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Team:Braunschweig/Notebook
From 2013.igem.org
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The distribution kit arrived!<br> | The distribution kit arrived!<br> | ||
We resuspended the DNA of 19 BioBricks, measured the DNA concentrations via NanoDrop and did transformed each BioBrick. This is the full list of BioBricks we plan on using in our project:<br> | We resuspended the DNA of 19 BioBricks, measured the DNA concentrations via NanoDrop and did transformed each BioBrick. This is the full list of BioBricks we plan on using in our project:<br> | ||
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<th>BioBrick</th> | <th>BioBrick</th> | ||
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<th>BioBrick</th> | <th>BioBrick</th> | ||
Revision as of 23:45, 10 September 2013
Labjournal
This is the documentation of our lab work. For detailed protocols of certain procedures please refer to Protocols. Attributions are given for each day, however please check our Attributions section for efforts beyond the lab work.
Week 1: May 19 - May 26, 2013
We set up our labspace and started some preparatory work.
Tuesday, May 21, 2013
We finally moved in our lab. A bit of dust here and some rubbish to dispose there, but after a few hours of combined strength our lab was ready to go. Wet experiments can begin!
Thursday, May 23, 2013
Investigators: Kevin, Kerstin, Laura
We prepared some chemically competent XL1 blue E. Coli cells for all the transformations we are going to have to do during the project.
Friday, May 24, 2013
Investigators: Kevin, Kerstin, Laura
Today we transformed our freshly prepared competent cells for test purposes. We used the plasmids pUC 18 and pUC19 to calculate the transformation efficiency. 100 pg of DNA were used for each transformation with 50 µl cells.
Additionally, competent cells were plated on ampicillin, kanamycin and chloramphenicol to ensure that the strain is not carrying any corresponding resistances. The plates were incubated at 37 °C over night.
Saturday, May 25, 2013
Investigators: Kevin, Kerstin, Laura
111 colonies were counted for pUC18 transformation, 50 for pUC19 transformation.
Transformation efficiency:
pUC18: ~ 106 µg-1
pUC19: ~5x105 µg-1
The plates with the additional antibiotics were empty, therefore the strain was not carrying any resistences.
Week 2: May 27 - June 1, 2013
The distribution kit arrived and we started with the actual labwork. 19 biobricks had to be transformed into our E. coli to secure the parts. Additionally we developed the cloning strategy for the next weeks.
Monday, May 27, 2013
Investigators: All
Today we developed our cloning strategy.
Tuesday, May 28, 2013
Investigators: Tabea, Jan, Laura
The distribution kit arrived!
We resuspended the DNA of 19 BioBricks, measured the DNA concentrations via NanoDrop and did transformed each BioBrick. This is the full list of BioBricks we plan on using in our project:
| BioBrick | B0010 5.13 D pSB1C3 Terminator |
B0012
3.1A pSB1C3 Terminator |
B0032 3.1I pSB1C3 RBS |
B1009
3.6J pSB1C3 Terminator |
C0060 3.3G pSB1C3 aiiA + LVA |
C0061 3.3I pSB1C3 luxI + LVA |
C0062 3.3K pSB1C3 luxR |
C0070 3.8N pSB1C3 rhII + LVA |
C0071 3.8P pSB1C3 rhIR + LVA |
|
|---|---|---|---|---|---|---|---|---|---|---|
| DNA concentration [ng/µL] |
91.4 | 56.7 | 53.4 | 48.4 | 50.2 | 61.8 | 62.2 | 52.7 | 50.0 | |
| BioBrick | C0078 3.10N pSB1C3 lasI + LVA |
C0079 3.10P pSB1C3 lasR + LVA |
E0240 3.23A pSB1C3 GFP |
E0430 3.20K pSB1C3 YFP |
J06702 3.16B pSB1C3 RFP |
J23106 5.18O BBa_J61002 Pcon medium |
J23100 5.18C BBa_J61002 Pcon strong |
J23112 5.20E BBa_J61002 Pcon weak |
R0062 3.5G pSB1C3 pLux |
R0071 3.6E pSB1C3 pRhl |
| DNA concentration [ng/µL] |
49.3 | 53.6 | 50.7 | 52.7 | 52.7 | 70.4 | 73.1 | 78.1 | 53.0 | 51.6 |
Wednesday, May 29, 2013
Investigators: Oliver, Jan
No growth could be observed on plates with the BioBricks
B0012
B0010
C0060
C0062
J23106
J23100
We repeated the transformation of all the above listed BioBricks. However, we noticed that many of these parts have an amp backbone.
Thursday, May 30, 2013
Investigator: Jan
Unfortunately, some plates still remained empty:
B0010
J23106
J23100
Thursday, May 31, 2013
Investigators: Kerstin, Roman
We did a colony PCR with the parts we secured so far. We got bands for all parts except for BioBricks C0061 and C0062.
Week 3: June 2 - June 8, 2013
This week we successfully cloned and transformed some of our first combination Bricks. We also managed to obtain some BioBricks that could not be transformed via Phusion-PCR directly from resuspended DNA.
Sunday, June 2, 2013
Investigators: Roman
Since our transformations for the Bricks B0010, C0060, C0061, C0062, C0070, J23100 and J23106 were not successful, they amplified via Phusion-PCR directly from the resuspended DNA from the Distribution Kit.
We prepared 5 mL liquid cultures of Bricks B0012, B0032, B1009, C0060, C0061, C0062, C0070, C0071, C0078, C0079, E0240, E0430, J06702, R0062, R0071 in order to miniprep them the next day.
Monday, June 3, 2013
Investigators: Oliver, Kerstin, Laura
We miniprepped the Bricks B0012, B0032, B1009, C0060, C0061, C0062, C0070, C0071, C0078, C0079, E0240, E0430, J06702, R0062 and R0071. As stated earlier the Bricks C0061 and C0062 showed no visible bands in the colony PCR but still were prepped. Furthermore we prepared glycerol stocks of all strains for further use.
Our gel electrophoresis of the PCR-amplified Bricks showed a suitable bands for BioBricks C0060, C0061, C0070, J23100, J23106 which were recovered successfully from the gel. The Bricks B0010, C0062 showed no bands on the gel and therefore could not be isolated.
Tuesday, June 4, 2013
Investigators: Jan, Kerstin
We received the new set of BioBricks we ordered from the registry: B0015 (this one is going to be our new terminator, replacing the combination of B0010 and B0012), B0017, E0420, J23100, J23106. These were tranformed via heatshock in E. coli XL1Blue MRF cells. We also prepared following of the miniprepped Bricks for sequencing: C0061, B0012, B1009. The results are outlined in the following table:
