Team:NTNU Trondheim/Notebook

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<h1>Notebook <small>Unfiltered notes from the lab</small></h1>
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__NOTOC__
__NOTOC__
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=Notebook=
 
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{{:Team:NTNU_Trondheim/Templates/Calendar}}
{{:Team:NTNU_Trondheim/Templates/Calendar}}
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For information on plasmids and DNA mentioned in the notebook, see [http://2012.igem.org/Team:NTNU_Trondheim/Stocks Stocks] and [http://2012.igem.org/Team:NTNU_Trondheim/Constructs Constructs].
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Click on a month name or date to view the respective notebook entries. For information on plasmids and DNA mentioned in the notebook, see [http://2012.igem.org/Team:NTNU_Trondheim/Stocks Stocks] and [http://2012.igem.org/Team:NTNU_Trondheim/Constructs Constructs]. For explanations of abbreviations used, click [http://2012.igem.org/Team:NTNU_Trondheim/Abbreviations here].
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For explanations of abbreviations used, click [http://2012.igem.org/Team:NTNU_Trondheim/Abbreviations here].
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<!--
 +
===Thursday 09.08.12===
 +
----
 +
 
 +
Did a restriction digest as described in the [http://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 [http://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 [http://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===
 +
----
 +
 
 +
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 [http://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 19: Line 188:
The inserts LacI <partinfo>BBa_C0012</partinfo> and constitutive promoter <partinfo>BBa_J23119</partinfo> were run on gel after the restriction digest.  The problem with the constitutive promoter is that it is only 35 b.p long, and is therefore difficult to use as an insert. We tried to stop the gel early to see if we could detect the constitutive promoter, but we could not see anything at all. We therefore ran the gel further and cut the LacI <partinfo>BBa_C0012</partinfo> out of the gel.
The inserts LacI <partinfo>BBa_C0012</partinfo> and constitutive promoter <partinfo>BBa_J23119</partinfo> were run on gel after the restriction digest.  The problem with the constitutive promoter is that it is only 35 b.p long, and is therefore difficult to use as an insert. We tried to stop the gel early to see if we could detect the constitutive promoter, but we could not see anything at all. We therefore ran the gel further and cut the LacI <partinfo>BBa_C0012</partinfo> out of the gel.
-
{|border="0"
+
[[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.]]
+
 
-
|}
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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 [http://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.
 +
 
 +
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 [http://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===
----
----
-
Plasmid DNA was isolated from pllD-1B and BBa_B0014 cultures were isolated by miniprep. DNA concentrations were measured as 41,2/42,5 and 53,3/52,8 ng/uL respectively (two measurements per sample).
+
Plasmid DNA was isolated from pllD-1B and <partinfo>BBa_B0014</partinfo> cultures were isolated by miniprep. DNA concentrations were measured as 41,2/42,5 and 53,3/52,8 ng/uL respectively (two measurements per sample).
Inspected agar plates in incubator inoculated on 3/8:
Inspected agar plates in incubator inoculated on 3/8:
Line 54: Line 231:
Ligated pBad (<partinfo>Bba_K206000</partinfo>) together with YFP+DTT (<partinfo>BBa_E0030</partinfo> and <partinfo>BBa_B0015</partinfo>), and VGB+RBS (<partinfo>BBa_K561001</partinfo> and <partinfo>BBa_B0034</partinfo>) and VHB+RBS (<partinfo>BBa_K258005</partinfo> and <partinfo>BBa_B0034</partinfo>) with Lysis (<partinfo>BBa_K124017</partinfo>). These were then transformed into competent E. coli DH5α cells and plated out on petri dishes with Ampicillin.
Ligated pBad (<partinfo>Bba_K206000</partinfo>) together with YFP+DTT (<partinfo>BBa_E0030</partinfo> and <partinfo>BBa_B0015</partinfo>), and VGB+RBS (<partinfo>BBa_K561001</partinfo> and <partinfo>BBa_B0034</partinfo>) and VHB+RBS (<partinfo>BBa_K258005</partinfo> and <partinfo>BBa_B0034</partinfo>) with Lysis (<partinfo>BBa_K124017</partinfo>). These were then transformed into competent E. coli DH5α cells and plated out on petri dishes with Ampicillin.
-
Terminator <partinfo>BBa_B0014</partinfo> and re-transformed pllD in pSB1C3 ("pllD 1-B") were transferred to liquid cultur.
+
Terminator <partinfo>BBa_B0014</partinfo> and re-transformed pllD in <partinfo>pSB1C3</partinfo> ("pllD 1-B") were transferred to liquid culture.
Ran gel on the PCR-products from yesterday.
Ran gel on the PCR-products from yesterday.
-
Inoculated 2 x 5 mL, 1 x 10 mL and 1 x 25 mL LB + Cm with pllD + lysis.
+
Inoculated 2 x 5 mL, 1 x 10 mL and 1 x 25 mL LB + Cm with plld + lysis.
===Thursday 02.08.12===
===Thursday 02.08.12===
Line 64: Line 241:
5 µl of the PCR product from yesterday was run on gel, but it did not give any results. We looked at the primers and thought we had done something wrong, but we did not. Will try to do the PCR one more time, but with a lower annealing temperature (55°C).
5 µl of the PCR product from yesterday was run on gel, but it did not give any results. We looked at the primers and thought we had done something wrong, but we did not. Will try to do the PCR one more time, but with a lower annealing temperature (55°C).
-
 
The constructs containing plld ligated with the lysis device(<partinfo>BBa_K112808</partinfo>), and plld in plasmid <partinfo>PSB1C3</partinfo> were miniprepped. The concentration were measured using nano drop.
The constructs containing plld ligated with the lysis device(<partinfo>BBa_K112808</partinfo>), and plld in plasmid <partinfo>PSB1C3</partinfo> were miniprepped. The concentration were measured using nano drop.
Line 78: Line 254:
|42,3
|42,3
|}
|}
-
 
Inoculated 1 colony from each of three plates containing LacI (<partinfo>BBa_C0012</partinfo>), RBS* <partinfo>BBa_B0030</partinfo> and pllD + lysis (all inoculated 1/8), into 5 mL LB + Amp (BBa_B0030, LacI)/ LB + Cm (pllD + lysis).  
Inoculated 1 colony from each of three plates containing LacI (<partinfo>BBa_C0012</partinfo>), RBS* <partinfo>BBa_B0030</partinfo> and pllD + lysis (all inoculated 1/8), into 5 mL LB + Amp (BBa_B0030, LacI)/ LB + Cm (pllD + lysis).  
Line 89: Line 264:
----
----
-
Gel purification was performed on the gel piece from yesterday, the lysis device cut with X+P. The concentration is 10,1 ng/µl. The lysis device and plld were ligated together, plld (with pSB1C3) as backbone.
+
Gel purification was performed on the gel piece from yesterday, the lysis device cut with X+P. The concentration is 10,1 ng/µl. The lysis device and plld were ligated together, plld (with <partinfo>pSB1C3</partinfo>) as backbone.
The ligated lysis and plld were transformed into competent ''E.coli'' DH5α cells. This was also done for the biobricks lacI repressor from E. coli (<partinfo>BBa_C0012</partinfo>) and RBS (<partinfo>BBa_B0030</partinfo>).
The ligated lysis and plld were transformed into competent ''E.coli'' DH5α cells. This was also done for the biobricks lacI repressor from E. coli (<partinfo>BBa_C0012</partinfo>) and RBS (<partinfo>BBa_B0030</partinfo>).
-
The petri dishes from yesterday showed colonies on the dishes with the pSB1C3+Colisin construct and the K+RBS+Colisin construct. One colony from each petri dish were transferred to liquid medium.
+
The petri dishes from yesterday showed colonies on the dishes with the pSB1C3+Colisin construct (<partinfo>pSB1C3</partinfo> and <partinfo>BBa_K150009</partinfo>) and the K+RBS+Colisin construct (<partinfo>BBa_K081005</partinfo> and <partinfo>BBa_K150009</partinfo>). One colony from each petri dish were transferred to liquid medium.
-
Sent a sample of the E. coli pllD promoter in pSB1C3 backbone to the iGEM team at RHIT.
+
Sent a sample of the E. coli pllD promoter in <partinfo>pSB1C3</partinfo> backbone to the iGEM team at RHIT.
PCR was performed on pBad (<partinfo>Bba_K206000</partinfo>) and DTT (<partinfo>BBa_B0015</partinfo>), the primers used, PCR-mix and programmed times are listed below.
PCR was performed on pBad (<partinfo>Bba_K206000</partinfo>) and DTT (<partinfo>BBa_B0015</partinfo>), the primers used, PCR-mix and programmed times are listed below.
Line 130: Line 305:
|72
|72
|}
|}
-
 
Mix 1:
Mix 1:
Line 143: Line 317:
* 5 µl buffer with MgCl<sub>2</sub>
* 5 µl buffer with MgCl<sub>2</sub>
* 0,75 µl Expand High Fidelity enzyme
* 0,75 µl Expand High Fidelity enzyme
-
 
This PCR mix was found here: http://francois.schweisguth.free.fr/protocols/High_fildelity_roche.pdf
This PCR mix was found here: http://francois.schweisguth.free.fr/protocols/High_fildelity_roche.pdf
 +
Thermal timetables:
-
The thermal timetable for pBad:
+
{|
-
 
+
| style="padding-right:7px;" |
-
{|border="0"
+
{|class="wikitable" style="margin: 1em auto 1em auto;"
 +
|+pBAD
 +
! Step
 +
! Action
 +
! Temperature
 +
! Duration
 +
|-
|1
|1
|Heated lid:  
|Heated lid:  
Line 203: Line 383:
|}
|}
 +
| style="padding-left:7px;" |
-
The thermal timetable for DTT:
+
{|class="wikitable" style="margin: 1em auto 1em auto;"
-
 
+
|+ DTT
-
{|border="0"
+
! Step
 +
! Action
 +
! Temperature
 +
! Duration
 +
|-
|1
|1
|Heated lid:  
|Heated lid:  
Line 257: Line 442:
|4°C
|4°C
|∞
|∞
 +
|}
|}
|}
Line 317: Line 503:
Further, the constitutive promoter containing RBS (<partinfo>BBa_K081005</partinfo>) was cut with S+P, and the purified colicin PCR product were cut with X+P, to be ligated tomorrow. Another colicin PCR product sample was cut with E+P, to be ligated into the already cut pSB1C3 tomorrow.
Further, the constitutive promoter containing RBS (<partinfo>BBa_K081005</partinfo>) was cut with S+P, and the purified colicin PCR product were cut with X+P, to be ligated tomorrow. Another colicin PCR product sample was cut with E+P, to be ligated into the already cut pSB1C3 tomorrow.
-
We also test cutted P<sub>lld</sub> with BpmI and AflIII. This was performed because the number of religations for the pSB1C3 plasmid was higher than it should have been. One of the samples to be test cutted was taken from the miniprepped plasmids of one of the colonies on the religation plate, to be used as negative controll, and three parallel samples from the ligation plate were test cutted.  
+
We also test cutted P<sub>lld</sub> with BpmI and AflIII. This was performed because the number of religations for the pSB1C3 plasmid was higher than it should have been. One of the samples to be test cutted was taken from the miniprepped plasmids of one of the colonies on the religation plate, to be used as negative controll, and three parallel samples from the ligation plate were test cutted. The gel pictures are shown below:
-
The gel pictures are shown below:
+
-
{|border="0"
+
-
|[[File:AflIII-testkutt_Plld_i_pSB1C3_30.07.12.png|400px|Religated pSB1C3, and three parallels of ligated P<sub>lld</sub> and pSB1C3 investigated by cutting with AflIII. AflIII cuts both inside the promoter region, and in the backbone, so if the promoter is present in the plasmid, the expected result for this test cut is two fragments; one on 277 bp, and one on 2136. All parallels from the ligation shows two bonds at the expected positions.]]
+
-
[[File:BpmI-testkutt_Plld_i_pSB1C3_30.07.12.png‎|400px|Religated pSB1C3, and three parallels of ligated P<sub>lld</sub> and pSB1C3 investigated by cutting with BpmI. BpmI also cuts both inside the promoter region and in the backbone, so if the promoter is present in the plasmid, the expected result for this test cut is two fragments; one on 898 bp, and one on 1515. Only one band is present on the gel.]]
+
{| style="text-align:center;"
 +
|[[File:AflIII-testkutt_Plld_i_pSB1C3_30.07.12.png|thumb|center|450px| Religated pSB1C3, and three parallels of ligated P<sub>lld</sub> and pSB1C3 investigated by cutting with AflIII. AflIII cuts both inside the promoter region, and in the backbone, so if the promoter is present in the plasmid, the expected result for this test cut is two fragments; one on 277 bp, and one on 2136. All parallels from the ligation shows two bonds at the expected positions.]]
 +
|[[File:BpmI-testkutt_Plld_i_pSB1C3_30.07.12.png|thumb|center‎|450px|Religated pSB1C3, and three parallels of ligated P<sub>lld</sub> and pSB1C3 investigated by cutting with BpmI. BpmI also cuts both inside the promoter region and in the backbone, so if the promoter is present in the plasmid, the expected result for this test cut is two fragments; one on 898 bp, and one on 1515. Only one band is present on the gel.]]
|}
|}
 +
The results from the test cutting were ambigous. The test cut performed with AflIII show the expected fragments, while the test cut performed with BpmI do not. It seems that the promoter is there in all thre parallels; if not it would have been impossible to yield two fragements in the AflIII test cut. But still, if the promoter sequence is as it should have been, also the test cut with BpmI should have yielded two fragments. It is, however, possible, that the BpmI restriction site inside the promoter is ok, but that the restriction site in the plasmid is damaged.
The results from the test cutting were ambigous. The test cut performed with AflIII show the expected fragments, while the test cut performed with BpmI do not. It seems that the promoter is there in all thre parallels; if not it would have been impossible to yield two fragements in the AflIII test cut. But still, if the promoter sequence is as it should have been, also the test cut with BpmI should have yielded two fragments. It is, however, possible, that the BpmI restriction site inside the promoter is ok, but that the restriction site in the plasmid is damaged.
Line 330: Line 516:
Made LB medium in preparation for experiment with the oxygen-sensitive promoters tomorrow.
Made LB medium in preparation for experiment with the oxygen-sensitive promoters tomorrow.
-
Inspeccted the pSB1C3 religation plate in the incubator. Many colonies that were not visible at the time of the colony count yesterday has now appeared. The religation and the pllD plate now contains about the same number of colonies, casting doubt on the sucess of the transformation. Moved the plate to the fridge.
+
Inspeccted the <partinfo>pSB1C3</partinfo> religation plate in the incubator. Many colonies that were not visible at the time of the colony count yesterday has now appeared. The religation and the pllD plate now contains about the same number of colonies, casting doubt on the sucess of the transformation. Moved the plate to the fridge.
-
Extracted plasmid DNA by miniprep from the pllD (unconfirmed) and pSB1C3 religation cultures inoculated yesterday, and the culture of <partinfo>BBa_E0040</partinfo> (A. vic. GFP) inoculated 26/7. DNA concentrations were measured with NanoDrop, as follows (two parallel measurements were made for each sample):
+
Extracted plasmid DNA by miniprep from the pllD (unconfirmed) and <partinfo>pSB1C3</partinfo> religation cultures inoculated yesterday, and the culture of <partinfo>BBa_E0040</partinfo> (A. vic. GFP) inoculated 26/7. DNA concentrations were measured with NanoDrop, as follows (two parallel measurements were made for each sample):
{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"
{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"
Line 347: Line 533:
|22,8/23,9
|22,8/23,9
|-
|-
-
|pSB1C3 1
+
|<partinfo>pSB1C3</partinfo> 1
|15,4/15,5
|15,4/15,5
|-
|-
-
|pSB1C3 2
+
|<partinfo>pSB1C3</partinfo> 2
|20,7/20,7
|20,7/20,7
|-
|-
-
|BBa_E0040
+
|<partinfo>BBa_E0040</partinfo>
|38,2/37,8
|38,2/37,8
|}
|}
Line 359: Line 545:
DNA from all the above samples except GFP was digested with Xba1 and Spe1. The digested samples were analyzed by gel electrophoresis to find out whether the desired insert was obtained in any of the samples. 75 V was applied for 45 min, and the gel was imaged. After imaging, electrophoresis was continued at 40 V for a further 30 minutes before imaging again. The result of the second imaging is shown below.
DNA from all the above samples except GFP was digested with Xba1 and Spe1. The digested samples were analyzed by gel electrophoresis to find out whether the desired insert was obtained in any of the samples. 75 V was applied for 45 min, and the gel was imaged. After imaging, electrophoresis was continued at 40 V for a further 30 minutes before imaging again. The result of the second imaging is shown below.
-
[[file:ChemiDoc 2012-07-29 22hr 41min pllD pSB1C3.jpg|center]]
+
[[file:ChemiDoc 2012-07-29 22hr 41min pllD pSB1C3.jpg|thumb|450px|center]]
Results of gel electrophoresis: All three samples from the pllD plate showed a short band identical between these samples, but it is difficult to determine the exact size - there seems to be an inconsistency in the migration of the 50 bp ladder on the left side of the gel and the 1 kb ladder on the right. Looking at the 50 bp ladder, the short band in the three first samples seems to be between the third and the fourth band from the bottom, representing 150 and 200 bp respectively. However, looking at the 1 kb ladder on the right, the short bands lie between the first and second ladder bands, equaling 250 and 500 bp respectively. The position of the short bands with respect to the 1 kb ladder appears to be consistent with the expected size of the pllD insert.
Results of gel electrophoresis: All three samples from the pllD plate showed a short band identical between these samples, but it is difficult to determine the exact size - there seems to be an inconsistency in the migration of the 50 bp ladder on the left side of the gel and the 1 kb ladder on the right. Looking at the 50 bp ladder, the short band in the three first samples seems to be between the third and the fourth band from the bottom, representing 150 and 200 bp respectively. However, looking at the 1 kb ladder on the right, the short bands lie between the first and second ladder bands, equaling 250 and 500 bp respectively. The position of the short bands with respect to the 1 kb ladder appears to be consistent with the expected size of the pllD insert.
-
Taken together, it seems possible that the short band represents the desired insert, but the samples should be analyzed again to get more data. The samples from the pSB1C3 religation plate does not show this band and is overall much fainter.  
+
Taken together, it seems possible that the short band represents the desired insert, but the samples should be analyzed again to get more data. The samples from the <partinfo>pSB1C3</partinfo> religation plate does not show this band and is overall much fainter.  
-
The top bands in the pllD wells are on heigth with the seventh band from the bottom, equaling 2500 bp, in the Generuler 1kb ladder on the right. This is approximately the size of the pSB1C3 plasmid and the pllD insert together. The two wells on the right containing DNA from the pSB1C3 religation plate show two faint bands each: One slightly above the top bands in the pllD wells, and one shorter band slightly above the 1000 bp reference band in the 1 kb ladder.
+
The top bands in the pllD wells are on heigth with the seventh band from the bottom, equaling 2500 bp, in the Generuler 1kb ladder on the right. This is approximately the size of the <partinfo>pSB1C3</partinfo> plasmid and the pllD insert together. The two wells on the right containing DNA from the <partinfo>pSB1C3</partinfo> religation plate show two faint bands each: One slightly above the top bands in the pllD wells, and one shorter band slightly above the 1000 bp reference band in the 1 kb ladder.
===Saturday 28.07.12===
===Saturday 28.07.12===
 +
----
Inspected the plates with PllD in <partinfo>pSB1C3</partinfo> and <partinfo>pSB1C3</partinfo> religation transformants inoculated yesterday. Growth on both plates, but more on the PllD plate. No growth on negative control (non-transformed cells). Approximate colony cound:
Inspected the plates with PllD in <partinfo>pSB1C3</partinfo> and <partinfo>pSB1C3</partinfo> religation transformants inoculated yesterday. Growth on both plates, but more on the PllD plate. No growth on negative control (non-transformed cells). Approximate colony cound:
Line 519: Line 706:
|}
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 +
The following PCR programs were used:
 +
{|style="text-align:center; margin: 1em auto 1em auto;" |
-
The following PCR programs were used:
+
| style="padding-right:10px"; |
-
lld promoter:
+
{|class="wikitable" style="margin: 1em auto 1em auto;"
-
{|border="0"
+
|+ lld promoter
 +
! Step
 +
! Action
 +
! Temperature
 +
! Duration
 +
|-
|1
|1
|Heated lid:  
|Heated lid:  
Line 581: Line 775:
|}
|}
 +
| style="padding-left:10px;" |
-
lldR:
+
{|class="wikitable" style="margin: 1em auto 1em auto;"
-
{|border="0"
+
|+ Colicin
 +
! Step
 +
! Action
 +
! Temperature
 +
! Duration
 +
|-
|1
|1
|Heated lid:  
|Heated lid:  
Line 607: Line 807:
|Elongation;
|Elongation;
|72°C
|72°C
-
|24 s
+
|31 s
|-
|-
|6
|6
-
|Go to step 3, repeat 25 x
+
|Go to step 3, repeat 10 x
|
|
|
|
|-
|-
-
|7
+
|7
 +
|Denaturation;
 +
|98°C
 +
|10 s
 +
|-
 +
|8
 +
|Elongation;
 +
|72°C
 +
|31 s
 +
|-
 +
|9
 +
|Go to step 7, repeat 15 x
 +
|
 +
|
 +
|-
 +
|10
|Final elongation;
|Final elongation;
|72°C
|72°C
|10 min
|10 min
|-
|-
-
|8
+
|11
|Hold
|Hold
|4°C
|4°C
Line 625: Line 840:
|}
|}
 +
|-
-
Colicin:
+
|colspan="2" |
-
{|border="0"
+
 
 +
{|class="wikitable" style="margin: 1em auto 1em auto;"
 +
|+ lldR
 +
! Step
 +
! Action
 +
! Temperature
 +
! Duration
 +
|-
|1
|1
|Heated lid:  
|Heated lid:  
Line 651: Line 874:
|Elongation;
|Elongation;
|72°C
|72°C
-
|31 s
+
|24 s
|-
|-
|6
|6
-
|Go to step 3, repeat 10 x
+
|Go to step 3, repeat 25 x
|
|
|
|
|-
|-
-
|7
+
|7
-
|Denaturation;
+
-
|98°C
+
-
|10 s
+
-
|-
+
-
|8
+
-
|Elongation;
+
-
|72°C
+
-
|31 s
+
-
|-
+
-
|9
+
-
|Go to step 7, repeat 15 x
+
-
|
+
-
|
+
-
|-
+
-
|10
+
|Final elongation;
|Final elongation;
|72°C
|72°C
|10 min
|10 min
|-
|-
-
|11
+
|8
|Hold
|Hold
|4°C
|4°C
|∞
|∞
 +
|}
 +
|}
|}
Line 689: Line 899:
----
----
-
Transformed the pBAD+RBS+YFP+DTT, VGB+RBS+YFP+DTT, and VHB+RBS+YFP+DTT constructs ligated yesterday to supercompetent ''E.coli'' Dh5α cells.
+
Transformed the pBAD+RBS+YFP+DTT (<partinfo>Bba_K206000</partinfo>, <partinfo>BBa_E0030</partinfo> and <partinfo>BBa_B0015</partinfo>), VGB+RBS+YFP+DTT (<partinfo>BBa_K561001</partinfo>, <partinfo>BBa_B0034</partinfo>, <partinfo>BBa_E0030</partinfo> and <partinfo>BBa_B0015</partinfo>), and VHB+RBS+YFP+DTT (<partinfo>BBa_K258005</partinfo>, <partinfo>BBa_B0034</partinfo>, <partinfo>BBa_E0030</partinfo> and <partinfo>BBa_B0015</partinfo>) constructs ligated yesterday to supercompetent ''E.coli'' Dh5α cells.
We also started making our iGEM matchmaker, which will be useful for iGEM teams wanting collaboration with other teams.  
We also started making our iGEM matchmaker, which will be useful for iGEM teams wanting collaboration with other teams.  
Line 700: Line 910:
----
----
-
We continnued the assembly of the promoter test constructs. As the test gel run on friday showed the expected fragment, the YFP+DTT samples were separated using gel electrophoresis. The inserts were cut out of the gel, as shown below, and purified using the QIAquick Gel Extraction Kit.  
+
We continnued the assembly of the promoter test constructs. As the test gel run on friday showed the expected fragment, the YFP+DTT (<partinfo>BBa_E0030</partinfo> and <partinfo>BBa_B0015</partinfo>) samples were separated using gel electrophoresis. The inserts were cut out of the gel, as shown below, and purified using the QIAquick Gel Extraction Kit.  
[[File:YFP_insert_23.07.12 exp.png|thumb|center|400px|The picture is showing two parallels of the YFP+DTT sample, containing an insert to be used in ligation with promoter+RBS constructs. The inserts, shown in red boxes, were cut out of the gel and purified.]]
[[File:YFP_insert_23.07.12 exp.png|thumb|center|400px|The picture is showing two parallels of the YFP+DTT sample, containing an insert to be used in ligation with promoter+RBS constructs. The inserts, shown in red boxes, were cut out of the gel and purified.]]
-
The pBad+RBS, Jen1+RBS, Vgb+RBS and Vhb+RBS backbones were purified using the QIAquick PCR Purification Kit. Concentrations of both inserts and backbones after purification are given below:
+
The pBad+RBS (<partinfo>Bba_K206000</partinfo>), Jen1+RBS (<partinfo>BBa_K284002</partinfo> and <partinfo>BBa_B0034</partinfo>), Vgb+RBS (<partinfo>BBa_K561001</partinfo> and <partinfo>BBa_B0034</partinfo>) and Vhb+RBS (<partinfo>BBa_K258005</partinfo> and <partinfo>BBa_B0034</partinfo>) backbones were purified using the QIAquick PCR Purification Kit. In all of the above it is the plasmid from RBS that's the backbone (<partinfo>psB1A2</partinfo>). Concentrations of both inserts and backbones after purification are given below:
{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"
{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"
Line 732: Line 942:
|}
|}
-
VGB+RBS, VHB+RBS and pBAD+RBS were all ligated with YFP+DTT.
+
VGB+RBS (<partinfo>BBa_K561001</partinfo> and <partinfo>BBa_B0034</partinfo>), VHB+RBS (<partinfo>BBa_K258005</partinfo> and <partinfo>BBa_B0034</partinfo>) and pBAD+RBS (<partinfo>Bba_K206000</partinfo>) were all ligated with YFP+DTT (<partinfo>BBa_E0030</partinfo> and <partinfo>BBa_B0015</partinfo>).
For the YFP + DTT fragment, DNA concentration measurements with NanoDrop after isolation with the quiagen kit were inconsistent. For the samples made from the two gel bands, values ranged from 3.7 to 14.8 and 4.0 to 24.4 ng/uL, respectively. Conclusion: Several measurements should always be made.
For the YFP + DTT fragment, DNA concentration measurements with NanoDrop after isolation with the quiagen kit were inconsistent. For the samples made from the two gel bands, values ranged from 3.7 to 14.8 and 4.0 to 24.4 ng/uL, respectively. Conclusion: Several measurements should always be made.
Line 1,522: 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!
 +
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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
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August
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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"