Team:Calgary/Lab/Blog

Every Wednesday (started at the end of June), our lab team uses our Synthetic BLOGology blog to update both the public and the rest of the team of their progress in the lab project. For more details regarding Synthetic BLOGology, please click HERE.

-July
-August

These updates have been posted with newest ones at the top.

Hi everyone, it’s Carol again! I won’t re-introduce myself again since I wrote the modelling blog a few days ago. I don’t have much to report since I’ve been having bad luck in the lab lately, actually from the start! I am working with Kevin (the nice individual) to construct the reporter circuit for the project; however, due to my lack of lab skills, I’m delaying the whole project and leaving Kevin with nothing to do! I’m just kidding. I spent a few weeks trying to concentrate DNA plasmids for LuxCDABE sequence in topo vector. After many failed maxi-prep and many mini-preps (with the help of my favourite lab equipment, the vacifuge), I was able to concentrate my sample. As well, before Biobrick construction, I was left with a difficult task of single site mutagenesis. For some reason (with my luck) after one trial I was able to mutate a specific site within the LuxCDABE gene. This past week, I was unable to successfully clone and transform the LuxCDABE into the Biobrick vector. The lab team are trying to think of other ‘innovative’ ways to get this large gene cloned into Biobrick vector. Hopefully, next week, I’ll be able to show some better results, as of now, only time will tell! Talk to you guys on Monday!

I, Kevin Shin, being a nice individual, am also involved in wet lab part of our team. Carol and I are responsible for our Reporter circuit, which involves Qrr4 promoter and LuxCDABE gene. As of now, I have completed verifying and glycerol stocking last year’s Pqrr4 part, and am waiting for LuxCDABE part (which is much longer, meaning harder to work with) to be done.

While waiting, I am conducting an exciting side project involving a variety of FLUORESCENT proteins!!(and dry ice...) Woot! Although I am really disappointed at not being able to gain access to purple, orange, and blue fluorescent proteins, we have secured a supply of red, green, yellow, and cyan for me to have fun with. Today, I will be transforming plasmids with these genes in them and by tomorrow, I should be able to draw some bacterial pictures! We will keep you posted on those glowing masterpieces.

Hi, I'm Emily and I’m going into second year Biomedical Sciences. When I’m not fighting with my LuxOD47E gene, I’m usually highland dancing or playing the Oboe. In the lab, I’m in charge of the mutant LuxOD47E circuit This week I am excited to say that sequencing has confirmed that I've finally biobricked my gene of interest, LuxOD47E. Yay! This has taken a lot longer than anticipated due to several negative control contamination issues (negative controls are not my friends) and a battle with strange reappearing bands in restriction digest and PCR products. Nevertheless, this gene is now biobricked and it is on to the construction of my circuit! This week I will be trying to get the J13002 promoter in front of LuxOD47E as well as the BOO15 terminator behind. I’ll be doing this through restriction digest with EcoRI, XbaI and SpeI followed by an awful lot of verification. Maybe, just maybe I’ll see some clean negative controls! If this is successful, my circuit should be completed and sequenced by Monday!

My name is Jeremy Kubik and I am part of iGEM Calgary’s wet lab and marketing team. This is my first time participating in iGEM and I am having a great time working with the other students on this awesome team. Outside of the lab, I enjoy competitive sports, all the way from swimming with the Varsity team at the U of C to ripping it up on the streets of Calgary for a little hockey action. I also enjoy long walks on the beach, sunrises and fine wines.

Within the lab, I am taking care of part of the signaling circuit. Our overall project is to create a Quorum Sensing (QS) system with the signaling molecule autoinducer-II (AI-2) as the input to the system. My collaboration with Jamie will eventually lead to the construction of a circuit with the genes LuxPQ and LuxOU, all of which code for important proteins with respect to the transmitting the AI-2 to induce a specified response (which was recently decided to be a protein output that degrades biofilms!). So where am I as of today? Stuck. Well, not really stuck, but I have had some difficulties with LuxPQ. The part is verified to be in the TOPO vector and to have a length of 3.8kb, and I have had quite some trouble simply cloning LuxPQ into a BioBrick Vector (psB1AC3). I performed a construction last week and finally got some colonies, of which I ran a colony PCR, isolated plasmid, and have sent plasmids of two colonies down to sequencing. I will find out in a few hours whether LuxPQ has been successfully cloned into the BioBrick vector. If it is, I can begin construction with Jamie’s circuit (LuxOU with promoter and terminators). If it has not been successfully cloned, it is back to the drawing board: trying different conditions to transfer LuxPQ from TOPO vector into a BioBrick vector.

So I decided to be lazy and outdo everyone on my team by doing some snazzy Web 2.0 related thing: VIDEO BLOGGING!!!!! Amazing stuff.

One downfall of video blogging is it is HARD to edit stuff out. :( And after reading everyone else's blog post and realizing I forgot to introduce myself OUTSIDE of iGEM (do I even exist???) and being to lazy to re-record anything I will include the following disclosure:

Jamie enjoys doing iGEM from 9-5 Monday to Fridays. Sometime 10-6, dependent on how late I sleep in :D I will include my extracurriculars next week (I will have had enough time by then to think of at least two extracurriculars so I do not look like a complete loser). Anyways, enjoy the video.

Hi! You heard from me last week in my third-person commentary and now I’m back in the first-person flesh. My name is Vicki and I recently graduated from the Engineering Science program at the University of Toronto, with a major in biomedical engineering. I grew up in Calgary and am very excited to help my hometown kick some synthetic biology butt. When I am not indulging in the satisfaction that only a properly-sequenced synthetic BioBricked circuit can provide, I am usually either swimming, skating, biking, learning German or a combination of any of the above (ever try riding a bicycle whilst on skates? It’s as perilous as it sounds!).

Over the last few weeks, I have been deeply entrenched in converting a mutant protein sequence in a TOPO plasmid into a fully functional and biobricked sequence in a pSB1AK3 plasmid, complete with the appropriate and properly-integrated promoter, RBS and terminator sequences. Indeed, it has been a most gruelling month of gradient PCRs, colony PCRs, plasmid isolations, restriction digests, ligations and – craziest of all – understanding what I’m doing and explaining it to the lab group in coherent sentences! Because even though the principles of synthetic biology and biobricking are supposed to make genetic engineering so easy that even an engineer like me can work with it, it’s really quite challenging when you cannot see what is really happening in terms of molecular interactions on the nano-scale and smaller. Although many parts of the project have flummoxed me, I am developing a better appreciation of what I am doing as I gain more experience in the lab.

So, here is my work to date.

Battle 1: Vicki vs gradient PCR of LuxOD47A in TOPO. The purpose of this step was to use biobrick gene-specific primers to make copies of a biobricked version of LuxOD47A.
Winner: Vicki

Battle 2: Vicki vs ligation of LuxOD47A into the pSB1AC3 plasmid. This is so that the gene sequence can be integrated into competent TOP 10 bacteria. Prefix-end cuts were made with EcoRI and XbaI (in separate samples).
Winner (VK vs EcoRI cuts): EcoRI cuts L. The restriction digests of these look like someone painted white-out in the 12 kb range just to spite me.
Winner (VK vs XbaI cuts): Vicki. The EcoRI enzyme sample that wasn’t up to par is spending its days in solitary confinement at room temperature. Indeed, things are so much cleaner when enzymes decide to cooperate.

Battle 3: Vicki vs the sequencing machine, part I. After a successful restriction digest of the XbaI-cut samples, I sent them down for confirmation that my PCR and restriction digest results weren’t just lying to me.
Winner: Vicki

Battle 4: Vicki vs integration of promoter and terminator sequences, attempt I. I tried both to see which would work best.
Winner (VK vs J13002 promoter): J13002 promoter L. That one didn’t integrate in any of the colonies that I PCR’d and RD’d
Winner (VK vs B0015 terminator): Vicki. We’ll move forward with a newly-made LuxOD47A-B0015 construct, while the attempted J13002-LuxOD47A samples can go roast in the autoclave.

Battle 5: Vicki vs the sequencing machine, part II. Yea, I know I skipped a lot of steps here. It wouldn’t tell you anything interesting beyond what you’ve already read. After good restriction digests and colony PCR results that would have been fine if not for a superfluous negative control band that didn’t show up anywhere else, we sequenced anyway. We had a scare when a simple [Crtl+F _ (B0015 sequence)] didn’t yield any results on the text file, but were reassured when we blasted the sequences against each other as that algorithm accounts for the reverse sequence given by the reverse primers that I used.
Winner: Vicki, with credit to BLAST

Battle 6: Vicki vs the integration of the nefarious promoter sequence. Lest the J13002 samples go the same way as our uncooperative EcoRI enzyme, we decided to attempt this integration thing one last time. Two colonies behaved appropriately, and the J-part lived to see another day.
Winner: Vicki

Battle 7: Vicki vs the sequencing machine, part III. Although I had to wait four days for my results, they were what we expected.
Winner: Vicki

This takes us to now. I need to make more of the biobricked sequence in question by letting it grow in bacteria, which I’ll do later today so that it can grow overnight. Results to come!

The last few days have been flavoured by a plethora of successes, not-quite-successes, and other incidences of interest and amusement.

THE GOOD: Jamie has usurped the throne of AI-2 Circuitland. With a complete and properly-sequenced LuxOU component of the circuit, complete with the necessary promoter and terminator sequences, Jamie gets to sit back, chillax and admire his work as he helps the rest of the lab group complete their work. Congrats, Jamie, and B0015-R0040-LuxOU-B0015 FTW!

THE BAD: Negative controls FTL! Emily and Vicki have both been plagued by unsightly bands in the negative control lane, despite using new and purportedly uncontaminated equipment in their PCRs and restriction digests every time. Of particular bamboozlement, Vicki’s negative control in her latest colony PCR had a band at 1kb that did not appear anywhere else in the gel. Nevertheless, the two will continue to move forward with their experiments. The sequencing results of Vicki’s LuxOD47A BBk circuit showed that she does indeed have the proper product present. And Emily is set to conquer the colony PCR, so hopefully we’ll see the awesome results of that at the end of the day.

THE UGLY: Vicki spat into a K-laced plate and placed it in the incubator to see what would happen. She was most disappointed to see that the plate was cleaner when she returned than when she first left it there. She’ll try again with a plate free of antibiotics next week. Results to come!