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, Kevin, Roman, Tabea
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, Laura
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: Laura, 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: Laura, Kerstin
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: Laura, Kerstin
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: Tabea, Oliver
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: Laura, Kerstin, Jan
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.

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.

Monday, June 17, 2013

Investigators: Laura, Oliver, Jan
Today, we first digested the inducible promoters, followed by purification and clean-up. Then we equipped the inducible and previously digested constitutive promoters with a RBS (B0032) by ligating respective parts. Subsequently, these ligated constructs were used to transform competent bacteria. We also inoculated overnight suspension cultures with B0015- and B0032-transformed E. coli cells from cryo stocks for DNA preparation.

Tuesday, June 18, 2013

Investigators: Kevin, Jan
We performed a colony-PCR to test if cloning from the previous day was successful. Positively tested clones were used to inoculate suspension cultures for DNA preparation. Overnight suspension cultures of B0015- and B0032-transformed cells were used for DNA preparation.
In order to separate the ampicillin resistance gene from its native promoter we performed a Phusion-PCR with appropriate primers on the P1002 brick.

Wednesday, June 19, 2013

Investigators: Roman, Laura, Kerstin
In order to harvest the successfully ligated bricks, DNA preparation of suspension cultures from positively tested clones was done. Samples from this DNA were prepared for sequencing to confirm correct ligation. We also repeated the colony-PCR of the last 4 ligations as they showed questionable restriction patterns. After the second colony-PCRs were also found to be negative, new digestions of these parts were performed, including gele extraction and purification. Furthermore, the previously amplified ampR gene was purified by gel electrophoresis. However, gel extraction did not yield enough DNA to work with.

Thursday, June 20, 2013

Investigators: Laura, Oliver
First, we performed another Phusion-PCR on ampR (P1002 brick) since the first PCR did not yield enough DNA. We also tried to amplify the luxR gene (C0062) through Phusion-PCR from pSB1A2 plasmid. However, not enough DNA was yielded and digestion of the purified PCR product did not show expected bands.
More digestions were set up to harvest the separated ampicillin resistance gene and prepare DNA for the next cloning round. Additionally, lactonase (C0060) was ligated with a RBS (B0032), ampR gene was cloned behind our inducible promoters and we ligated each autoinducer synthetase with a RBS (B0032) over night.

Friday, June 21, 2013

Investigators: Laura, Jan, Oliver, Roman
Overnight ligation was followed by transformation of competent e.coli cells. In parallel, PCR for C0062 from pSB1A2 was done using Phusion and Q5 polymerase. However, both preparations were negative, so we went ahead and purified the other two repressor/activator genes (C0071, C0079) by gele extraction, which was followed by ligation of these bricks with a constitutive promoter. As usual, we transformed competent bacteria with the ligated vectors. Furthermore, Phusion- and Q5-PCR was repeated with different annealing temperatures, which were also negative. We also changed our cloning strategy as we thankfully received new chromoproteins from iGEM team Uppsala.

Saturday, June 22, 2013

Investigators: Laura, Kerstin
Today was a PCR day. We performed a colony PCR of 10 clones of each ligation we set up yesterday, resulting in 100 PCRs! We had to use all electrophoresis chambers that we could find in order to screen them all at the same time.

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. Unfortunately 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!

Monday, July 1, 2013

Investigators: Roman, Laura
In order to combine lactonase and our lactonase-terminator construct as well as LasI und the LasI-terminator construct with the ribosome binding site and a constitutive protomor we digested B0032, C0060, C0061-B0015, J23112-B0032 and C0060-B0015. Afterwards gel extraction was performed for the insert parts C0060, C0061-B0015 and C0060-B0015 and DNA purification for the vector parts. Inserts and vectors were ligated using T4-DNA Ligase (NEB) over night at 16°C.

Tuesday, July 2, 2013

Investigators: Kerstin, Laura
The ligations from yesterday were transformed into E. coli XL1 by heatshock and plated on 2xYT agar containing glucose and chloramphenicol.
Since the chromoprotein DNA from Uppsala iGEM Team 2011 arrived today we now switch to our new cloning strategy starting with resuspending and transforming the newly arrived DNA into E. coli XL1 by heatshock. Cells were as well plated on 2xYT agar containing glucose and chloramphenicol.
Even if we switched to our new strategy we still keep working on the other fluorescent markers. Therefore we digested the bricks E0420 (eCFP), E0430 (YFP) and J06702 (mCherry) as well as our promotor-RBS-LasR and promotor-RBS-RhlR-constructs. Still lacking the construct containing LuxR we decided to proceed with the promotor-RBS-construct instead to ligate it to the YFP-Brick. Gelextraction was performed for the inserts and DNA purification for the vector parts. Inserts and bricks were ligated overnight using T4 DNA ligase (NEB) at 16°C.

Since we now have our first constructs containing the inducible promotors as well as the the ampicillin resistence gen we started our first leakyness experiments. All constructs (P_las, P_rhl, P_lux in combination with the RBS and ampR) were cultivated overnight in 2xYT medium containing various ampicillin concentrations.

Wednesday, July 3, 2013

Investigators: Kerstin, Laura
Before we started working on our own chromoprotein-constructs, we screened for the optimum cultivation conditions. We cultivated E. coli XL1 containing a new construct from Uppsala iGEM containing the device J23110-B0034-aeBlue. We tested expression of the blue chromoprotein at different temperatures, oxygen supply and rpm. From that experiments we came to the conclusion that low oxygen supply and a temperature of 37°C leads to higher expression rates.

The evaluation of the experiment for the inducible promotors showed that only the Prhl is not leaky. Plux as well as Plas were leaky and showed growth of E. coli XL1 at all tested ampicillin concentrations. Therefore we repeated the experiment using higher ampicillin conentrations.

The bricks containing the fluorescent markers ligated yesterday were transformed in E. coli XL1 by heatshock.

Colony PCR of the constructs containing lactonase and LuxI which were transformed yesterday was performed. Since all screened colonies showed religated vectors we decided to try an alternativ restriction strategy to combine the bricks by using the endonuclease NcoI.
B0032, C0060 and C0061-B0015 were digested with NcoI and corresponding SpeI and XbaI. Gelextraction was performed for the vector DNA as well as the inserts.
Overnight liquid cultures were inoculated with E. coli XL1 containing the chromoprotein in 2xYT containing chloramphenicol.

Thursday, July 4, 2013

Investigators: Kerstin, Laura
Ligation of the constructs B0032-C0060 (containing lactonase) and B0032-C0061-B0015 (containing LuxI) was performed for 30 minutes at room temperature using T4 DNA Ligase (NEB). Constructs were transformed in E. coli XL1 by heatshock and plated on agar plates.
As we were still having trouble with the brick C0062 (LuxR) we amplified the brick from the device F2620 using Q5 Polymerase (NEB). Gelextraction was performed for the PCR products.




The chromoprotein cultures were minipreped and DNA was directly used for digestion to combine the chromoproteins with our promotor-RBS construct. The insert parts were extracted from agarose gel while the vector part was dephosphorylated and purified using DNA clean-up columns.
Colony PCR was conducted for the constructs with the fluorescent proteins which were transformed yesterday. We were able to identify colonies with the right brick size for all tested constructs.

Friday, July 5, 2013

Investigators: Kerstin, Laura, Jan
Checking the agar plates for transformed colonies with the LuxI and lactonase containing constructs we did not have any colonies.
The digested chromoprotein DNA was ligated with the RBS and the promotor-RBS construct at room temperature for 30 minutes. The ligated DNA was transformed in E. coli XL1 by heatshock and plated on agar plates. We cannot wait to see colorful colonies on Sunday :-)

Since we almost ran out off our competent cells it was time for new ones. New compentent cells were made and transformation efficiency was tested.
A new idea to cope with the leakyness of the Plas and Plux was to try a different antibiotic which cannot be metabolised. We used carbenicillin instead of ampicillin at different concentrations in liquid culture with 2xYT medium. Unfortunatly no effect could be observed on the leakyness of both inducible promotors.

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

Week 13: August 11 - August 17, 2013

This week was dominated by testing our constructs in continuous as well as in batch culture for inducibility and regulation. Unregulated growth in chloramphenicol containing medium and regulated growth in ampicillin containing medium was tested in continuous culture. The growth kinetics observed for our Rhl regulated could be verified in a second growth curve for induced and uninduced growth. Further we received E. coli JM109 strains containing a reporter plasmid expressing luxCDABE in the presence of specific activating N-acyl homoserine lactones (AHLs) (Wilsen, M.K. et al. 1998) resulting in bioluminescence of these reporter strains. We will be using these strains in order to verify the production of the specific homoserine lactones by our constructs.

Sunday, August 11, 2013

Investigators: Roman
We prepared pre-cultures of our chromoprotein expression cassettes (promoter-RBS-chromoprotein) in E. coli XL1. For each chromoprotein (amilGFP, eforRed, aeBlue) 50 ml 2xYT containing chloramphenicol were inoculated from -80°C glycerol stocks and grown at 37°C and 250 rpm over night.

Monday, August 12, 2013

Investigators: Kerstin, Laura
Pre-cultures of chromoprotein constructs were mixed in one main culture in order to see how they behave during cultivation over several hours. OD₅₂₀ of pre-cultures was measured in order to inoculate the main culture with 33% of each strain with a final OD₅₂₀=0.3 For the main culture, 25 ml 2xYT containing chloramphenicol were inoculated with the three different strains and grown at 37°C and 250 rpm in a non-baffled flask. Samples from the culture were taken at several time points, diluted and plated on 2xYT agar-plates containing chloramphenicol. Agar plates where incubated at 37°C over night.
During the day we noticed that the 2xYT medium used for the pre-cultures yesterday showed contamination. Thus, there is a high possibility of contamination in the pre-culture as well as in the main culture. Therefore we decided to repeat the experiment. New pre-cultures of the three chromoprotein expression cassettes where inoculated in 50 ml 2xYT containing chloramphenicol directly from -80°C.
In order to have our constructs available in different E. coli strains for the fluorescence microscopy experiments, the eforRed expression cassette was transformed into E. coli Top10F' by electroporation. Transformed cells were plated on 2xYT agar containing chloramphenicol and incubated over night at 37°C.
A continuous cultivation of the Rhl inducible construct in E. coli Top 10 prepared, including preparation of pre-cultures of our inducible construct in 50 ml 2xYT containing chloramphenicol and grown at 37°C and 250 rpm over night.

Tuesday, August 13, 2013

Investigators: Kevin, Melanie
Unfortunately, the agar plates from yesterday’s mixed culture didn’t show any colour which might be due to contamination. Therefore we did not evaluate these plates any further. We also had some problems with the shaker and lost the culture containing the aeBlue construct. A test was run on how to dilute a mixed culture of the remaining eforRed and amilGFP constructs in order to be able to count colonies of each colour on large agar plates.
The transformation of the eforRed construct did not work (no colonies on agar plate) and has to be repeated.
The first continuous cultivations were carried out in order to get to know the routines and problems of a continuous cultivation. Due to problems with the regulation of pumps no valuable results were produced but the setting was improved based on these experiences.

Wednesday, August 14, 2013

Investigators: Anna, Kevin
Transformation of the eforRed expression cassette in E. coli Top10 was performed by electroporation.
The reporter strains for the Las and Rhl systems (E. coli JM109 pSB1075 and E. coli JM109 pSB406 respectively) arrived today. Liquid cultures in 2xYT containing ampicillin were inoculated and grown at 37°C and 250 rpm overnight.
Pre-cultures of E. coli Top10F’ containing finale pRhl inducible construct and E. coli XL1 containing finale pLas inducible construct in 50 ml 2xYT containing chloramphenicol for continuous cultures were grown at 37°C and 250 rpm overnight.