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:

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:

Wednesday, June 5, 2013

Investigators: Kerstin, Kevin, Oliver, Laura
Since we were not able to transform the BioBricks B0015, J23100 and J23106 we decided to obtain these Bricks via Phusion-PCR directly from resuspended DNA from the Distribution Kit.
A gel electrophoresis was conducted with the freshly acquired PCR products of B0015, J23100 and J23106 as well as C0060, C0061, C0070, J23100 and J23106 from our last batch of PCR amplificates. Besides J23100 from today's PCR all Bricks showed bands of the expected size that were isolated via gel extraction.

Thursday, June 6, 2013

Investigators: Oliver, Laura
We prepared to clone some of our first and essential parts. The Bricks were digested according to the table below. Parts labeled as vector were dephosphorylated to prevent religation and purified.
In order to obtain the inserts a gel electrophoresis was conducted and the appropriate band was isolated by gel extraction.The ligations were conducted according to the following table.

Friday, June 7, 2013

Investigators: Tabea, Oliver, Laura, Kevin
We transformed our ligations from yesterday using our competent glycerol stocks without prior heat inactivation of T4-ligase. Transformed cells were plated on agar plates containing glucose and Chloramphenicol and were grown at 37 °C over night.
To test the success of our ligation beforehand we conducted a colony-PCR (see protocoll colony-PCR, Extension time 30 s) using 1 µL of our untransformed ligation mix of C0061+B0015 and B0032+J23100 as template. The conducted gel electrophoresis was visualized and showed a band of the expected size for B0032+J23100. The band for C0061+B0015 was too small. Further investigation revealed the chosen extension was too short.

Week 4: June 9 - June 15, 2013

This week we confirmed the successful cloning of the constitutive promoter + RBS. Furthermore we confirmed the addition of the TT to the autoinducer synthases and the lactonase. More biobrick sequences were verified. However, we observed some point mutations in the prefix/suffix sequences of some bricks as well as major annotation differences in the biobrick C0061.

Week 5: June 16 - June 22, 2013

This week was dominated by a lot of cloning. We equipped our constitutive and inducible promoters with ribosome binding sites, terminators, an ampicillin resistance gene and a lactonase (not all of them got each of these elements). Additionally, we separated the ampicillin resistance gene from its native promoter in order to make its expression inducible. Although cloning was mostly successful, we were struggling to amplify and purify the luxR brick (C0062), which cost us a lot of time and effort. Unfortunately, it also wasn’t rewarded with success in the end.

Another important step was to adapt our cloning strategy towards new chromoproteins which we received from iGEM team Uppsala. These allow us to evaluate our cultuvation experiments solely with own equipment, rather than having to find a cell sorter with suitable excitement lasers for all fluorescent proteins.

Week 6: June 23 - June 29, 2013

Week 6

Week 7: June 30 - July 6, 2013

In week 7 we started our first experiments with our inducible promotors. Unfortunatly we had trouble caused by the leakyness of two of our three promotors. We did further experiments but could not find a solution yet. Good news is that we received the chromoproteins from Uppsala! So we started working on our new cloning strategy by combining the chromoprotein DNA with our constitutive promotor-RBS construct. We hope to see nice and colorful colonies next week!

Week 8: July 7 - July 13, 2013

Week 8

Week 9: July 14 - July 20, 2013

Week 9

Week 10: July 21 - July 27, 2013

Week 10

Week 11: July 28 - August 3, 2013

Week 11

Week 12: August 4 - August 10, 2013

Week 11