Lab books
Hover over a week for a protein to see a summary. Click to view the actual lab book (a PDF in a new page). See our Abbreviations to view and understand them throughout the lab books!
Difference between revisions of "Team:Stockholm/Labbook"
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| − | <p>Hover over a week for a protein to see a summary. Click to view the actual lab book (a PDF in a new page). </p> | + | <p>Hover over a week for a protein to see a summary. Click to view the actual lab book (a PDF in a new page). See our <a href="https://static.igem.org/mediawiki/2016/a/a8/T--Stockholm--abbreviations_list.pdf" target="_blank">Abbreviations</a> to view and understand them throughout the lab books! </p> |
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<td summary="The goal of this week was to confirm the BioBrick ordered from iGEM HQ by preparing for sequencing."><a href="https://static.igem.org/mediawiki/2016/8/8e/T--Stockholm--nuc_week_1.pdf" target="_blank">Week 1</a></td> | <td summary="The goal of this week was to confirm the BioBrick ordered from iGEM HQ by preparing for sequencing."><a href="https://static.igem.org/mediawiki/2016/8/8e/T--Stockholm--nuc_week_1.pdf" target="_blank">Week 1</a></td> | ||
| − | <td summary=" | + | <td summary="To be able to actively work with lysostaphin, we first amplified the biobrick present in the distribution kit by transformation of *E.coli* TOP10."><a href="https://static.igem.org/mediawiki/2016/2/2f/T--Stockholm--lys_week_1.pdf" target="_blank">Week 1</a></td> |
<td summary="We used PCR to confirm the size of the insert and sent samples for sequencing."><a href="https://static.igem.org/mediawiki/2016/2/2e/T--Stockholm--esp_week_1.pdf" target="_blank">Week 1</a></td> | <td summary="We used PCR to confirm the size of the insert and sent samples for sequencing."><a href="https://static.igem.org/mediawiki/2016/2/2e/T--Stockholm--esp_week_1.pdf" target="_blank">Week 1</a></td> | ||
| − | <td summary=" | + | <td summary="The defensin BioBrick from iGEM HQ was prepared and sent for sequencing."><a href="https://static.igem.org/mediawiki/2016/4/49/T--Stockholm--def_week_1.pdf" target="_blank">Week 1</a></td> |
<td summary="Testing conjugation between two proteins using commercial available Sortase."><a href="https://static.igem.org/mediawiki/2016/e/ea/T--Stockholm--Sortase_week_1.pdf" target="_blank">Week 1</a></td> | <td summary="Testing conjugation between two proteins using commercial available Sortase."><a href="https://static.igem.org/mediawiki/2016/e/ea/T--Stockholm--Sortase_week_1.pdf" target="_blank">Week 1</a></td> | ||
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<td summary="Preparing digestion of T7 and Nuc for 3A assembly. The Nuc gel ran for too long and was lost, so we had to re-do the PCR of Nuc."><a href="https://static.igem.org/mediawiki/2016/4/49/T--Stockholm--nuc_week_3.pdf" target="_blank">Week 3</a></td> | <td summary="Preparing digestion of T7 and Nuc for 3A assembly. The Nuc gel ran for too long and was lost, so we had to re-do the PCR of Nuc."><a href="https://static.igem.org/mediawiki/2016/4/49/T--Stockholm--nuc_week_3.pdf" target="_blank">Week 3</a></td> | ||
| − | <td summary=" | + | <td summary=" |
| + | To obtain a construct that could be transcribed into a fully functional protein, a promoter must be placed prior to the lysostaphin coding sequence. To accomplish this, we have chosen to place a T7 promoter by means of 3A assembly with pSB1K3 as the new backbone. | ||
| + | "><a href="https://static.igem.org/mediawiki/2016/a/a3/T--Stockholm--lys_week_3.pdf" target="_blank">Week 3</a></td> | ||
<td summary="We performed 3A assembly to incorporate T7 promoter with Esp (both EB and EC) and put into kanamycin backbone. The new construct was transformed and prepared for sequencing. | <td summary="We performed 3A assembly to incorporate T7 promoter with Esp (both EB and EC) and put into kanamycin backbone. The new construct was transformed and prepared for sequencing. | ||
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<td></td> | <td></td> | ||
| − | <td summary="We aimed make glycerol stocks of S.aureus newman strain and to extract the genome of the bacteria."><a href="https://static.igem.org/mediawiki/2016/5/5f/T--Stockholm--Sortase_week_3.pdf" target="_blank">Week 3</a></td> | + | <td summary="We aimed make glycerol stocks of S.aureus newman strain and to extract the genome of the bacteria."><a href="https://static.igem.org/mediawiki/2016/5/5f/T--Stockholm--Sortase_week_3.pdf" target="_blank">Week 3</a></td></tr> |
| − | + | ||
| − | </tr> | + | |
<tr> | <tr> | ||
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"><a href="https://static.igem.org/mediawiki/2016/6/6e/T--Stockholm--nuc-week-4.pdf" target="_blank">Week 4</a></td> | "><a href="https://static.igem.org/mediawiki/2016/6/6e/T--Stockholm--nuc-week-4.pdf" target="_blank">Week 4</a></td> | ||
| − | <td summary=" | + | <td summary="Performed digestions and re-assembled the pSB1K3-T7-Lys construct."><a href="https://static.igem.org/mediawiki/2016/a/a0/T--Stockholm--lys_week_4.pdf" target="_blank">Week 4</a></td> |
<td summary="PCR showed that both T7-EB and T7-EC had correct size but after sequencing, only EB was found to be correctly assembled. We abandoned CB and tried to induce the EB protein expression by IPTG induction. | <td summary="PCR showed that both T7-EB and T7-EC had correct size but after sequencing, only EB was found to be correctly assembled. We abandoned CB and tried to induce the EB protein expression by IPTG induction. | ||
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"><a href="https://static.igem.org/mediawiki/2016/c/c7/T--Stockholm--nuc_week_5.pdf" target="_blank">Week 5</a></td> | "><a href="https://static.igem.org/mediawiki/2016/c/c7/T--Stockholm--nuc_week_5.pdf" target="_blank">Week 5</a></td> | ||
| − | <td summary=" | + | <td summary="Performed further analysis of the pSB1K3-T7-Lys construct previously assembled. Began with overhang PCR to add BamHI site for addition of linker tags."><a href="https://static.igem.org/mediawiki/2016/8/83/T--Stockholm--lys_week_5.pdf" target="_blank">Week 5</a></td> |
<td summary="We re-did the transformation, induced the protein expression at different conditions and performed the first SDS-page. We found a slightly increased band after induction, which had similar size and might be the EB. The first overhang PCR was tried as the initial step for connecting with linker tag. But it failed."><a href="https://static.igem.org/mediawiki/2016/b/b2/T--Stockholm--esp_week_5.pdf" target="_blank">Week 5</a></td> | <td summary="We re-did the transformation, induced the protein expression at different conditions and performed the first SDS-page. We found a slightly increased band after induction, which had similar size and might be the EB. The first overhang PCR was tried as the initial step for connecting with linker tag. But it failed."><a href="https://static.igem.org/mediawiki/2016/b/b2/T--Stockholm--esp_week_5.pdf" target="_blank">Week 5</a></td> | ||
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3A assemblies performed were successful. However, the first attempted transformation of pSB1K3-T7-Nuc in TOP10 cells was not successful. To increase the chances of transforming we aimed to increase the concentration of Nuc in our final samples, After several attempts we transformed successfully. However, when running a gel on the transformed DNA, it seemed to EB."><a href="https://static.igem.org/mediawiki/2016/8/8f/T--Stockholm--nuc_week_6.pdf" target="_blank">Week 6</a></td> | 3A assemblies performed were successful. However, the first attempted transformation of pSB1K3-T7-Nuc in TOP10 cells was not successful. To increase the chances of transforming we aimed to increase the concentration of Nuc in our final samples, After several attempts we transformed successfully. However, when running a gel on the transformed DNA, it seemed to EB."><a href="https://static.igem.org/mediawiki/2016/8/8f/T--Stockholm--nuc_week_6.pdf" target="_blank">Week 6</a></td> | ||
| − | <td summary=" | + | <td summary="After having transformed pSB1K3-T7-Lys in the previous week, protein expression was started this week. Gel purification of overhang PCR product with BamHI was performed so that the linker-tag sequence could be added in. To ensure the correct gene expression, the products were sent for sequencing."><a href="https://static.igem.org/mediawiki/2016/1/14/T--Stockholm--lys_week_6.pdf" target="_blank">Week 6</a></td> |
<td summary="Troubleshooting of the overhang PCR, different conditions were tested. Unfortunately, none of them succeeded, the bands were either faint or unspecific. | <td summary="Troubleshooting of the overhang PCR, different conditions were tested. Unfortunately, none of them succeeded, the bands were either faint or unspecific. | ||
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<td summary="We realized that the primers were mixed up between EB and EC. Then we did overhang PCR with the correct primers and got strong bands at correct size. The problem with overhang PCR of EB was solved. We continued with digestion and ligation with linker tag but It didn’t succeed."><a href="https://static.igem.org/mediawiki/2016/d/d9/T--Stockholm--esp_week_7.pdf" target="_blank">Week 7</a></td> | <td summary="We realized that the primers were mixed up between EB and EC. Then we did overhang PCR with the correct primers and got strong bands at correct size. The problem with overhang PCR of EB was solved. We continued with digestion and ligation with linker tag but It didn’t succeed."><a href="https://static.igem.org/mediawiki/2016/d/d9/T--Stockholm--esp_week_7.pdf" target="_blank">Week 7</a></td> | ||
| − | <td summary=" | + | <td summary="The pSB1C3-Def was extracted from liquid cultures and PCR amplified."><a href="https://static.igem.org/mediawiki/2016/1/1d/T--Stockholm--defensin_week_7.pdf" target="_blank">Week 7</a></td> |
<td summary="Focused on changing the Backbone to a pSB1C3 one, but also fixing details in the sequence such as adding the full prefix and suffix. We also transformed the new ligation product. The ligation was deemed unsuccessful after analysis."><a href="https://static.igem.org/mediawiki/2016/7/74/T--Stockholm--sort_week_7.pdf" target="_blank">Week 7</a></td> | <td summary="Focused on changing the Backbone to a pSB1C3 one, but also fixing details in the sequence such as adding the full prefix and suffix. We also transformed the new ligation product. The ligation was deemed unsuccessful after analysis."><a href="https://static.igem.org/mediawiki/2016/7/74/T--Stockholm--sort_week_7.pdf" target="_blank">Week 7</a></td> | ||
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<td summary="We digested pSB1C3-Nuc from week 5, and even though the concentrations were low we could use it for 3A-assembly of pSB1C3-T7-Nuc. We later found out that digestion had been done with incorrect digestion enzymes, resulting in invalid results this week."><a href="https://static.igem.org/mediawiki/2016/c/ca/T--Stockholm--nuc_week_8.pdf" target="_blank">Week 8</a></td> | <td summary="We digested pSB1C3-Nuc from week 5, and even though the concentrations were low we could use it for 3A-assembly of pSB1C3-T7-Nuc. We later found out that digestion had been done with incorrect digestion enzymes, resulting in invalid results this week."><a href="https://static.igem.org/mediawiki/2016/c/ca/T--Stockholm--nuc_week_8.pdf" target="_blank">Week 8</a></td> | ||
| − | <td summary=" | + | <td summary="Retried 3A assembly of pSB1C3-T7-Lys with success. The construct was subjected to overhang PCR to add BamHI and was also expressed in BL21(DE3)."><a href="https://static.igem.org/mediawiki/2016/d/d6/T--Stockholm--lys_week_8.pdf" target="_blank">Week 8</a></td> |
<td summary="Kirby-Bauer test showed EB might have bactericidal activity. Troubleshooting for digestion and ligation of T7-EB with linkertag, none of them worked. We tried to improve the EB biobrick by making truncated version, it was unsuccessful unfortunately."><a href="https://static.igem.org/mediawiki/2016/d/d7/T--Stockholm--esp_week_8.pdf" target="_blank">Week 8</a></td> | <td summary="Kirby-Bauer test showed EB might have bactericidal activity. Troubleshooting for digestion and ligation of T7-EB with linkertag, none of them worked. We tried to improve the EB biobrick by making truncated version, it was unsuccessful unfortunately."><a href="https://static.igem.org/mediawiki/2016/d/d7/T--Stockholm--esp_week_8.pdf" target="_blank">Week 8</a></td> | ||
| − | <td summary=" | + | <td summary="Attempts of 3A assembling pSB1C3-T7-Def constructs were successful."><a href="https://static.igem.org/mediawiki/2016/a/aa/T--Stockholm--defensin_week_8.pdf" target="_blank">Week 8</a></td> |
<td summary="We continued to focus on changing the backbone in which the overlap extension PCR product is in and then further transform. Colonies were picked and analysed. Negative PCR results were apprehended from the colony PCR and sequencing results were not as expected."><a href="https://static.igem.org/mediawiki/2016/1/1b/T--Stockholm--sort_week_8.pdf" target="_blank">Week 8</a></td> | <td summary="We continued to focus on changing the backbone in which the overlap extension PCR product is in and then further transform. Colonies were picked and analysed. Negative PCR results were apprehended from the colony PCR and sequencing results were not as expected."><a href="https://static.igem.org/mediawiki/2016/1/1b/T--Stockholm--sort_week_8.pdf" target="_blank">Week 8</a></td> | ||
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<td summary="Gel of pSB1C3-T7-Nuc seems correct. Will be sent for sequencing to identify the construct. A BamHI restriction site was successfully added to the construct using overhang PCR amplification. Results from assembly of a pSB1C3-T7-Nuc-BamHI-LT construct were inconclusive."><a href="https://static.igem.org/mediawiki/2016/d/d6/T--Stockholm--nuc_week_9.pdf" target="_blank">Week 9</a></td> | <td summary="Gel of pSB1C3-T7-Nuc seems correct. Will be sent for sequencing to identify the construct. A BamHI restriction site was successfully added to the construct using overhang PCR amplification. Results from assembly of a pSB1C3-T7-Nuc-BamHI-LT construct were inconclusive."><a href="https://static.igem.org/mediawiki/2016/d/d6/T--Stockholm--nuc_week_9.pdf" target="_blank">Week 9</a></td> | ||
| − | <td summary=" | + | <td summary="This week we mainly focused on characterizing the expressed construct. This was done by an SDS Page and a Kirby Bauer test on *E.coli* TOB1. More attempts of adding the linker tag was also performed by means of constructing pSB1C3-T7-Lys-LT."><a href="https://static.igem.org/mediawiki/2016/8/82/T--Stockholm--lysostaphin_week_9.pdf" target="_blank">Week 9</a></td> |
<td summary="Kirby-Bauer test on EB. Troubleshooting for digestion and ligation, one colony developed but with incorrect sequence. "><a href="https://static.igem.org/mediawiki/2016/7/74/T--Stockholm--esp_week_9.pdf" target="_blank">Week 9</a></td> | <td summary="Kirby-Bauer test on EB. Troubleshooting for digestion and ligation, one colony developed but with incorrect sequence. "><a href="https://static.igem.org/mediawiki/2016/7/74/T--Stockholm--esp_week_9.pdf" target="_blank">Week 9</a></td> | ||
| − | <td summary=" | + | <td summary="Assembled pSB1C3-T7-Def construct was transformed into TOP10 cells and successfully confirmed through several analyzes."><a href="https://static.igem.org/mediawiki/2016/3/30/T--Stockholm--defensin_week_9.pdf" target="_blank">Week 9</a></td> |
<td summary="Several attempts to ligate the joined gene fragments into the pSB1C3 Backbone. Transformation was tried out in electro-competent cells. Colonies were grew and were analyzed through colony PCR and sequencing which gave negative results"><a href="https://static.igem.org/mediawiki/2016/f/fc/T--Stockholm--sortase_week_9.pdf" target="_blank">Week 9</a></td> | <td summary="Several attempts to ligate the joined gene fragments into the pSB1C3 Backbone. Transformation was tried out in electro-competent cells. Colonies were grew and were analyzed through colony PCR and sequencing which gave negative results"><a href="https://static.igem.org/mediawiki/2016/f/fc/T--Stockholm--sortase_week_9.pdf" target="_blank">Week 9</a></td> | ||
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<td></td> | <td></td> | ||
| − | <td summary=" | + | <td summary="BL21(DE3) cells containing pSB1C3-T7-Lys was further characterized by colony picking and expression. "><a href="https://static.igem.org/mediawiki/2016/7/79/T--Stockholm--lysostaphin_week_10.pdf" target="_blank">Week 10</a></td> |
<td></td> | <td></td> | ||
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<td></td> | <td></td> | ||
| − | <td summary=" | + | <td summary="We had confirmed that pSB1C3-T7-Lys-LT that had been successfully assembled had not contained the LT, therefore we re-checked the sequence of this construct with PCR. "><a href="https://static.igem.org/mediawiki/2016/8/81/T--Stockholm--lys_week_12.pdf" target="_blank">Week 12</a></td> |
<td></td> | <td></td> | ||
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<td summary="New method attempted for the assembly of a pSB1C3-T7-Nuc-BamHI-LT construct."><a href="https://static.igem.org/mediawiki/2016/e/ec/T--Stockholm--nuc_week_14.pdf" target="_blank">Week 14</a></td> | <td summary="New method attempted for the assembly of a pSB1C3-T7-Nuc-BamHI-LT construct."><a href="https://static.igem.org/mediawiki/2016/e/ec/T--Stockholm--nuc_week_14.pdf" target="_blank">Week 14</a></td> | ||
| − | <td summary=" | + | <td summary="Attempts of of adding the BamHI site by performing the overhang PCR strategy 2 was performed with different conditions. "><a href="https://static.igem.org/mediawiki/2016/9/9d/T--Stockholm--lys_week_14.pdf" target="_blank">Week 14</a></td> |
<td summary="T7-EB was changed to the chloramphenicol backbone. "><a href="https://static.igem.org/mediawiki/2016/c/c7/T--Stockholm--esp_week_14.pdf" target="_blank">Week 14</a></td> | <td summary="T7-EB was changed to the chloramphenicol backbone. "><a href="https://static.igem.org/mediawiki/2016/c/c7/T--Stockholm--esp_week_14.pdf" target="_blank">Week 14</a></td> | ||
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<td summary="Additional eight attempts with the new method of assembling a pSB1C3-T7-Nuc-BamHI-LT construct. A Kirby-Bauer test was performed of earlier expressed nuclease."><a href="https://static.igem.org/mediawiki/2016/1/17/T--Stockholm--nuc_week_15.pdf" target="_blank">Week 15</a></td> | <td summary="Additional eight attempts with the new method of assembling a pSB1C3-T7-Nuc-BamHI-LT construct. A Kirby-Bauer test was performed of earlier expressed nuclease."><a href="https://static.igem.org/mediawiki/2016/1/17/T--Stockholm--nuc_week_15.pdf" target="_blank">Week 15</a></td> | ||
| − | <td summary=" | + | <td summary="Attempts of of adding the BamHI site was optimised by varying several parameters, however with the attempts we had tried it had been successful. "><a href="https://static.igem.org/mediawiki/2016/e/ec/T--Stockholm--lys_week_15.pdf" target="_blank">Week 15</a></td> |
<td summary="We performed overhang PCR on EB with new strategy with different annealing temperature. But none of them succeeded. First biofilm assay on EB, It showed positive effect in dispersing the biofilm."><a href="https://static.igem.org/mediawiki/2016/7/76/T--Stockholm--Esp_week_15.pdf" target="_blank">Week 15</a></td> | <td summary="We performed overhang PCR on EB with new strategy with different annealing temperature. But none of them succeeded. First biofilm assay on EB, It showed positive effect in dispersing the biofilm."><a href="https://static.igem.org/mediawiki/2016/7/76/T--Stockholm--Esp_week_15.pdf" target="_blank">Week 15</a></td> | ||
| − | <td summary=" | + | <td summary="The first step, adding a BamHI restriction site through overhang PCR amplification, in the assembly of the pSB1C3-T7-Def-BamHI-LT construct was successfully performed."><a href="https://static.igem.org/mediawiki/2016/1/14/T--Stockholm--def_week_15.pdf" target="_blank">Week 15</a></td> |
<td summary="Overlap extension PCR of to join the two fragments of interest. They were later digested on the ends and ligated into a pSB1C Backbone, followed by a successful transformation."><a href="https://static.igem.org/mediawiki/2016/b/b0/T--Stockholm--sortase_week_15.pdf" target="_blank">Week 15</a></td> | <td summary="Overlap extension PCR of to join the two fragments of interest. They were later digested on the ends and ligated into a pSB1C Backbone, followed by a successful transformation."><a href="https://static.igem.org/mediawiki/2016/b/b0/T--Stockholm--sortase_week_15.pdf" target="_blank">Week 15</a></td> | ||
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<tr> | <tr> | ||
| − | <td summary="Biofilm dispersal and inhibition by expressed nuclease was investigated with biofilm assays."><a href="https://static.igem.org/mediawiki/2016/e/e5/T--Stockholm--nuc-week-16.pdf" target="_blank">Week 16</a></td> | + | <td summary="Biofilm dispersal and inhibition by expressed nuclease was investigated with biofilm assays."><a href="https://static.igem.org/mediawiki/2016/e/e5/T--Stockholm--nuc-week-16.pdf" target="_blank">Week 16</a></td> |
| − | <td></td> | + | |
| + | <td summary="The biofilm assay was performed on cell lysates containing lys and this was done to observe the inhibitory and dispersal capability of Lys. "><a href="https://static.igem.org/mediawiki/2016/2/2a/T--Stockholm--lys_labbook_week16.pdf" target="_blank">Week 16</a></td> | ||
| + | |||
<td summary="We modified the method and redid the biofilm assay. The bactericidal effect of EB was not clear but EB was potent in dispersing biofilm. | <td summary="We modified the method and redid the biofilm assay. The bactericidal effect of EB was not clear but EB was potent in dispersing biofilm. | ||
We tried overhang PCR with new strategy on EB but failed."><a href="https://static.igem.org/mediawiki/2016/c/cf/T--Stockholm--esp_labbook_week16.pdf" target="_blank">Week 16</a></td> | We tried overhang PCR with new strategy on EB but failed."><a href="https://static.igem.org/mediawiki/2016/c/cf/T--Stockholm--esp_labbook_week16.pdf" target="_blank">Week 16</a></td> | ||
| − | <td summary=" | + | <td summary="The activity of earlier expressed defensin in biofilm dispersal and inhibition was analysed with biofilm assays. Also, the pSB1C3-T7-Def-BamHI-LT construct was successfully assembled. Defensin with added Sortase and His tag was successfully expressed in BL21(DE3) cells, extracted and purified. The tagged defensin was further successfully conjugated to spider silk protein."><a href="https://static.igem.org/mediawiki/2016/c/c6/T--Stockholm--def-week-16_%281%29.pdf" target="_blank">Week 16</a></td> |
<td summary="Testing conjugation between two proteins using commercial available Sortase. | <td summary="Testing conjugation between two proteins using commercial available Sortase. | ||
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</section> | </section> | ||
</article> | </article> | ||
</main></div></html>{{Template:Stockholm/Footer}} | </main></div></html>{{Template:Stockholm/Footer}} | ||
Revision as of 18:11, 19 October 2016
