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{{:Team:NTNU_Trondheim/Templates/Calendar}}

{{:Team:NTNU_Trondheim/Templates/Calendar}}

===Monday 06.08.12===

===Monday 06.08.12===

In order to start making the construct for the biobrick we are going to improve, the parts needed for the construct were cut using the restriction digest protocol [https://2012.igem.org/Team:NTNU_Trondheim/Protocols].

In order to start making the construct for the biobrick we are going to improve, the parts needed for the construct were cut using the restriction digest protocol [https://2012.igem.org/Team:NTNU_Trondheim/Protocols].

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.

===Sunday 05.08.12===

===Sunday 05.08.12===

Inspected agar plates in incubator inoculated on 3/8:

Inspected agar plates in incubator inoculated on 3/8:

===Friday 03.08.12===

===Friday 03.08.12===

Isolated plasmid DNA from the cultures of RBS* (<partinfo>BBa_B0030</partinfo>) and LacI (<partinfo>BBa_C0012</partinfo>) inoculated yesterday.

Isolated plasmid DNA from the cultures of RBS* (<partinfo>BBa_B0030</partinfo>) and LacI (<partinfo>BBa_C0012</partinfo>) inoculated yesterday.

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.

Ran gel on the PCR-products from yesterday.

Ran gel on the PCR-products from yesterday.

===Thursday 02.08.12===

===Thursday 02.08.12===

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.

|42,3

|42,3

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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).  

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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>).

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.

|72

|72

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Mix 1:

Mix 1:

* 5 µl buffer with MgCl₂

* 5 µl buffer with MgCl₂

* 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

|1

|1

|Heated lid:  

|Heated lid:  

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|1

|1

|Heated lid:  

|Heated lid:  

|4°C

|4°C

|∞

|∞

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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.

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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.

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.

{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"

{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"

|22,8/23,9

|22,8/23,9

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|15,4/15,5

|15,4/15,5

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|20,7/20,7

|20,7/20,7

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|38,2/37,8

|38,2/37,8

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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.

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.

===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:

===Friday 27.07.12===

===Friday 27.07.12===

The PCR batches run yesterday were investigated on gel electrophoresis, and the gel picture shows that colicin has been made!

The PCR batches run yesterday were investigated on gel electrophoresis, and the gel picture shows that colicin has been made!

New primers were ordered for LldR, this time containing the biobrick prefix and suffix.

New primers were ordered for LldR, this time containing the biobrick prefix and suffix.

Discussed experimental design for testing of oxygen-sensitive promoters with Martin. Made a plan for testing both promoters on Monday.

Discussed experimental design for testing of oxygen-sensitive promoters with Martin. Made a plan for testing both promoters on Monday.

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|Heated lid:  

|Heated lid:  

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|1

|1

|Heated lid:  

|Heated lid:  

|Elongation;

|Elongation;

|72°C

|72°C

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|6

|6

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|Final elongation;

|Final elongation;

|72°C

|72°C

|10 min

|10 min

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|Hold

|Hold

|4°C

|4°C

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|1

|1

|Heated lid:  

|Heated lid:  

|Elongation;

|Elongation;

|72°C

|72°C

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|6

|6

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|Final elongation;

|Final elongation;

|72°C

|72°C

|10 min

|10 min

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|Hold

|Hold

|4°C

|4°C

|∞

|∞

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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.  

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[[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.]]

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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.

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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