Assembly of endosomal detection operon

Marcin

Task 1:

• Ligations of biobricks

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):

1. BBa_B0032+BBa_c0040
2. BBa_B0032+BBa_C0051
3. BBa_B0032+BBa_E0032
4. BBa_B0032 (ligation of empty vector as a negative control of the experiment)

Methods:

• 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

Task 2:

• Transformation of chemocompetent E. coli strain DH5α

Methods:

• 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

Cloning of p53 coding sequence

Marcin

Task:

• Isolation and evaluation of pKS/p53 plasmid

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:

1. Prepare LB medium with kanamycin
2. Add 3.5 ml of the medium to the probes
3. Add one bacterial colony to each probe
4. Breed the bacteria about 8 hours

Construction of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 K177012] operon1_part2

Franek

Jarek

Ania

Tasks:

• 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.

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:

• Seting new liquid cultures to obtain of pure [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] construct

Methods:

• 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

Results:

• Will be determined next day by cell presence in tubes

Cloning of the mgtc promoter into the pKSII+ plasmid

Kamil

Tasks:

• Transformant selection

Methods:

• 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.

Results:

• All of the newly transferred colonies turned blue overnight as well and not suprisingly the purified plasmids yealded no mgtc promoter.

Conclusions:

• 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.