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Team:Warsaw/Calendar-Main/17 July 2009
From 2009.igem.org
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<h3><div style="text-align: center;">Assembly of endosomal detection operon</div></h3> | <h3><div style="text-align: center;">Assembly of endosomal detection operon</div></h3> | ||
<h4>Marcin</h4> | <h4>Marcin</h4> | ||
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<p>Task 1:</p> | <p>Task 1:</p> | ||
| + | <ul> | ||
| + | <li>Gel-out of digested biobricks</li> | ||
| + | </ul><br/> | ||
| + | <p>Methods</p><ul> | ||
| + | <li>After the digest samples were loaded into gel and electrophoretically separated</li><br/> | ||
| + | <p>Image of the digested biobricks:</p> | ||
| + | <center> | ||
| + | <img src="https://static.igem.org/mediawiki/2009/c/c2/Biobricks_digest_16_07_09.png" width="50%" heigth="50%"><center> | ||
| + | <p><div style="text-align: center;">verification of the effectiveness of the restriction digest</div><p> | ||
| + | <ul> | ||
| + | <li>Fragments of agarose gel were carefully cut out and in the next step DNA was extracted from the gel using the A&A gel-out kit. Detailed procedure is described <a href="http://aabiot.com/products/dna_purification/dna_fragments/gel_out/protocol_gel_out.pdf">here</a></li> | ||
| + | <li>Quantification of amount of p53 DNA after restriction digest:</li> | ||
| + | <ul> | ||
| + | <p>1 μl of the digest mixture was diluted to 10 μl and loaded into the gel.</p> | ||
| + | <br/> | ||
| + | <center> | ||
| + | <p>Task 2:</p> | ||
<ul> | <ul> | ||
<li>Ligations of biobricks</li> | <li>Ligations of biobricks</li> | ||
Revision as of 04:32, 28 July 2009
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Assembly of endosomal detection operon
Marcin
Task 1:
- Gel-out of digested biobricks
Methods
- After the digest samples were loaded into gel and electrophoretically separated
- Fragments of agarose gel were carefully cut out and in the next step DNA was extracted from the gel using the A&A gel-out kit. Detailed procedure is described here
- Quantification of amount of p53 DNA after restriction digest:
- Ligations of biobricks
- BBa_B0032+BBa_c0040
- BBa_B0032+BBa_C0051
- BBa_B0032+BBa_E0032
- BBa_B0032 (ligation of empty vector as a negative control of the experiment)
- Ligation mixture composition: 3 μl digested plasmid with RBS, 3 μl digested CDS, 5 μl ligation buffer (Fermentas, PEG4000 have been added previously), 1 μl ligase T4, 13 μl MQ water
- Negative control mixture composition: 3 μl digested plasmid with RBS, 5 μl ligation buffer (Fermentas, PEG4000 have been added previously), 1 μl ligase T4, 16 μl MQ water
- Duration of ligation was about 12 hours
- Transformation of chemocompetent E. coli strain DH5α
- Ligation reaction was stopped via thermal inactivation in 80°C for 20 minutes
- Detailed protocol of ligation is described here. The only modification is total volume of ligation mixture prepared 16.07.09
- petri dish were hold in 37°C for 36 hours
- Isolation and evaluation of pKS/p53 plasmid
- Prepare LB medium with kanamycin
- Add 3.5 ml of the medium to the probes
- Add one bacterial colony to each probe
- Breed the bacteria about 8 hours
- Samples of ligated[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032 - RBS.3] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor] on the pSB1A2 ampicillin resistant plasmid cut with EcoRI and PstI were run on the gel.
- Seting new liquid cultures to obtain of pure [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] construct
- 2 test tubes with 5ml LB and ampicillin were inoculated with colonies containing [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] on [http://partsregistry.org/Part:pSB1A2 pSB1A2] plasmid. The cultures were left for overnight incubation at 37°C. This time cultures were taken from sparse plate
- Will be determined next day by cell presence in tubes
- Transformant selection
- White colonies that appeared after 48h of incubation were picked up and transferred to a new petri dish.
- Liquid cultures were established along the way for plasmid purification.
- Both the dish and the liquid cultures were incubated at 37°C overnight.
- All of the newly transferred colonies turned blue overnight as well and not suprisingly the purified plasmids yealded no mgtc promoter.
- It is possible that the X-gal used is not of top quality which may result in the late colorisation of the colonies.
- It is also possible that the overwhelming autoligation rate is due to the fact that the SmaI enzyme has only 50% activity in 37°C, a fact that was not taken into account previously.
Image of the digested biobricks:
verification of the effectiveness of the restriction digest
1 μl of the digest mixture was diluted to 10 μl and loaded into the gel.
Task 2:
Comment:
Due to obtain set of biobricks which each of them contain RBS and particular coding sequence I designed the following elemets (the order of biobricks is from the 5'end to 3' end):
Methods:
Task 2:
Methods:
Cloning of p53 coding sequence
Marcin
Task:
Comment:
Besides high effectivity of transformation all of the bacterial colonies were blue. It is possible that excess of X-Gal may cause some problems with the selection system. Due to this uncentainty I decide to prepare a few bacterial breedings to isolate the plasmids and digest them.
Methods:
Construction of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 K177012] operon1_part2
Franek
Jarek
Jarek and Franek run the gelAnia
only described it. Thank you guys:)Tasks:
Results: Obtain results confirm ligation of the http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032 - RBS.3] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor], the undigested plasmid is visible on the gel. It is probably due to incorrect digestion buffer concentration.
Testing different E. coli strains regarding [http://partsregistry.org/Part:BBa_R0010 lacI] and [http://partsregistry.org/Part:BBa_R0080 AraC] repressors
Franek
Tasks:
Methods:
Results:
Cloning of the mgtc promoter into the pKSII+ plasmid
Kamil
Tasks:
Methods:
Results:
Conclusions:
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