Team:NTNU Trondheim/Notebook

From 2012.igem.org

(Difference between revisions)
(Tuesday 07.08.12)
 
(33 intermediate revisions not shown)
Line 1: Line 1:
{{:Team:NTNU_Trondheim/Templates/Header}}
{{:Team:NTNU_Trondheim/Templates/Header}}
 +
<html>
 +
<div class="container">
 +
<div class="page-header-top">
 +
<h1>Notebook <small>Unfiltered notes from the lab</small></h1>
 +
</div>
 +
</div>
 +
 +
<div class="container main-container">
 +
 +
 +
<div class="row">
 +
<div class="span10 offset1">
 +
</html>
__NOTOC__
__NOTOC__
-
=Notebook=
 
 +
<html><div class="well well-small"></html>
{{:Team:NTNU_Trondheim/Templates/Calendar}}
{{:Team:NTNU_Trondheim/Templates/Calendar}}
 +
<html></div>
-
For information on plasmids and DNA mentioned in the notebook, see [https://2012.igem.org/Team:NTNU_Trondheim/Stocks Stocks] and [https://2012.igem.org/Team:NTNU_Trondheim/Constructs Constructs].
+
</div>
 +
</div>
 +
<div class="row-fluid">
 +
<div class="span12">
 +
</html>
 +
Click on a month name or date to view the respective notebook entries. For information on plasmids and DNA mentioned in the notebook, see [https://2012.igem.org/Team:NTNU_Trondheim/Stocks Stocks] and [https://2012.igem.org/Team:NTNU_Trondheim/Constructs Constructs]. For explanations of abbreviations used, click [https://2012.igem.org/Team:NTNU_Trondheim/Abbreviations here].
-
For explanations of abbreviations used, click [https://2012.igem.org/Team:NTNU_Trondheim/Abbreviations here].
+
<!--
 +
===Thursday 09.08.12===
 +
----
 +
 
 +
Did a restriction digest as described in the [https://2012.igem.org/Team:NTNU_Trondheim/Protocols protocol] on the following parts. On the pieces to be used as inserts, the number of base pairs are also listed.
 +
 
 +
{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"
 +
!Biobrick/Construct
 +
!BioBrick
 +
!Enzymes
 +
!Buffer
 +
!bp insert
 +
|-
 +
|LuxI+term
 +
|<partinfo>Bba_C0061</partinfo>, <partinfo>BBa_B0015</partinfo>
 +
|XbaI+PstI
 +
|3
 +
|747
 +
|-
 +
|YFP+term
 +
|<partinfo>BBa_E0030</partinfo>, <partinfo>BBa_B0015</partinfo>
 +
|XbaI+PstI
 +
|3
 +
|852
 +
|-
 +
|Lysis
 +
|<partinfo>BBa_K112808</partinfo>
 +
|XbaI+PstI
 +
|3
 +
|1785
 +
|-
 +
|LuxI
 +
|<partinfo>Bba_C0061</partinfo>
 +
|XbaI+PstI
 +
|3
 +
|618
 +
|-
 +
|RBS*
 +
|<partinfo>BBa_B0030</partinfo>
 +
|SpeI+PstI
 +
|1
 +
| -
 +
|-
 +
|P<sub>luxR+HSL</sub>
 +
|<partinfo>BBa_R0062</partinfo>
 +
|SpeI+PstI
 +
|1
 +
| -
 +
|-
 +
|RBS
 +
|<partinfo>BBa_B0034</partinfo>
 +
|SpeI+PstI
 +
|1
 +
| -
 +
|-
 +
|pSB1A3
 +
|<partinfo>pSB1A3</partinfo>
 +
|EcoRI+PstI+DpnI
 +
|3
 +
| -
 +
|-
 +
|plld
 +
| -
 +
|EcoRI+SpeI
 +
|4
 +
|433
 +
|-
 +
|}
 +
 
 +
The parts lysis <partinfo>BBa_K112808</partinfo>, YFP+term (<partinfo>BBa_E0030</partinfo>, <partinfo>BBa_B0015</partinfo>), <partinfo>pSB1A3</partinfo> and plld will be assembled using the 3A assembly, so made double cutting mix with these parts.
 +
 
 +
Gel electrophoresis was done with luxI+term (<partinfo>Bba_C0061</partinfo>, <partinfo>BBa_B0015</partinfo>), lysis <partinfo>BBa_K112808</partinfo>, YFP+term (<partinfo>BBa_E0030</partinfo>, <partinfo>BBa_B0015</partinfo>), LuxI <partinfo>Bba_C0061</partinfo> and plld.
 +
 
 +
P<sub>lld</sub>+lysis and pSB1A3 cut yesterday with E+P was investigated on gel. Two fragments were obtained for P<sub>lld</sub>+lysis, but the sizes of the fragments were not as expected. Since we are not sure which fragment is which, both of the fragments were cut out of the gel and purified using the QIAquick gel extraction kit. In case one of them is uncut plasmid, the unligated DNA from both bonds will be transformed tomorrow, and we will assume that if the cells transformed with DNA from one of the samples won't grow, that sample will contain digested plasmid. This sample will be ligated with pSB1A3, plated out on a ampicillin plate, and then, several cell cultures from this plate will be plated out on a chloramphenicol plate. The colonies that won't grow on chloramphenicol, is assumed to contain the correct plasmid.
 +
 
 +
Vgb+RBS religated, Vgb+RBS+LuxR+DTT #1, Vgb+RBS+LuxR+DTT #2, LuxI+DTT, K+RBS+LacI+DTT and his-tagged LldR was miniprepped using the Promega SV miniprep kit. The concentrations of isolated plasmid are given below:
 +
 
 +
{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"
 +
!Plasmid
 +
!Concentration [ng/µl]
 +
|-
 +
|Vgb+RBS religated
 +
|27.3
 +
|-
 +
|Vgb+RBS+LuxR+DTT #1
 +
|26.2
 +
|-
 +
|Vgb+RBS+LuxR+DTT #2
 +
|31.7
 +
|-
 +
|LuxI+DTT
 +
|35.3
 +
|-
 +
|K+RBS+LacI+DTT
 +
|7.9
 +
|-
 +
|his-LldR
 +
|51.0
 +
|}
 +
 
 +
 
 +
===Wednesday 08.08.12===
 +
----
 +
 
 +
Tranformed Vgb+RBS+YFP+term (<partinfo>BBa_K561001</partinfo>, <partinfo>BBa_B0034</partinfo>, <partinfo>BBa_E0030</partinfo> and <partinfo>BBa_B0015</partinfo>) and Vhb+RBS+YFP+term (<partinfo>BBa_K258005</partinfo>, <partinfo>BBa_B0034</partinfo>, <partinfo>BBa_E0030</partinfo> and <partinfo>BBa_B0015</partinfo>) in competent DH5ɑ cells as described in the [https://2012.igem.org/Team:NTNU_Trondheim/Protocols protocol]. The plasmid in both of the constructs is <partinfo>psB1A2</partinfo>. They were left in the incubator over night.
 +
 
 +
Inspected the petri dishes with plld+RBS (lactate induced promoter and <partinfo>BBa_B0030</partinfo>) and the religation of RBS <partinfo>BBa_B0030</partinfo>, had many more colonies on the plld+RBS than the religation, which is good. Transferred a colony of the plld+RBS (lactate promoter and <partinfo>BBa_B0030</partinfo>) and a colony of RBS <partinfo>BBa_B0030</partinfo> to liquid media, with ampicillin resistance.
 +
 
 +
Cut LuxI+term (<partinfo>Bba_C0061</partinfo> and <partinfo>BBa_B0015</partinfo>) and LuxR+term (<partinfo>BBa_C0062</partinfo> and <partinfo>BBa_B0015</partinfo>) with XbaI and PstI, so that they can be used as inserts. Used the [https://2012.igem.org/Team:NTNU_Trondheim/Protocols protocol] with NEBuffer 3.
 +
 
 +
Did a gel electrophoresis with LuxI+term (<partinfo>Bba_C0061</partinfo> and <partinfo>BBa_B0015</partinfo>) and LuxR+term (<partinfo>BBa_C0062</partinfo> and <partinfo>BBa_B0015</partinfo>).
 +
 
 +
Purified RBS and LuxR+DTT. Resulting concentrations were 4.8 and 11.2 ng/µl, respectively.
===Tuesday 07.08.12===
===Tuesday 07.08.12===
----
----
-
Inspected the petri dishes from yesterday, LacI (<partinfo>BBa_C0012</partinfo>) + terminator (<partinfo>BBa_B0015</partinfo>) showed colonies, the religation of the terminator (<partinfo>BBa_B0015</partinfo>) did not. Transferred to LB with ampicillin and inoculated at 37C with shaking.
+
Inspected the petri dishes from yesterday, LacI <partinfo>BBa_C0012</partinfo> + terminator <partinfo>BBa_B0015</partinfo> showed colonies, the religation of the terminator <partinfo>BBa_B0015</partinfo> did not. Transferred a LacI <partinfo>BBa_C0012</partinfo> + terminator <partinfo>BBa_B0015</partinfo> colony to LB with ampicillin and inoculated at 37C with shaking.
 +
 
 +
Did a gel electrophoresis on plld, cut it out and extracted it, according to [https://2012.igem.org/Team:NTNU_Trondheim/Protocols protocols]. The concentration after gel extraction was: c<sub>plld</sub> = 10,6 ng/µl.
 +
 
 +
[[File:Plld, cut E+S, 07.08.12exp.png‎|thumb|center|400px|Picture of gel electrophoresis with plld. It was found at approximately 340 bp, as expected (344 bp).]]
 +
 
 +
Ligation was performed with plld as insert and RBS <partinfo>BBa_B0034</partinfo> as backbone. That means that the construct has a <partinfo>psB1A2</partinfo> plasmid. Also did a religation of the RBS <partinfo>BBa_B0034</partinfo> without any insert.
 +
 
 +
Transformed plld and RBS <partinfo>BBa_B0034</partinfo> in competent DH5ɑ cells, as well as the religation of RBS <partinfo>BBa_B0034</partinfo>. They were transferred to petri dishes with ampicillin resistance and left in the incubator over night.
 +
 
 +
Miniprepped VHB+RBS+lysis (<partinfo>BBa_K258005</partinfo>, <partinfo>BBa_B0034</partinfo> and <partinfo>BBa_K112808</partinfo>), VGB+RBS+lysis (<partinfo>BBa_K561001</partinfo>, <partinfo>BBa_B0034</partinfo> and <partinfo>BBa_K112808</partinfo>), pllD+lysis (lactate promoter and <partinfo>BBa_K112808</partinfo>) and three parallels of pBad+YFP (<partinfo>Bba_K206000</partinfo> and <partinfo>BBa_E0030</partinfo>)
 +
 
 +
{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"
 +
!Sample
 +
!Concentration [ng/µl]
 +
|-
 +
|VHB+RBS+lysis
 +
|38,2
 +
|-
 +
|VGB+RBS+lysis
 +
|50,5
 +
|-
 +
|pllD+lysis
 +
|40,2
 +
|-
 +
|pBad+YFP #1
 +
|46,6
 +
|-
 +
|pBad+YFP #2
 +
|40,5
 +
|-
 +
|pBad+YFP #3
 +
|38,9
 +
|}
===Monday 06.08.12===
===Monday 06.08.12===
Line 26: Line 190:
[[File:LacI-E+S_K-E+X_06.08.12_(1).png‎|thumb|center|400px|Gel picture of the two inserts ran on gel. Used two different ladders. To the left we used a 1kb ladder to identify the Lac I insert, and to the right we used a 50 kb ladder to identify the constitutive promoter. Expected the LacI band to be approximately 1100 bp which seems to be about right. The constitutive promoter was expected to be 35 bp, but was not detected on the gel at all.]]
[[File:LacI-E+S_K-E+X_06.08.12_(1).png‎|thumb|center|400px|Gel picture of the two inserts ran on gel. Used two different ladders. To the left we used a 1kb ladder to identify the Lac I insert, and to the right we used a 50 kb ladder to identify the constitutive promoter. Expected the LacI band to be approximately 1100 bp which seems to be about right. The constitutive promoter was expected to be 35 bp, but was not detected on the gel at all.]]
-
Did a ligation with LacI (<partinfo>BBa_C0012</partinfo>) and double terminator (<partinfo>BBa_B0015</partinfo>), where the double terminator (<partinfo>BBa_B0015</partinfo>) was used as a backbone. That means, the construct has a <partinfo>psB1AK3</partinfo> backbone and resistance Ampicillin and Kanamycin. A religation of the backbone (<partinfo>BBa_B0015</partinfo>) was also performed.
+
Used the PCR purification kit and protocol from www.qiagen.com [http://www.google.no/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=0CFUQFjAB&url=http%3A%2F%2Fwww.qiagen.com%2FHB%2FQIAquickGelExtractionKit_EN&ei=RhYhUPiCA4iL4gS64oD4CA&usg=AFQjCNFwed-RxSRRgTFSJgtn7Fl5qisiWw&sig2=-pxyzkbLpVKt4DLlcSzQcw], on RBS <partinfo>BBa_B0030</partinfo>, pBad <partinfo>Bba_K206000</partinfo> and terminator <partinfo>BBa_B0014</partinfo>. Did a gel extraction using the Gel Extraction kit and [https://2012.igem.org/Team:NTNU_Trondheim/Protocols Protocol] on LacI <partinfo>BBa_C0012</partinfo>.
 +
 
 +
Did a ligation with LacI <partinfo>BBa_C0012</partinfo> and double terminator <partinfo>BBa_B0015</partinfo>, where the double terminator <partinfo>BBa_B0015</partinfo> was used as a backbone. That means, the construct has a <partinfo>psB1AK3</partinfo> backbone and resistance Ampicillin and Kanamycin. A religation of the backbone <partinfo>BBa_B0015</partinfo> was also performed.
-
The ligated LacI (<partinfo>BBa_C0012</partinfo>) and double terminator (<partinfo>BBa_B0015</partinfo>) was transformed in competent DH5ɑ cells, along with the religation of the backbone (<partinfo>BBa_B0015</partinfo>). The petri dishes are left to inoculate through the night.  
+
The ligated LacI <partinfo>BBa_C0012</partinfo> and double terminator <partinfo>BBa_B0015</partinfo> was transformed in competent DH5ɑ cells, along with the religation of the backbone <partinfo>BBa_B0015</partinfo>. The petri dishes are left to inoculate through the night.  
A new batch of LA-medium was made and used to make ampicillin petri dishes.
A new batch of LA-medium was made and used to make ampicillin petri dishes.
-
A restriction digest was done on the lld promoter, as described in the [https://2012.igem.org/Team:NTNU_Trondheim/Protocols protocols]. It was cut with EcoRI and SpeI, and will be an insert with RBS (<partinfo>BBa_B0030</partinfo>) as backbone. This will then make the plasmid <partinfo>pSB1A2</partinfo> the backbone.
+
A restriction digest was done on the lld promoter, as described in the [https://2012.igem.org/Team:NTNU_Trondheim/Protocols protocols]. It was cut with EcoRI and SpeI, and will be an insert with RBS <partinfo>BBa_B0030</partinfo> as backbone. This will then make the plasmid <partinfo>pSB1A2</partinfo> the backbone.
===Sunday 05.08.12===
===Sunday 05.08.12===
Line 541: Line 707:
The following PCR programs were used:
The following PCR programs were used:
-
{|style="text-align:center; margin: 1em auto 1em auto;"
+
 
-
| style="padding-right: 20px;"; |
+
{|style="text-align:center; margin: 1em auto 1em auto;" |
 +
 
 +
| style="padding-right:10px"; |
 +
 
{|class="wikitable" style="margin: 1em auto 1em auto;"
{|class="wikitable" style="margin: 1em auto 1em auto;"
|+ lld promoter
|+ lld promoter
Line 606: Line 775:
|}
|}
-
| style="padding-left:20px;" |
+
| style="padding-left:10px;" |
{|class="wikitable" style="margin: 1em auto 1em auto;"
{|class="wikitable" style="margin: 1em auto 1em auto;"
Line 670: Line 839:
|∞
|∞
|}
|}
 +
|-
|-
-
| colspan="2" |
+
 
 +
|colspan="2" |
 +
 
{|class="wikitable" style="margin: 1em auto 1em auto;"
{|class="wikitable" style="margin: 1em auto 1em auto;"
|+ lldR
|+ lldR
Line 719: Line 891:
|∞
|∞
|}
|}
 +
|}
|}
Line 1,559: Line 1,732:
We recieved emails from Eivind letting us know that we are the ones that have been selected to represent NTNU in iGEM 2012. Everybody is happy!
We recieved emails from Eivind letting us know that we are the ones that have been selected to represent NTNU in iGEM 2012. Everybody is happy!
 +
-->
 +
<html>
</div>
</div>
 +
</html>
 +
{{:Team:NTNU_Trondheim/Templates/Sponsors}}
 +
<html>
 +
</div></div></html>
 +
{{:Team:NTNU_Trondheim/Templates/Footer}}

Latest revision as of 17:15, 25 September 2012

NTNU IS B.A.C.K.
Bacterial Anti-Cancer-Kamikaze

Notebook Unfiltered notes from the lab


February
Mon Tue Wed Thu Fri Sat Sun
1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 25
27 28 29
March
Mon Tue Wed Thu Fri Sat Sun
1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30
April
Mon Tue Wed Thu Fri Sat Sun
1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30
May
Mon Tue Wed Thu Fri Sat Sun
1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30 31
June
Mon Tue Wed Thu Fri Sat Sun
1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
July
Mon Tue Wed Thu Fri Sat Sun
1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30 31
August
Mon Tue Wed Thu Fri Sat Sun
1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
September
Mon Tue Wed Thu Fri Sat Sun
1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30

Click on a month name or date to view the respective notebook entries. For information on plasmids and DNA mentioned in the notebook, see Stocks and Constructs. For explanations of abbreviations used, click here.



Retrieved from "http://2012.igem.org/Team:NTNU_Trondheim/Notebook"