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Team:Braunschweig/Notebook
From 2013.igem.org
Labjournal
This is the documentation of our lab work. An overview on how we approached this project can be found under Approach. 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 E. coli XL1Blue MRF' cells for all the transformations we are going to perform 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 pUC18 and pUC19 to calculate the transformation efficiency. 100 pg of DNA were used for each transformation with 50 µl of compentent cells.
Additionally, competent cells were plated on agar plates containing ampicillin, kanamycin and chloramphenicol respectively to ensure that the strain is not carrying any corresponding resistances. The plates were incubated at 37 °C overnight.
Saturday, May 25, 2013
Investigators: Kevin, Kerstin, Laura
Cells from yesterday's transformation only grew on agar plates containing ampicillin. The plates with the additional antibiotics were empty, thus we can conclude that our competent E. coli XL1 Blue MRF' cells do not carry other antibiotic resistances.
111 colonies were counted for pUC18 transformation, 50 for pUC19 transformation on ampicillin containing agar plates. According to these numbers the transformation Efficiency was calculated.
Transformation efficiency:
pUC18: ~ 106 µg-1
pUC19: ~5x105 µg-1
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 XL1 Blue MRF' strain 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 transformed each BioBrick. This is the full list of BioBricks we planned 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 had an ampR 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 of 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 the transformations of the BioBricks B0010, C0060, C0061, C0062, C0070, J23100 and J23106 were not successful, These Bricks were amplified via Phusion-PCR directly from the resuspended DNA from the distribution kit instead.
We prepared 5 mL liquid cultures of Bricks B0012, B0032, B1009, C0060, C0061, C0062, C0070, C0071, C0078, C0079, E0240, E0430, J06702, R0062, R0071 in 2xYT medium containing respective antibiotics in order to miniprep the plasmid DNA tomorrow.
Monday, June 3, 2013
Investigators: Oliver, Laura, Kerstin
We miniprepped the plasmids containing Bricks B0012, B0032, B1009, C0060, C0061, C0062, C0070, C0071, C0078, C0079, E0240, E0430, J06702, R0062 and R0071. As stated earlier the BioBricks C0061 and C0062 showed no visible bands in the colony PCR but we still prepped them. Furthermore we prepared glycerol stocks of all strains for further use.
Our gel electrophoresis of the PCR-amplified BioBricks showed a suitable bands for BioBricks C0060, C0061, C0070, J23100, J23106 which were recovered successfully from the gel. The BioBricks 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 transformed via heatshock in E. coli XL1 Blue MRF' cells. We also prepared following of the miniprepped BioBricks for sequencing: C0061, B0012, B1009. The results are outlined in the following table:
Wednesday, June 5, 2013
Investigators: Kerstin, Laura, Kevin, Oliver
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 BioBricks showed bands of the expected size and therefore were isolated via gel extraction.
Thursday, June 6, 2013
Investigators: Oliver, Laura
We prepared to clone some of our first and essential parts. The BioBricks were digested according to the table below.
Parts labeled as vector were dephosphorylated to prevent religation and then 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 overnight.
To test the success of our ligation beforehand we conducted a 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 BioBricks 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 double terminator 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 two 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 gel electrophoresis for the expected fragment sizes. Lokking at the gel all clones seem to be positive, although 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 evaluated 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 template. The PCR was successful (expected fragment size was 443 bp).
A colony PCR of two 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 evaluated 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 Registry of Standard Parts annotation and the BioBrick.
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 gel extraction 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 purified 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 cultivation 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 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 XL1 Blue MRF' cells from glycerol 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 2xYT liquid 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 of liquid cultures from positively tested clones was prepared for sequencing.
We also repeated the colony PCR of the last four ligations as they showed questionable restriction patterns.
After the second colony PCR was also found to be negative, these parts were once again digested, including gel 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 obtained and digestion of the purified PCR product did not show the 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), the ampR gene was cloned behind our inducible promoters and we ligated each autoinducer synthetase with a RBS (B0032) overnight.
Friday, June 21, 2013
Investigators: Laura, Jan, Oliver, Roman
The overnight ligation was followed by transformation of competent E. coli XL1Blue MRF cells. In parallel, PCR for C0062 from pSB1A2 was done using Phusion and Q5 polymerase. However, both preparations were negative, so we purified the other two repressor/activator genes (C0071, C0079) by gel extraction. Subsequently, we ligated 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 all 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
The focus of this week’s work was cloning further constructs and the evaluation of their quality by colony PCR and sequencing. Gel extraction was supposed to yield a functional structural gene for a beta lactamase. Additionally we took soil samples for the iGEM Team Norwich.
Sunday, June 23, 2013
Investigator: Laura
The ligations of J23100-B0032-C0071/C0079 and B0032-C0070/C0078-B0015 were positive in the colony PCR, so that we inoculated cultures for biomass production of the clones. We then conducted a colony PCR with ten clones of the B0032-C0061-B0015 construct to identify a correctly ligated clone. Cells with R0071/R0062/K091117-B0032-ampR were inoculated on agar plates with ampicillin supplemented media since we had evidence that our promoters might be leaky.
Monday, June 24, 2013
Investigators: Roman, Laura
Yesterday’s colony PCR of B0032-C0061-B0015 unfortunately was negative for all ten clones, as can be seen on the gel. Roman made minipreps of J23100-B0032-C0071/C0079 and B0032-C0070/C0078-B0015. These parts and furthermore C0061 and C0062 were prepared and sent to be sequenced. Since purification of ligated ampR in a column was not successful we tried to obtain it by gel extraction. The ligations of R0071-B0032-ampR, R0062-B0032-ampR and K091117-B0032-ampR were successful as indicated by colony PCR, therefore we inoculated cultures with bacteria carrying this construct for further processing.
Tuesday, June 25, 2013
Investigators: Jan, Kerstin
We made minipreps and Glycerin stocks of R0071/R0062/K091117-B0032-ampR. For validation of K091117’s sequence (2 basepairs differed from the sequence given in the registry) Kerstin conducted another Phusion-PCR. After ligation the product was evaluated by colony PCR. Together with R0071/R0062/K091117-B0032-ampR, K091117 was prepared and sent for sequencing. K091117-B0032-ampR was a double clone. We also took soil samples for the iGEM Team Norwich.
Wednesday, June 25, 2013 to Friday, June 28, 2013
The iGEM Team Braunschweig went to Berlin to join the Strategy Congress Biotechnology 2020+ of the Federal Ministry of Education and Research.
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 leakiness 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 promotor we digested B0032, C0060, C0061-B0015, J23112-B0032 and C0060-B0015. Afterwards gel extraction was performed for the inserts C0060, C0061-B0015 and C0060-B0015 and DNA purification for the vectors. Inserts and vectors were ligated using T4-DNA Ligase (NEB) overnight at 16°C.
Tuesday, July 2, 2013
Investigators: Kerstin, Laura
Yesterday's ligations transformed into E. coli XL1 MRF' by heatshock and plated on 2xYT agar containing glucose and Chloramphenicol.
Since the chromoprotein DNA from Uppsala iGEM Team 2011 arrived today we now switched to our new cloning strategy starting with resuspending and transforming the newly arrived DNA into E. coli XL1 Blue MFR' by heatshock. Cells were as well plated on 2xYT agar containing glucose and chloramphenicol.
Even if we switched to our new strategy we still kept working on the fluorescence markers. Therefore we digested the BioBricks 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. Gel extraction 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 leakiness experiments. All constructs (Plas, Prhl, Plux in combination with the RBS and ampR) were cultivated overnight in 2xYT medium containing various ampicillin concentrations.
Wednesday, July 3, 2013
Investigators: Laura, Kerstin
Before we started working on our own chromoprotein-constructs, we screened for the optimum cultivation conditions. We cultivated E. coli XL1 Blue MRF' containing a new construct from Uppsala iGEM encoding the device J23110-B0034-aeBlue. We tested expression of the blue chromoprotein at different temperatures, oxygen supply and rpm. These experiments led to the conclusion that low oxygen supply and a temperature of 37°C result in higher expression rates.
The evaluation of the leakiness of 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 Blue MRF' at all tested ampicillin concentrations. Therefore we repeated the experiment using higher ampicillin conentrations.
The bricks containing the fluorescencw markers ligated yesterday were transformed in E. coli XL1 Blue MRF' 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 alternative restriction strategy to combine the BioBricks by using the endonuclease NcoI.
B0032, C0060 and C0061-B0015 were digested with NcoI and corresponding SpeI and XbaI. Gel extraction was performed for the vector DNA as well as the inserts.
Overnight liquid cultures were inoculated with E. coli XL1 Blue MRF' containing the chromoprotein in 2xYT supplemented with chloramphenicol.
Thursday, July 4, 2013
Investigators: Laura, Kerstin
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 Blue MRF' 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 miniprepped and DNA was directly used for digestion to combine the chromoproteins with our promotor-RBS construct. The insert parts were extracted from an 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 construct size for all tested constructs.
Friday, July 5, 2013
Investigators: Kerstin, Laura, Jan
When we checked 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 Blue MRF' 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 leakiness 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 10: July 21 - July 27, 2013
This week we managed to construct our Rhl-inducicle Ampicillin resistance cassette (R0071-B0032-AmpR-B0015-J23100-B0032-C0071) and our Las-inducible Ampicillin resistance cassette (K091117-B0032-AmpR-B0015-J23100-B0032-C0079). Furthermore, the constructs of the amilGFP cassette (J23100-B0032-K592010-B0015), the Las-inducer and eforRed cassette (B0032-C0076-B0015-J23100-B0032-K592012-B0015), the Rhl-inducer and aeBlue cassette (B0032-C0070-B0015-J23100-B0032-K864401-B0015) and terminator-modified Rhl-inducible Amp-resistance cassette (B0015-R0071-B0032-AmpR-B0015-J23100-B0032-C0071) are nearly finished. Additionally we started our first test runs with the new chromoproteins.
Monday, July 22, 2013
Investigators:
Week 11: July 28 - August 3, 2013
This week was all about cloning our final constructs – unfortunately without success – and fighting our leaky promotors. We managed to modify the Las-inducible ampicillin resistance cassette with a terminator. A test in liquid culture to confirm if this makes the promotor less leaky was undermined by contamination. We also modified our constructs by adding new reporters.
Monday, July 29, 2013
Investigators: Kerstin, Laura
We transformed our inducible Ampicillin resistance into a new E. coli strain (JM109) in order to test whether the promotor being leaky is strain specific.
We did a colony PCR to test the result of our latest cloning experiments for the amilGFP cassette (J23100-B0032-K592010-B0015), the inducible ampicillin resistance and LasR transcription factor cassette (B0015-K091117-B0032-ampR-B0015-J23100-B0032-C0079), the Las-synthase with eforRed reporter cassette (B0032-C0076-B0015-J23100-B0032-K592012) and the Rhl-synthase with aeBlue reporter cassette (B0032-C0070-B0015-J23100-B0032-K864401-B0015).
The amilGFP cassette (J23100-B0032-K592010-B0015), the Las-Synthase with eforRed reporter cassette (B0032-C0076-B0015-J23100-B0032-K592012) and the Rhl-synthase with aeBlue reporter cassette (B0032-C0070-B0015-J23100-B0032-K864401-B0015) showed the expected bands on the gel and were prepped. The inducible ampicillin resistance and LasR transcription factor cassette (B0015-K091117-B0032-ampR-B0015-J23100-B0032-C0079) was prepped as well despite a second colony PCR showing no positive results.
We also tested the inducibility of our final red construct (BBa_K1073035) in liquid culture with ampicillin, which showed normal growth without being induced, showing us that the promotor is still leaky.
Tuesday, July 30, 2013
Investigators: Kerstin, Laura
The amilGFP cassette (J23100-B0032-K592010-B0015), the inducible Ampicillin resistance and LasR transcription factor cassette (B0015-K091117-B0032-ampR-B0015-J23100-B0032-C0079), the Las-Synthase with eforRed reporter cassette (B0032-C0076-B0015-J23100-B0032-K592012) and the Rhl-synthase with aeBlue reporter cassette (B0032-C0070-B0015-J23100-B0032-K864401-B0015) were sent to GATC for sequencing.
The sequencing revealed the following: The amilGFP cassette showed a point mutation in prefix A → T in front of XbaI. The sequence of the inducible ampicillin resistance and LasR transcription factor cassette (B0015-K091117-B0032-ampR-B0015-J23100-B0032-C0079) was verified. The Las-Synthase with eforRed reporter cassette (B0032-C0076-B0015-J23100-B0032-K592012) was sequence verified as well as the Rhl-synthase with aeBlue reporter cassette (B0032-C0070-B0015-J23100-B0032-K864401-B0015).
We also cloned the PCR amplificates of BioBricks C0062 and C0061 + B0015 into the sequence verified pSB1C3.
Wednesday, July 31, 2013
Investigators: Roman, Kevin, Anna
We started a restriction digest of several bricks to construct three new parts: The Inducible promotor with Rhl expression cassette (R0071-B0032-B0015-J23100-B0032-C0071) and the Las-Synthase with eforRed reporter cassette (B0032-C0076-B0015-J23100-B0032-K592012) were combined to our first complete construct (BBa_K1073035).
The Rhl-synthase with amilGFP reporter cassette (B0032-C0070-B0015-J23100-B0032-K592010-B0015) was derived from the Rhl-expression cassette (B0032-C0070-B0015) and the amilGFP cassette (J23100-B0032-K592010-B0015) as the blue chromoprotein was not usable for fluorescence detection.
The second final construct (BBa_K1073034), which is Las inducible, was constructed from the in-ducible Ampicillin resistance and LasR transcription factor cassette (B0015-K091117-B0032-ampR-B0015-J23100-B0032-C0079) and the Rhl-synthase with aeBlue reporter cassette (B0032-C0070-B0015-J23100-B0032-K864401-B0015).
Parts E0420 and J23100 were combined as another alternative for fluorescence experiments.
We tested the results of yesterday's cloning experiments via colony PCR. C0062 and C0061-B0015 did not show the expected bands.
Thursday, August 1, 2013
Investigators: Roman, Melanie
We continued yesterday's cloning experiment by purifying the insert DNA via gel extraction and dephosphorylation of vector DNA followed by ligation and transformation.
Friday, August 2, 2013
Investigators: Kevin, Anna, Melanie
To test the success of the last days’ cloning experiment we conducted a colony PCR. Unfortunately all clones of both our supposed final constructs were negative. Therefore the PCR was repeated later with new colonies.
The Rhl-synthase with amilGFP reporter cassette (B0032-C0070-B0015-J23100-B0032-K592010-B0015) and the eCFP cassette (J23100-E0420) were both positive.
We also conducted a test in 5 mL liquid cultures with different concentrations of ampicillin and autoinducers to test if our new constructs were leaky. Cells bearing the inducible ampicillin resistance and LasR transcription factor (B0015-K091117-B0032-ampR-B0015-J23100-B0032-C0079) were tested with ampicillin supplements between 1 and 8 µg/mL. We expected no growth as the ampicillin resistance was not induced.
Cells carrying the inducible ampicillin resistance with Rhl expression cassette (R0071-B0032-B0015-J23100-B0032-C0071) were incubated on medium containing between 1 and 2 µg/mL ampicillin and additionally 5 to 50 µmol/L Butyryl-HSL (Rhl autoinducer). As we induce the ampicillin resistance we expect some of the cultures to grow, depending on which concentration is optimal.
Cells carrying the constructs inducible ampicillin resistance combined with LasR transcription factor (B0015-K091117-B0032-ampR-B0015-J23100-B0032-C0079) and the inducible ampicillin resistance with RhlR expression cassette (R0071-B0032-B0015-J23100-B0032-C0071) respectively were grown in media supplemented with chloramphenicol were prepared as positive control. A culture carrying the inducible ampicillin resistance with RhlR expression cassette (R0071-B0032-B0015-J23100-B0032-C0071) was cultivated on media with different ampicillin concentrations as negative control.
The next day, all cultures were heavily grown indicating a contamination in our medium.
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) (Winsen, 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 Blue MRF'. 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 to 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 were 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 PRhl inducible in E. coli Top10F' was 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, Judith, Jan, Roman
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 Top10F' 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 final PRhl inducible construct and E. coli XL1 Blue MRF' containing final PLas inducible construct in 50 ml 2xYT containing chloramphenicol for continuous cultures were grown at 37°C and 250 rpm overnight.
Thursday, August 15, 2013
Investigators: Laura, Kerstin, Roman, Kevin, Jan, Judith
Pre-culture of erforRed expression cassette in E. coli Top10F' in 50 ml 2xYT containing chloramphenicol was inoculated from the agar plate and grown at 37°C and 250 rpm overnight.
For the reproduction of the growth curve a pre-culture of the PRhl inducible construct in E. coli Top10F' in 30 mL 2xYT containing chloramphenicol and incubated at 37°C overnight.
To test the production of homoserine lactones (HSL) by our final constructs we prepared a pre-culture of each reporter strain in 30 ml 2xYT containing ampicillin and incubated them overnight at 37°C and 250 rpm. We also made glycerol cell stocks of the reporter strains for further use.
Cells containing the finale constructs were grown in 30 ml 2xYT containing ampicillin as well at 37°C and 250 rpm. We inoculated these cultures with a high cell density as growth depends on the leakiness of the inducible promoters. These cultures were grown over 24 h in order to reach a high HSL concentration in the culture broth. As negative controls strains bearing constructs with the inducible promoters but not the HSL synthesis were grown in 30 ml 2xYT over night at 37°C as well.
The first attempt to cultivate regulated and unregulated mixed cultures in continuous culture was made today. For regulated growth ampicllin was added to the medium, for unregulated growth chloramphenicol was used as selection marker. Samples of each culture were taken at several time points. OD520 was measured and dilutions of samples were plated on 2xYT agar plates containing chloramphenicol.
We had problems to find the right dilution of cell suspension for the agar plates (too many or too little colonies on plates). The results of these cultivations were therefore not statistically relevant and no conclusions about the regulation of growth by our constructs could be drawn.
Friday, August 16, 2013
Investigators: Laura, Kerstin
The eforRed expression cassette in E. Coli TOP10F’ was miniprepped and glycerol stocks of this strain were made.
The main cultures (induced and not induced) of the final PRhl inducible construct were inoculated to a start OD520 of 0.05 in 75 ml 2xYT containing ampicillin with cells of the pre-culture. In order to induce the expression of ampR N-3-buturyl homoserine lactone was added to a final concentration of 10 µM. Samples were taken at appropriate times depending on the growth phase until the induced culture reached the stationary phase. OD was determined at 520 nm to avoid absorptions by chromoproteins.
To verify production of HSLs by our constructs the pre-cultures containing the finale constructs as well as the negative controls were centrifuged for 10 min at 6000 rpm and 4°C. Supernatant was transferred to a new Falcon tube and sterilized by filtration.
Dilution series of the supernatants and the synthetic HSLs as standards were pipetted in 96-well microtiter plates. Wells were inoculated with the corresponding reporter strain and grown for 3 h at 37°C. Bioluminescence produced by the luxCDABE of the reporter strains was detected by a microplate reader. We were able to show that our constructs produced the specific HSLs. However due to the high background especially in the N-3-oxododecanoyl-HSL producing strain we might need to modify the experimental layout in order to get a stronger signal.
References:
Winson, M. K., S. Swift, L. Fish, J. P. Throup, F. Jørgensen, S. R. Chhabra, B. W. Bycroft, P. Williams, and G. S. A. B. Stewart. 1998. Construction and analysis of luxCDABE-based plasmid sensors for investigating N-acyl homoserine lactone-mediated quorum sensing. FEMS Microbiol. Lett. 163:185–192.
Week 17: September 8 - September 14, 2013
We did some unseccessful growth curve experiments, but gain a lot of new infomation about our final constructs. The experiments showed again that bacteria carrying the final constructs can grow on a low level on medium containing ampicillin without inducing the ampicillin resistance by respective HSLs. But we figured out how to deal with this problem: We have to add the beta-lactamase inhibitor clavulanic acid to the culture broth to inhibit the antibiotic resistance just enough to allow for bacteria with induced resistance to grow, but prevents growth of non-induced cultures. Thus the next step for us is to determine the right beta-lactamase inhibitor concentration for our experiments.
Tuesday, September 10, 2013
Investigators: Jan, Anna, Melanie
We prepared pre-cultures of E. Coli JM109::K1073034 for the growth curve experiments tomorrow.
Wednesday, September 11, 2013
Investigators: Jan, Anna, Melanie
We conducted a cultivation experiment with the E. Coli JM109::K1073034 to test the growth with and without synthetic HSL autoinducer added to the media. Cultivation was carried out in four 500 mL flasks with 75 mL 2xYT medium with Ampicillin. In two flasks n-oxo-HSL was added. Ideally the flask without HSL would not growth any growth in the beginning until the antibiotic has been depleted. The HSL in the other flask should induce the quorum-sensing controlled Ampicillin resistance. Unfortunately all cultures were growing equally fast from the start so the experiment was aborted.
Wednesday, September 12, 2013
Investigators: Jan, Anna, Melanie
We repeated the growth curve experiment to see if the unexpected growth was caused by a resistant contamination. The result was the same as yesterday, all culture showed normal growth. We needed a new strategy for future experiments.
Thursday, September 13, 2013
Investigators: Jan, Anna, Melanie
We figured that maybe our Ampicillin resistance was too potent because even the basal expression allowed for normal growth in medium with antibiotic.
We prepared 5mL liquid cultures of JM109::K1073034 and added varying amounts of a beta-lactamase inhibitor (Clavulanic acid) to suppress the background expression. Tubes with 1 µg/µL to 100 µg/µL Clavulanic acid showed no growth while tubes with 0.001 µg/µL to 0.1 µg/µL Clavulanic acid showed normal growth.
Week 18: September 15 - September 21, 2013
This week we were happy to find a workaround for a big troublemaker: the background expression of β-lactamase in non-induced state. We determined the critical concentration of β-lactamase inhibitor clavulanic acid to be around 1 µg/ml. Furthermore, we directly applied it to experiments which previously were problematic because of the resistance to ampicillin under non-induced conditions.
Monday, September 16, 2013
Investigators: Jan
This week started with a quite important experiment: Since we experienced background expression of β-lactamase, our transformed bacteria were resistant on a low level to ampicillin, although cultivated under non-inducing conditions. We prepared 5mL liquid cultures of JM109::K1073034 and added varying amounts of a beta-lactamase inhibitor (Clavulanic acid) to suppress the background expression. Tubes with 1 µg/µL to 100 µg/µL Clavulanic acid showed no growth while tubes with 0.001 µg/µL to 0.1 µg/µL Clavulanic acid showed normal growth.
Wednesday, September 18, 2013
Investigators: Jan
On Wednesday, we repeated the experiment we did on Monday with a different scale of concentration steps to more precisely determine the best concentration of clavulanic acid for future experiments. Unfortunately, all samples showed growth so no further distinction could be made.
Hence, we went ahead with the 1 µg/ml of clavulanic acid we determined on Monday to remove background expression of β-lactamase. We applied this information to a certain growth behavior of JM109 bacteria transformed with our final construct (K1073034). Therefore, we cultivated this strain in 2x YT media with ampicillin and clavulanic acid. Out of four cultures, only two contained the inducer necessary for successful induction of ampicillin resistance.
Thursday, September 19, 2013
Investigators: Jan
Liquid cultures of 2x YT media with ampicillin and 1 µg/ml clavulanic acid were inoculated at different ratios (based on OD520) of JM109::K1073034 and Top10F'::K1073035. Each culture was additionally performed with chloramphenicol instead of ampicillin as unregulated control. Unfortunately the concentration of Clavulanic acid was too high and no growth was observed.
Week 19: September 22 - September 28, 2013
This week started with a continuous cultivation, which in contrast to earlier ones was performed with added β-lactamase inhibitor (clavulanic acid). On Wednesday, we repeated last weeks experiment to investigate the ratio of bacterial growth if both strains (JM109::K1073034 and TOP10F’::K1073035) are cultivated together under different conditions and with varying inoculation ratios.
Thursday, September 22, 2013
Investigators: Jan
Overnight cultures were inoculated for the continuous cultivation on Monday. Furthermore, we set up the reactors and made sure we are ready to start early next day.
Wednesday, September 23, 2013
Investigators: Jan, Anna, Melanie, Kevin, Roman, Judith
This time we inoculated the reactors with a 70/30 ratio of JM109::K1073034/TOP10F’::K1073035 and cultivated for 22h under constant observation to make sure we didn’t miss potentially interesting growth behavior. Samples were taken every hour. After plating a diluted sample on agar plates with chloramphenicol, OD520 was measured for monitoring the growth behavior. In comparison to earlier reactor cultivations, this time we added 5 µg/L clavulanic acid to the cultivation media (2x YT + either ampicillin in first reactor or chloramphenicol in second reactor) to suppress the background expression of β-lactamase.
Friday, September 27, 2013
Investigators: Jan
We conducted another ratio test on Wednesday. Therefore 2x YT media with ampicillin and 0.01 µg/mL were inoculated with different ratios of E. Coli Top10F’::K1073035 and E. Coli JM109::K1073034 and cultivated for 14 hours. Additionally a control on Chloramphenicol was cultivated. Unfortunately this time the clavulanic acid concentration was too low and the cells grew as if cultivated without clavulanic acid.
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