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Team:UC Chile/Cyano/Notepad
From 2012.igem.org
In this page we shall write what is being done and what remains to be done. <- (yeah, right)
Ok, let's start by saying that the other guy (the one in charge of bactomithril section) is the serious one. I see myself more like a sort of crazy engineer type of person (thereby my code name is crazyengineer42). While reading the following pages you'll probably stumble on things that don't make much sense and many references to star wars, x files, lost and García Márquez. Oh, and dragons, there's gonna be lots of dragons. But don't despair, some ideas are intelligible if you make the effort to think as I think. If it's impossible for you to do so, well, at least I hope you have some fun (otherwise it may be better for you to stick with bactomithril section and never come back here!).
I make up for my lack of artistic style with entertaining writing, funny pics and unnecessary smilies.
Tasks
Week 1
Week 2
Week 6
Week 7
Week 8
Week 9
Week 10
Week 1
Check out our constructs!!!
Week 2
Hey, it's just me at SJ library.
Monday: weekly meeting. We came up with three solutions for the sustentability problem. The formal proposal must be ready next Tuesday afternoon. Also, we designed primers for the integration biobrick experiment.
Tuesday: we prepared synechocystis culture media and lots of other pretty things :) I ate ice cream and pizza for example (seriously, need some help here!).
Wednesday: I am supposed to be studying proba instead of doing this. Can't concentrate though, too boring for me still.
Week 6
Sat 04.14
Horrible day. Nothing worked.
The one good thing:
FLYIIIIIIIIIIIIIIIIIIIIIIIIIIIIIING PIIIIIIIIIIIIIIIIIIIIIIIIIIIIG!!!!!!!!!!!!!!!!!!!!!!
Week 7
Tuesday 04.17
We purified parts for Construct 1 and then assembled them by Gibson. Hope everythings works out. Simon was wearing his Gibson Les Paul T-shirt so we kinda hope that the power of rock leads us through the good side of the force.
Also, have you ever seen a centrifuge dance? It's pretty funny actually.In one step of the purification, the protocol stated "centrifuge with lid open"
So we did...and it danced...
Pic of the day
Thursday 04.19
Construct 1 is ready!!!
Conclusion: absolutely nothing is more powerful than rock!
Friday 04.20
Seven a.m., waking up in the morning. Gotta be fresh, gotta go downstairs...
hahaha i'm just messing with your head
Sat 04.21
Started preparing parts for Construct 2 by PCR.
Here is Simon heroically diminishing his life span for the success of Construct2.
Although he actually seems to be enjoying it...
To do:
Assemble C2.1 and C2.2. Characterization of psAB and psbA2 promoters.
Week 8
Tuesday 04.24
Did a restriction digest of construct 1 to check if it is really what we wanted to assemble. We believe it's not. We are strongly considering to start work from scratch. (Order primers again and so on).
Simon confessed that he didn't like classic rock, Bernardo tried to fix his headphones with a soldering gun and Carla quit Proba.
Getting ready for Ingenia fair at engineering faculty tomorrow!!!
Wednesday 04.25
Spent the morning talking about our project at engineering faculty. At least 30 people are interested in participating next year. For details check out our human practices section.
Today is Carla's birthday. In the spirit of celebration, we ordered pizza-cake and she could blow out her candles. Happy 20 years!
Finally, run 42 PCR's to check if the parts assembled by gibson were correct. We included the digested contructs. Waiting for tomorrow's results.
Thursday 04.26
Weird things are happening. The desired parts have wrong weights so clearly they are not what we want. The constructs don't have all the parts. Some have two or three and none is complete.
Second day at Ingenia fair.
Had a few gourmet drinks to cheer up the team!
Friday 04.27
Did one last PCR run with parts that correctly assembled. We are going to build our desired construct from the good parts of the former ones. Frankenson is on! (Frankenstein Gibson).
First rainny day of the season! Brace yourselves, winter is coming.
Saturday 04.28
Checked the PCR results and they are not what we expected. Parts that supposedly were together are not in the right weight. Still, we did the corresponding Frankenson.
Week 9
Monday 04.30
Did a massive PCR run. We amplified every part of Construct 1 using as template old PCR purifications and PCR products. We'll build C1 from scratch. Hoping everything works out well this time.
As an inspirational motif:
Muchos años después, al frente del pelotón de fusilamiento, el coronel Aureliano Buendía había de recordar aquella remota tarde en que su padre lo llevó a conocer el hielo.
Tuesday 05.01
We decided to transform e. coli with the expression plasmid pPMQAK1. Also, as the mentioned plasmid contains ccdb toxin, we took it out by a digestion/ligation procedure and transformed again. Transformed e. coli with GFP coding device.
During the afternoon, we checked yesterday's PCR's under UV light. Luckily, all parts amplified except for KanR u.u Did a PCR run for KanR alone. We tried with different master mix combinations, some containing GC buffer and some with DMSO.
Existential Question: WHAT DO YOU DO WHEN YOU HAVE A GREMLIN PLAGUE IN THE LABORATORY??
Got it!! The next protocol can be used,
Protocol to get rid of gremlins: http://christalawler.com/2010/06/05/bits-how-to-kill-a-gremlin-as-seen-in-gremlins/
- The previous protocol needs to be adapted to laboratory conditions!!!
Wednesday 05.02
Talked with one of our advisors today. He gave us interesting input on our failed PCR's.
Checked PCR runs. KanR didn't have the strenght we'd have liked, but still we used it. All parts were assembled by Gibson. Also, every part has its neg. control. After Gibson, we transformed coli in kan, chlor and both resistance plates.
gel image
Friday 05.04
Happy Star Wars day!!!! May the fourth be with you :)
Week 10
Monday 05.07
Well... we believe we have just found the root of all evil: our Gibson master mix. Its efficiency is way lower compared to the master mix used during the introductory course. So, we made new master mix, new competent colis (we ran out of them), tried gibson with new mix and transformed. Hope it works this time!!!!!!
Tuesday 05.08
NEW PRIMERS IN DA HOUSE!!!! Made PCR runs for the parts of the new constructs we are going to assemble (with the new primers). Also, digested pPMQAK1 to get rid of the incorporated toxin. We plan to transform coli with new constructs, pPMQAK1 and digested pPMQAK1.
Had another pizza orgy!!! Started making plans to buy our own mountain (apparently you can do that in this country u.u).
Finished last PCR run at 23.00 (new record).
Wednesday 05.09
- tried protocol to ged rid of gremlins in the lab*
AHHHHHH POR QUÉ? NOOOOOO MALDITOS GELES (poem by B. Pollak)
Thursday 05.10
Reinoculated synechocystis. Getting our bacteria ready for tomorrow's transformation :) Minipreped lux brick, digested pPMQAK1 and pPMQAK1. Transformed future useful parts from the registry. We have not yet transformed tuesday's PCR u.u Also, compared (again) old master mixes, failed master mix and fresh master mix with a Gibson assembly for sfGFP.
Finished with a measurement of competence for our e. colis using pSB1C3.
Week 11
In the last 2 weeks we have been working in a plasmid construction strategy in parallel to that of Gibson Assembly, This strategy has been based in cloning reporter biobricks (hereafter refered as BBs) to ligate in to pPMQAK1 -our E.coli-Synechocystis expression plasmid- by standard biobrick assembly. We followed this protocol http:[http://ginkgobioworks.com/support/]
Strategy´s main goals are: 1) getting reporter constructs lacking only the promoters, in order to make further caracterizations of Synechocystis promoters (wich we allready get from the registry) and 2) Test in synechocystis some of the so called "constitutive promoter family members" designed to E.coli(BB_J23100 - BB_J23119).
So, after cloning and minipreping the BB´s plasmids, we digested all of them, including pPMQAK1 with Ecor1 (hereafter refered as "E") and Pst1 (hereafter refered as "P") As the destination plasmid pPMQAK1 has the ccdB gene(gyrase toxin, lethal to the cells) flanked by the restriction sites of E and P, after the ligation the transformants were just selected with one antibiotic resistance encoded in the destination plasmid (AmpR or KanR) and not in the BB original plasmid. Some BBs, nevertheless, have both resistance genes, in those cases after de digestion we had to make a gel electrophoresis and then purify the BB band. It is worth to remember that when you use E and P to digest both the BB and the destination plasmid, during the ligation the chances of these molecules to reassemble as they were previous to the digestion are very similar to those of the BB beeing ligated in the destination plasmid.
OK, sorry for that boring introduction, now the RESULTS:
Digestions made: all with E and P, and with the name "digN°". Original plasmid resistance in bold
dig1: pPMQAK1 : broad host plasmid, kanR and ampR, ccdB toxin between prefix and suffix
C dig2: K398500 : constitutive promoter + rbs + GFP + double.terminator
A dig3: LuxBrick : check [http://partsregistry.org/Part:BBa_K325909]
A dig4: K216008 : rbs + LuxA + rbs + LuxB
A+K dig5: K081012 : rbs + GFP + terminator *
A+K dig6: K081014 : rbs + GFP + terminator *
K dig7: I20261 : constitutive promoter + rbs + GFP + terminator
K dig8: I20270 : constitutive promoter + rbs + GFP + terminator
A dig9: E0430 : rbs + YFP + double.terminator.
* as these had the same resistance casettes as pPMQAK1, a gel purification was performed.
Ligations made: Plates antibiotic selection in bold
K lig1 = dig1+dig2
K lig2 = dig1+dig3
K lig3 = dig1+dig4
A lig4 = dig1+dig7
K lig5 = dig1+dig8
K lig6 = dig1+dig5
K lig7 = dig1+dig6
K lig8 = dig1+dig9
Transformations:
All transformations were succesful exept those with lig4 and lig5. Picked colonies are growing in the shaker right now.
THINGS TO DO:
1) Altough lig4 and lig5 are "synonyms" with lig 1, we should have more reporters under E.coli constitutive promoters.Some strategies are 1)repeat without changing anything (yes, sadly that works sometimes) 2) try another "synonyms" BBs from the distribution kits and 3) repeat dig7 and dig8 but now purify the band, that excludes the possibility of the BB plasmid being re-ligated so it should yield more effective ligations.
2)Miniprep the cultures with the cloned ligations
3)Make analytical digestions of the purified plasmids
4)Check if the cells transformed with lig1 are expressing GFP as expected
5)Plate the cells containing the requested BBs and purify the DNA
6)Ligate the requested BBs with the reporter constructs (lig3,6,7 and 8)
TUESDAY
We recieved the following biobricks from the registry: (all of them are synechocystis pcc6803 promoters with native rbs) in agar-embebed bacteria, they were plated and colonies were selected
BBa_K390049 psB1C3
BBa_K390013 psB1C3
BBa_K390014 psB1C3
BBa_K390015 psB1C3
BBa_K390016 psB1C3
BBa_K390017 psB1C3
BBa_K390018 psB1C3
BBa_K390019 psB1C3
BBa_K390021 psB1C3
BBa_K390022 psB1C3
BBa_K390023 psB1C3
BBa_K390024 psB1C3
BBa_K390025 psB1C3
BBa_K390026 psB1C3
BBa_K390027 psB1C3
BBa_K390028 psB1C3
BBa_K390029 psB1C3
Competent cells were transformed with the following BBs from the 2012 distribution kit
Plasmid resistance in bold
C K515105 = constitutive promoter + rbs + sfGFP
A E0422 = rbs + CFP.lva.tag + term
A K145015 = GFP.lva.tag
A J04630 = GFP + double.term
A E0432 = rbs + YFP.lva.tag + double.term
A I763020 = rbs + GFP.lva.tag + doulbe.term
C K325902 = rbs + luxC + rbs + luxD
C K325903 = rbs + luxE + RBS + luxG
Transformations with K325902 & K325903 weren´t succesful.
