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Team:Braunschweig/Notebook
From 2013.igem.org
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. Details can be found in the Approach section
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.
We also send some of the Bricks miniprepped on June 3, 2013 for sequencing (primers VR and VF2).
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.
Sunday, June 9, 2013
Investigators:
We ran a colony PCR of 2 clones of each ligation (transformation from June 6, 2013) and the biobricks P1002 and K091117 (primers VF2 and VR).
We also inoculated overnight cultures for plasmid preparations the next day.
Monday, June 10, 2013
Investigators:
We analyzed yesterday’s colony PCR gelelectrophoresis for the expected fragment sizes. All clones were positive(clone 1 of J23100+B0032 and J23106+B0032 did not contain the promoters as later shown by DNA sequencing.
We isolated the plasmid DNA from all clones with a Miniprep Kit following the manufacturer’s instructions.
Tuesday, June 11, 2013
Investigators: Kerstin, Laura
We transformed the chemically competent E.coli XL1 Blue MRF’ with the ligation C0061+B0015 (prepared on June 6, 2013) and the biobrick E0420 (eCFP).
The plasmid DNA isolated the day before was sent to GATC for sequencing.
The DNA sequences from June 7, 2013 were analyzed by sequence alignment with the vector maps.
The biobricks C0079, C0078, C0070 and B0032 were sequence verified. However, the biobrick C0061 did not match the annotated sequence. The prefix was missing and there was an additional oligonucleotide sequence in the 5’ region of the biobrick. This led to the assumption that C0061 might be a composite part.
Wednesday, June 12, 2013
Investigators:
The biobrick B0015 was amplified by PCR using the resuspended DNA from the distribution kit as a template. The PCR was successful (expected fragment size was 443 bp).
A colony PCR of 2 clones of each of yesterday’s transformations (E0420 and C0061+B0015) was run (primers VF2 and VR). Overnight cultures of the analyzed clones were inoculated.
The remaining DNA sequences (from June 7,2013) were analyzed by sequence alignment. While the Biobricks J06702, B0012, C0071, R0062 and R0071 were sequence verified, the biobrick C0062 did not match the sequence, the DNA sequencing for clone 2 failed. Furthermore the sequence alignment of E0240 revealed additional 25 bp after the EcoRI restriction site.The DNA sequence of E0430 was confirmed except for a point mutation (T to A between Not/I and XbaI restriction site) as well as the biobrick J23112 which contained a point mutation in the prefix sequence (T to A).
Thursday, June 13, 2013
Investigators:
Today we received the NEB iGEM Support Kit. This will keep the labwork rolling ;-)
Yesterday’s colony PCR(E0420 and C0061+B0015) was analyzed on a 1% agarose gel.The amplified fragment for E0420 matched the expected fragment size of 1192 bp. The PCR for C0061+B0015 failed. A faint PCR product of 6-7kb could be detected for clone 2 indicating (again) an additional oligonucleotide sequence 5’ of the biobrick. This additional DNA sequence may have up to 6kb. We isolated the plasmid DNA from all clones of E0420 and C0061 + B0015 with a Miniprep Kit following the manufacturer’s instructions to send them to GATC.
In order to clone the inducible promoters Plux (R0062), Prhl (R0071) and Plas (K091117) 5’ of the RBS (B0032), we digested them with EcoRI and SpeI. At the same time the lactonase (C0060) and lactonase + TT were digested with XbaI and PstI to clone them 3’ of a RBS. The autoinducer synthases + TT (C0078+B0015, C0070+B0015, C0061+B0015) were digested with XbaI and PstI for cloning. Also we did a test restriction of C0061 since the DNA sequence indicated it contained an additional nucleotide sequence.
The expected fragments of the promoters are 82, 80 and 153 bp. We were not able to detect the fragments on the agarose gel. The restriction digest of C0060, C0078+B0015 and C0070+B0015 was not complete. However, the fragments matched the expected sizes, so they were extracted from the agarose gel.
For C0061, the restriction failed. The size of the linearized vector is about 10kb confirming differences in the partsregistry annotation and the brick.
The sequence data from June 11, 2013 was analyzed by sequence alignments. J23100+B0032 and J23105+B0032 did not contain the promoters. J23112+B0032, C0078+B0015, C0070+B0015, C0060+B0015 was sequence verified. Unfortunately the sequencing failed for K091117 and P1002.
Friday, June 14, 2013
Investigators: Laura, Kerstin, Oliver
Since cloning of the constructs J23100+B0015 and J23106+B0015 failed previously and gelextraction of the promoters is difficult due to their small fragment sizes, we tried a copy&paste cloning approach:
The promoters J23100 and J23106 were PCR amplified using resuspended DNA from the distribution kit as a template. The inducible promoters R0062, R0071 and K091117 were PCR amplified using plasmid DNA (miniprep) as a template.
The endonuclease digest on June 13, 2013 was repeated from scratch. This time the digest was successful and the fragments were extracted. The vectors were dephoshorylated.
