl of culture with 300 μl 50% glycerol. They are stored in the -80C freezer.

Week 4, 20.07.2012, Friday

1) Gel analysis of ligation sample


2) Multi-host vector characterization in Cf – results II
The Cf + ptg262 plasmid grew on all plates to some extent whereas the no plasmid control grew only below 8 μl chloramphenicol and there were fewer colonies on the plates compared to the Cf + ptg262. This suggests that the plasmid must work, but very weakly.

Retrieved from "http://2012.igem.org/Template:Team:Edinburgh/Notebook/Week04"