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Team:UC Chile/Cyano/Notepad/week11
From 2012.igem.org
May 14 - May 20
The following entry was written by: Simón
Wednesday
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)
IMPORTANT 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
