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                     Naringenin Operon Biosynthesis Notebook

                 <h1 class="display-2">Follow the Newcastle iGEM team on their journey</h1>

                     <p> The gblocks previously constructed in Benchling based on the parts used by the 2014 TU Darmstadt iGEM team. However we had to manually codon optimised the gene TAL from the operon to lower the G/C content, allowing all 4 gblocks to be ordered from IDT. These gblocks and the primers designed for detection of the assembled operon arrived, therefore lab work could commence. The pSB1C3 backbone for naringenin biosynthesis was amplified using PCR. As the plasmid came with a stock concentration of 25 ng/µl and the PCR reaction requires between 1pg – 1ng the plasmid was diluted by X50. A negative control with 1 µl H2O instead of plasmid DNA was made up. Thermocycling conditions of 71 °C for annealing based on the primers. Conditions for PCR are shown in the table below. Used agarose gel electrophoresis for detection of the 2kb amplified band, this was then purified however it resulted in a very low concentration of DNA around 3.58 µg/ml. Therefore it was planned to conduct this PCR again to increase the concentration.

Made an overnight culture of DH5 alpha cells to make more competent cells for subsequent transformations. These cells were then cultured in a 37°C shaking incubator until the OD600 reaches between 0.2 – 0.3, followed by additions of 75 mM MgCl2, 75mM CaCl2 and 15% glycerol to induce competency.

                     <p>A Positive control for Gibson assembly was undertaken by assembling 2 overlapping dsDNA fragments and the backbone pUC19 control DNA provided by NEB, using the NEBuilder HiFi DNA Assembly Cloning Kit. To check that Gibson Assembly using the master mix works and that the cells had been made competent. This positive control was then transformed into the competent cells made earlier in the week using NEBuilder HiFi DNA Assembly Transformation Protocol. These cells underwent heat shock before being spread onto ampicillin containing LB plates as the positive control plasmid carried resistance to ampicillin. All 3 of the positive control plates had growth of DH5 alpha cells, the negative control plate had no growth of DH5 alpha cells, as they contained no plasmid and were not ampicillin resistant. This showed that the Gibson master mix was effective and the competent cells had been successfully made competent.

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                     <p>Gblocks and the primers for the detection of the gblocks were resuspended following instructions by IDT. Gibson Assembly of the gblocks and the pSB1C3 backbone was conducted using the volumes worked out through NEB, as the vector should be 0.05 pmol and each insert between 0.1 – 0.2 pmol so that the total does not exceed 0.5 pmol. This was then transformed into the competent cells using chloramphenicol resistance plates instead of previously used ampicillin, as pSB1C3 has chloramphenicol resistance. Plated up positive and negative control plates; negative control plates with DH5 alpha cells without the operon. Following overnight incubation there was no colony growth on the positive plates.

                     <p>A second attempt at Gibson was conducted and transformation protocol the same as previously. No colony growth resulted from this. As it was thought that the backbone concentration was too low, which could be limiting transformation more of the backbone was amplified and purified to a stock concentration of 26.2 µg/ ml.

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                     <p>A third attempt of Gibson assembly was done using the increased backbone concentration resulting in a total reaction volume of 36.3 µl. This was then transformed into DH5 alpha cells resulting in 1 colony on each 200 µl plate. However these colonies only showed that the pSB1C3 backbone had been transformed due to its chloramphenicol resistance, in order to test for the Naringenin operon colony PCR was undertaken. Used primers 4CLF and CHSR to amplify the start and end of the operon with 66 °C annealing temperature. This reaction will amplify the inserts inside the backbone resulting in a PCR product of about 5kb, therefore 3 minutes extension times is required for the large segment. No 5kb band could be seen on the gel, so overnight cultures of the colonies were prepared to undergo miniprep. These two colonies were miniprepped in order to see a band at 7kb for the plasmid and the operon when gel electrophoresis was implemented.

                     <p>Following agarose gel electrophoresis the only bands present for multiple samples of the two colonies were at 2kb showing only the uptake of the plasmid. Next Gibson Assembly was conducted again, however instead of having an 18 µl reaction the mixture was spun down in a SpeedVac 120 for 5 minutes to increase the DNA concentration. As last attempt the total reaction volume was 36 µl when the protocol recommends 20 µl. 5µl of the Gibson Assembly was transformed multiple times into DH5 alpha cells instead of 2µl to see if this increased efficiency of plasmid uptake. However there was no colony growth on any of the plates. Constructed primers for amplification of the gblocks and ordered from IDT as concerned about the possibility of running out due to many Gibson assemblies.

                     <p>As the primers for gblock amplification arrived used the forward and reverse primers to amplify by PCR. Set up a gradient PCR to account for the different annealing temperatures with 1 minute extension time. Gel showed that gblocks 3 and 4 and the backbone amplified well resulting in a bright band, whereas 1 had two bands and appeared to be contaminated. Gblock 2 appeared to be degraded and no visible band could be observed. As gblock 2 did not amplify at all, conducted gradient PCR using different annealing temperatures for the primers resulting in 8 reactions. *insert table for annealing temps* Reaction 3 of gblock 2 showed the clearest band, therefore this was purified and quantified as having a stock concentration of 51.8 µg/ml. Gel extraction of the other gblocks was performed, as unsure which band was correct for gblock 1 cut both fragments and put in separate tubes. These stock concentrations were 13.6µg/ml for gblock1 higher band, 8.06µg/ml for gblock 1 lower band, 11.6µg/ml for gblock 3, 12.2µg/ml for gblock 4 and 14.3µg/ml for the backbone. Made up 4 separate Gibson Assemblies using variations of the gblock1 higher and lower bands and the gblock2 original and amplified. This was then transformed into commercial DH5 alpha competent cells and between 30-60 colonies grew on each plate.

<img src="https://static.igem.org/mediawiki/2018/7/7a/T--Newcastle--June17thPic.jpeg">

                     <p>Did a miniprep of 3 colonies from overnight cultures from transformed cells from the 4 plates, however only the backbone was transformed so set up an overnight culture of 10 more colonies. Ran another Gibson assembly in parallel with this using two tubes of 50µl Top10 commercial cells, transformed 2 µl into one and 5 µl into another, however this did not yield any colonies. The 10 colonies were ran on agarose gel, however only 2kb bands were observed therefore overnight cultures of colonies 11-20 were set up. Gel electrophoresis showed a promising band at 7kb for the miniprep of colony 19, which was the correct size of the plasmid and the operon. In order to obtain more colonies with a 7kb band the amplified plasmid backbone was treated with DPN1 and purified to a stock concentration of 9.84µg/ml. The 7th Gibson Assembly was then implemented using 6.53µl of the DPN1 digested backbone. To check the results of the miniprep of colony 19, PCR was conducted using the primers 4CLF and CHSR to observe if the 5kb operon could be amplified from the miniprep. Further PCR was done using the primers for each segment *insert primer conditions table*.¹.

<img src="https://static.igem.org/mediawiki/2018/a/aa/T--Newcastle--June18thPic.jpeg">

                     <p>Ran the PCR products for all the primers on a gel, however the primers did not amplify any of the segment. Despite this colony 19 was sent for sequencing as the band on the miniprep gel was the correct size. In case colony 19 was correct transformation into expression BL21 E. coli cells was done, these grew on chloramphenicol containing plates which could be used in future experiments for the extraction of naringenin. In concordance with this Gibson assembly with DPN1 was transformed into Top10 commercial cells and DH5 alpha commercial cells. This resulted in lots of colonies on the DH5 alpha cell plates and less than 5 on each Top10 plate. Colonies 21-25 from the Gibson assemblies using variants of gel extracted and amplified gblocks were transferred to overnight cultures, alongside colonies 27-34 from DPN1 treated cells. Conducted a miniprep of colonies 21-34 and found one band at 7kb for colony 23 which was subsequently sent for sequencing. Miniprepped overnight cultures 35-45 from Gibson Assemblies in commercial cells, however the only band was at 2kb.

<img src="https://static.igem.org/mediawiki/2018/f/f2/T--Newcastle--June19thPic.jpeg">