Team:York UK/Notebook.html

Overview

My part of the team - Gintare, Evaldas and Jonas - were responsible for cloning of the mtr complex, gold sensing device and preparing peptides for expression.

July 7th

Making of chemically competent DH5α cells. Protocol can be found in our protocols section.

July 10th

Purification of genomic DNA from Shewanella oneidensis and Salmonella sp.

July 25th

After a long break we finally started working on the actual assembly of parts - the mtr complex. Today Jonas ran a trial PCR on mtrB and mtrC part of the complex both of which are around the size of 2kb. GoTaq polymerase (Promega) was used, this is the polymerase we generally use for trials and colony PCRs as fidelity is not the main priority.

This is the gel from the PCR. Temperatures ranged 46-71˚C. It appears that the primers are sufficiently specific at even low temperatures. 1% agarose, SYBR-Safe(Invitrogen), QStep4 ladder.

July 26th

Trial PCR again, this time for three small (~500bp) synthetic DNA fragments - gBlocks (IDT), these fragments are highlighted below on the diagram as well as the overall view of our mtr gene.

The gel. Three different temperatures (51-61˚C)- all work. 1% agarose dyed with SYBR-Safe, however, due to poor practice the ladder is indistinguishable.

At this point Jonas arrived at the realisation that as long as the primers are specific enough, there is no point in doing PCR trials (silly Jonas). Therefore most of the PCRs done this day forth use a universal 58˚C temperature.

July 27th

This is the first day that we actually got some positive outcome from our cloning. Or so we thought.

All the fragments of our mtr complex were once again amplified via PCR but this time using Phusion polymerase for high fidelity (Thermo Fisher). Gel confirmed that everything went fine and specifically. Using QIAquick PCR purification kit (Qiagen) we clean up the PCR product, then measure the concentrations using NanoDrop1000, do the required calculations and then proceed to the main reaction:

In 60 minutes at 50 ˚C we joined all of the 5 fragments using the Gibson Assembly Master Mix provided by NEB. Confirmed by a PCR.

The PCR was not fully specific but it is unclear whether these fragments formed during Gibson reaction (main fragment joining sites are identical RBS) or because the temperature used was too low (51˚C & 58˚C). 3rd and 4th lanes are negative controls. 1% agarose dyed with SYBR-Safe, QStep4 ladder.

Before August 11th

During this time we tried putting all of our genes inside the pSB1C3 iGEM plasmid but with little luck. Everything seemed to be working fine up until the point when we realised that colony PCRs are not so trustworthy. It appeared clear that most of the colony PCRs came out as false positives after we decided to run the PCR on already purified plasmids. However, some samples were already on their way for sequencing (thinking that we actually got something within our pSB1C3 backbones we sent them). The sequencing results gave us an answer: we planned it all wrong! Graphical summary:

So all of our parts that were to be put inside the iGEM plasmid only had XbaI and SpeI cutting sites, but we were unaware that they actually had complementary sticky ends and it was nearly impossible to ligate something in between.

We fixed it with new primers which add the remaining part of the prefix and suffix. However, we lost 2 weeks worth of productive labwork.

August 12th

So after receiving the new primers we continue working. This time we're using Gibson's assembly to directly insert our mtr part into pSB1C3, this allows us to skip any problems that might occur during digestion or ligation.Note that during design of these primers we decided to produce two different mtr parts, one of which would lack the promoter and the first RBS(crR12) which we thought might not be as convenient for other teams.

So today we tried amplifying mtrAB(from the previous Gibson), mtrC(from previous PCR, should have used the genome) and pSB1C3 backbone(the linearised plasmid from iGEM HQ). Unfortunately, the backbone PCR failed.

We thought that perhaps dimers of the primers formed as the sequences of prefix and suffix are not that different. 1% agarose dyed with SYBR-Safe, 1kb ladder(Invitrogen)(on the right).

However, after playing a bit with DMSO and annealing temperatures we were still unable to amplify it until we changed the template. We took a random iGEM part we found in the fridge and tried the PCR on it and it worked. Conclusion: iGEM HQ should provide more information about their plasmid linearisation and how were the ends modified.

August 13th

2nd and 3rd Gibson Assembly. So we purified the assembly parts from previous PCR and did the calculations to maximise the amount of product. This reaction was also relatively simple as we reduced the number of fragments to 3 and it results in already circular plasmid.

So while we do the transformations, spending some usual late time in the labs, we also did a PCR using VF2 and VR primers. The gel confirms that reactions were successful. We can see the bands just above the 5kb mark,and as there is only about 130bp difference between these distinct mtr constructs it is impossible to see that on the gel. 1% agarose dyed with SYBR-Safe, 1kb ladder(Invitrogen)(on the right).

August 14th

So after having some sleep we head back to the lab and do the colony PCR on our transformations. But because Jonas is a very lazy person he used Phusion polymerase (which is as much as 4 times faster than GoTaq) and failed - there is nothing visible on the gel apart from ladder. It is not the first time this happens with Phusion colony PCR, so we try to avoid doing it, it appears though that Phusion requires relatively pure DNA template.

Nonetheless, we inoculate 2 cultures each of both mtr ORFs only and mtr+promoter constructs and grow them overnight. But it is not all for the day.

Also, today we got the new primers for the rest of the cloning, so we amplified the sequences of gold peptides and gold sensing device adding the rest of prefix and suffix. It all worked but the gel is really really hideous thus it will not appear up in here. We leave the rest for tomorrow.

August 15th

We extract the plasmids from overnight cultures using NucleoSpin Plasmid(Macherey-Nagel) and digest them with ThermoFisher's FastDigest enzymes:

Every triplet is: 1st the control, 2nd PstI digest and 3rd the EcoRI & SpeI digest. It is obvious that there is nothing near the size of 5kb visible. However, the 1kb fragment that is visible in double digest shows us that it is actually the template we used for pSB1C3 backbone amplification. 1% agarose dyed with SYBR-Safe, 1kb ladder(Invitrogen)(on the right).

So the solution is simple: we digest the Gibson reactions we had with DpnI(Promega) thus removing the background and then repeat the transformations.

At the same time we continued working with other sequences. Amplified gBlocks (peptides,the gold device) were purified with QIAquick kit, digested with EcoRI & PstI and then purified again using the same kit - this step frees our samples of small 2-6bp long fragments making the ligation easier. We also digest the linearised plasmid from iGEM HQ with the same enzymes and purify it as well. Even though this leads us to a very low concentration of pSB1C3, it is still enough for transformations as long as there are no problems with ligations. So we then make the reactions with T4 Ligase(Thermo Fisher) and proceed with the transformations.

August 16th

The day of GoTaq colony PCRs. So basically Gintare marched to the lab to do PCR of 44 colonies (and we are all aware of how annoying it is). Results were pretty straightforward with the peptides and gold sensing device parts. Two colonies each were cultivated overnight.

However, the results for mtr complexes were weird. So we picked a few cultures and grew them overnight for further analysis.

All gels were 1% agarose dyed with SYBR-Safe, 1kb ladder(Invitrogen)(on the right). We also noticed that something was not right with our gel viewing box as the images turned out to be very dark even at max exposure.

August 17th

Another busy day, so firstly about mtr complex. We purified the plasmids from yesterdays colonies using our usual NucleoSpin Plasmid kit and then digest them with FastDigest enzymes:

Triplet consists of: 1. Control 2. PstI digest 3. EcoRI & SpeI digest. First three triplets are of the mtr ORFs only construct and the last one (a lot of it!) is the one with promoter. Even though the gel is faint we can see the most important thing: double digests separate the pSB1C3(2kb) backbone and our part (5.3kb). The PstI digest also confirms the prohibited PstI cutting site within mtrC sequence (present in the genome). 1% agarose dyed with SYBR-Safe, 1kb ladder(Invitrogen)(on the right).

Using the primers we designed earlier we do the Site-Directed Mutagenesis on each of the mtr constructs, which should remove the PstI cutting site from where it should not be present but still retaining the aminoacid sequence. The protocol for which can be found here. We also finish it up with DpnI digestion to remove any background and then transform the cells.

In the meantime, we did similar to mtr digestions on the peptide parts we cloned earlier. Again on NucleoSpin purified plasmids with FastDigest enzymes.

Each plasmid was ran on the gel in triplets: 1. uncut control 2. XbaI & SpeI digest 3. EcoRI & PstI digest. The gel shows us that the peptides are actually there within the pSB1C3 with working prefixes and suffixes (there was only a slight nuisance with the 3rd peptide). 1% agarose gels dyed with either ethidium bromide(left) or SYBR-Safe(right), QStep4 ladder.

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The University of York

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