"
Team:Braunschweig/Notebook
From 2013.igem.org
| Line 486: | Line 486: | ||
We had problems to find the right dilution of 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. </p> | We had problems to find the right dilution of 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. </p> | ||
| + | <p style="font-size:14px; font-weight:bold; text-decoration:none; border:none; color:#be1e3c; margin-left:5px; margin-right:5px; margin-top:0px; margin-bottom:0px">Thursday, August 15, 2013</p> | ||
| + | <p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px"> | ||
| + | <b>Investigators: Laura, Kerstin</b><br> | ||
| + | The eforRed expression cassette in <i>E.Coli</i> TOP10F’ was miniprepped and glycerol stocks of this strain were made.<br> | ||
| + | |||
| + | The main cultures (induced and not induced) of the final Rhl inducible construct were inoculated to a start OD<sub>520</sub> of 0.05 in 75 ml 2xYT containing Ampicillin with cells of the pre-culture. In order to induce the expression of <i>ampR</i> N-3-buturyl homoserine lactone was added to a final concentration of 1 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.<br> | ||
| + | To verify production of AHLs 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. <br> | ||
| + | Dilution series of the supernatants and the synthetic AHLs 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 <i>luxCDABE</i> of the reporter strains was detected by a microplate reader. We were able to show that our constructs produced the specific AHLs. However due to the high background especially in the N-3-oxododecanoyl-HSL producing strain we need to modify the experimental layout in order to get a stronger signal.</p> | ||
</div> | </div> | ||
Revision as of 11:00, 2 October 2013
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 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 overnight.
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 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 our transformations of the BioBricks 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 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 XL1Blue 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, 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 BioBricks 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 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. All clones were 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 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 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 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 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 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 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 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: 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. 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 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 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 containing the chromoprotein in 2xYT supplemented with 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 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 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.
