Team:Wageningen UR/Notebook/QuorumSensing
General
When cells or bacteria are described in the population dynamics notebook, those bacteria were E. coli DH5alpha. Unless stated otherwise, all actions performed for the wetlab construction and testing of the population dynamics subproject were done by Thomas. Where Arabinose and/or Glucose are mentioned, we refer to L-Arabinose and D-Glucose respectively in concentrations of 2 g/L. Where sequencing of DNA is mentioned we refer to GATC lightrun tube sanger sequencing. Where it is mentioned that samples were sent for sequencing - unless stated otherwise - the sequencing results corresponded to the expected sequence.
Plate reader measurements:
The machine used for plate reader experiments is BioTek’s SynergyMx. Gen5 2.01 software was used to acquire the data and export these to microsoft excel files which were further organized with Microsoft Excel 2011. All plate reader measurements were done in black 96-wells plates with a transparent bottom. During measurements the lid was on the plate in all cases to prevent mixing of sample due to shaking during incubation. All reported experiments were performed at 37℃ with fast continuous shaking. Measurements described were always at least 10 minutes apart to allow for proper shaking of the samples and allow for sufficient aeration. GFP activity was measured with fluorescence measurements with an excitation at 485nm and emission at 510nm. mRFP activity was measured for excitation at 584nm and emission at 607nm. Bandwidth for fluorescence was always 9nm. Wells contained 200µl cultures. Absorbance was measured at both 600nm and 660nm to assess bacterial growth. Absorbance data for bacterial cultures were corrected with the absorbance of medium only wells. This caused absorbance to start at 0 for all cultures, the first measurements notwithstanding. Fluorescence values were corrected for the fluorescence of medium only wells. This sometimes caused negative values for the fluorescence of cultures at low OD600 values. This of course it not realistic and was considered an artifact caused by fluctuations in the measurement of low level fluorescence.
Flow Cytometry
The machine used for flow cytometry in Wageningen is the Flow cytometry sorter - BD FACS AriaTM III. This machine is in principle also able to sort cells, but we did not make use of this function. Marcel H. Tempelaars, Specialist Flow cytometry sorter - BD FACS AriaTM III at Wageningen UR operated the controls while Thomas was responsible for sample handling and insertion. Marcel meanwhile explained the functioning of the technique.
During the flow cytometry analysis we found that the choice of mRFP as reporter was a good one: we observed a clear difference in fluorescence between the mRFP producing cells and cells that did not have a mRFP gene. Differentiation between GFP expressing cells and cells unable to produce GFP is known to be more troublesome. Therefore we advise future iGEM team planning to do flow cytometry of FACS to (if at all possible) use mRFP as reporter rather than GFP.
Fluorescence Microscopy
The microscope used for fluorescence microscopy of the subpopulation clones is an Olympus BX41. The laser used to excite fluorophores is Excelitas Technologies X-cite series 120. A lumenera infinity 3 was used to capture the images. Steven Aalvink, technician at Wageningen UR molecular ecology, instructed me in the use of the microscope and other hardware.
May 9th - July 3th:
General
Belwina, Marijn and Thomas transformed biobrick parts from the registry to home-made chemically competent DH5alpha cells. They spread the transformations on LB agar plates with antibiotics corresponding to the resistance in the backbone of each plasmid. Transformed parts (table 1). We isolated (Machery-Nagel nucleospin kit) the plasmids from liquid cultures of these transformation plates.
July 4th - July 17th:
General
We verified the length (through taq PCR) and sequence (through GATC lightrun sanger sequencing) of the biobrick isolated (May 9th - July 3th) plasmid inserts. We also made -80℃ glycerol stocks for cells containing the parts.
434- and λ cI repressor operon construction
We designed primers with overhangs for amplification of biobricks in Benchling for subsequent Gibson assembly (GA) and ordered those primers from NEB: Primers designed for parts: BBa_B0015, BBa_C0052, BBa_I0500, BBa_K081007 and pSB1C3. Primers:897 - 908.
We used primers 897 - 906 for Q5 PCR to amplify the parts for GA. We was unable to amplify pSB1C3 with the flanked primers through numerous attempts. We then realized that (because of the overhangs in the other fragments) the fragments all already contain 20 + bp overlap with each other required for GA. We chose to Q5 PCR amplify pSB1C3 with primers suffix-forward and prefix-reverse and use this PCR product with the previously mentioned PCR products for the other fragments.
We did a first attempt to Gibson assemble BBa_K1913007 using (unpurified) PCR reactions of BBa_B0015, BBa_C0052, BBa_I0500, BBa_K081007 and pSB1C3 mentioned before. We used NEB GA mastermix and followed the corresponding protocol. We used 0.2 to 2.0 µl of 50 ng/µl dilutions of the PCR product to use 75 ng of pSB1C3 and 2.5 as much moles of the other fragments, except for B0015 where 5 times the backbone concentration was used. After assembly, we transformed the mixture to electro competent cells prepared beforehand by Belwina. The plates were kindly transferred from 37℃ to 4℃ by Alex (supervisor).
We did a colony taq PCR of 48 of the resulting colonies to screen for successful assembly clones. We found one clone (‘colony 35’) that showed a substantially bigger insert than the background found in the other colonies and in the negative control.
We made -80℃ glycerol stocks and isolated the plasmid from ‘colony 35’ using the Machery-Nagel plasmid isolation kit. We did a verification PCR on the isolated plasmid and found that the insert size is shorter than it should be.
July 25th - August 7th
434- and λ cI repressor operon construction
We sent ‘colony 35’ for GATC lightrun Sanger sequencing. The sequencing revealed that all fragments except BBa_K081007 were assembled in pSB1C3. We designed (and ordered from NEB) primers with overhangs for amplification for a second step of GA to include BBa_K081007 in the construct. Primers: 109 - 110.
We used the primers for Q5 PCR to amplify BBa_K081007 and the plasmid from ‘colony 35’ for GA. Both reactions yielded band of the expected sizes in agarose gel electrophoresis. We purified the reactions using the Machery-Nagel kit for PCR purification and eluted in milli-Q water.
We used the purified PCR reaction in GA with NEB HiFi mastermix according to the corresponding protocol. We saved part of the reaction after 15m at 50℃ and left the rest to react for 60m 50℃. We purified the GA reaction with the Zymo Research DNA clean and concentrator kit. We transformed 5µl the 15m reaction to NEB chemically competent cells according to the manufacturer's protocol. Lisa transferred the plates from 37℃ to 4℃.
mRFP cI protein balance reporter construction
We designed primers with overhangs for amplification of biobricks in Benchling for subsequent GA and ordered those primers from NEB: Primers designed for parts: BBa_I12006, BBa_K081007, BBa_K081014 and pSB1C3. Primers: 113 - 118.
We used the primers for Q5 PCR to amplify the fragments for GA. All reactions yielded band of the expected sizes in agarose gel electrophoresis. pSB1C3 again could only successfully be amplified with primers suffix-forward and prefix-reverse. We purified the reactions using the Machery-Nagel kit for PCR purification and eluted in milli-Q water.
We used the purified PCR reaction in GA with NEB HiFi mastermix according to the corresponding protocol. We saved part of the reaction after 15m at 50℃ and left the rest to react for 60m 50℃. We purified the GA reaction with the Zymo Research DNA clean and concentrator kit. We transformed 5µl the 60m reaction to NEB chemically competent cells according to the manufacturer's protocol. Lisa transferred the plates from 37℃ to 4℃.
Quorum sensing GFP reporter construction
We designed two flanked forward primers to elongate (in two PCR steps) the 5’ end of BBa_K081012 with the sequence of promoter BBa_R0062. We ordered the primers from IDT.
We did Q5 PCR on BBa_K081012 with the primers 111 and VR, got a band of the expected size (around 1kb). We purified the PCR product with the Machery-Nagel PCR purification kit. We then used a dilution hereof for a seconds step with purification with primers 112 and VR. We also amplified (Q5 PCR) pSB1C3 with primers suffix-forward and prefix-reverse to linearize the backbone. Both PCRs yielded bands of the expected sizes in agarose gel electrophoresis. We then purified both PCR products with the Machery-Nagel kit and protocol.
Quorum sensing 2 plasmid system construction
We Q5 PCR amplified BBa_K546000 and pSB4K5 with primers VF2 + VR and suffix-f + prefix-r. Both PCRs yielded bands of the expected size on agarose gel. We purified both PCR products with the Machery-Nagel PCR purification kit and corresponding protocol.
We, then started digestion of 1µg of both purified products with EcoRI-HF and PstI-HF, both from NEB and according to the NEB digestion protocol 15m at 37℃. We purified both digestion products with the Machery-Nagel PCR purification kit and protocol.
We started ligation with NEB T4 according to the NEB T4 ligase protocol. We used 50ng of pSB4K5 and 3 times as much moles of the BBa_K546000 insert. Ligation took place overnight at 16℃. We then purified the ligation reaction with the Machery-Nagel PCR purification kit according to the manufacturer's protocol.
The ligation was transformed using homemade electrocompetent cells. The transformations were spread on LB agar plates with Kanamycin. The transformation yielded plates with moderate amount of colonies.
August 8th - August 21st
434- and λ cI repressor operon construction
Transformation of the GA for assembly of the 434- and λ cI repressor operon plasmid yielded many colonies. We screened for clones with the correct insert through taq colony PCR with primers biobrick-forward and biobrick-reverse. Prior to colony PCR, the colonies were grown in 40µl LB medium inside a 96-wells plate for 30m. 1µl of the medium was used as template for the colony PCR. All screened colonies were found to contain an insert of the correct size.
Sept 1 - Okt 10
Expression of Cas9-expresso constructs in C321ΔA
An expression experiment was performed with 3 ml cultures induced overnight with rhamnose, arabinose and synthetic amino acids when applicable. This yielded no visible Cas9 bands.
The same happened when 5 ml cultures where induced for 4 hours. What worked, was the protocol with bigger volumes followed by Ni-NTA purification (the majority of the actual work with the FPLC was performed by a supervisor)
First, samples were purified as described, but with addition of DNAse. This gave good yields, but DNAse remained in the purified fractions as was later found out during in vitro Cas9 assays. However, without DNAse also a good yield was obtained.
The green line indicates the amount of His buffer B that is passed through the column.
in vitro Cas9 cleaving assays
Assays were performed with all produced guide RNAs, according to the protocol.
References
1. ajoie, M. J., Rovner, A. J., Goodman, D. B., Aerni, H. R., Haimovich, A. D., Kuznetsov, G., ... & Rohland, N. (2013). Genomically recoded organisms expand biological functions. Science, 342(6156), 357-360. ↩
