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Team:BostonU/Results
From 2012.igem.org
Results Summary
Below are the step by step results we obtained in making all of our destination vectors:
We first transformed the destination vectors into cells on to plates with IPTG/X-Gal for blue white screening. In this case, we wanted to see blue cells, indicative of the presence of LacZ. Our destination vectors also varied in whether the backbone is phosphorylated or dephosphorylated. In this picture, the dephosphorylated showed higher transformation efficiency by preventing recircularization of the backbone, yielding more cells transformed with the LacZ as part of the destination vector.
We checked whether the destination vectors contained the LacZ fragment amplified with the moclo fusion sites by running digest of cut vs uncut. In the gel picture, each SM refers to a different destination vector and the C and UC means cut and uncut, respectively.
The final step is sequencing the destination vectors to confirm the correct orientation of LacZ. In the nucleotide sequence file and the complementary diagram, the LacZ sequence, 2 fusion sites and restriction sites are highlighted in different colors.
The trace file shows confidence of the sequencing.
We first added fusion sites to Biobrick parts through PCR. The primers contained the sequence for the fusion sites as part its overhang. In the process we used regular PCR amplification and also ligation PCR.
The first picture refers to promoters R0010 and R0079 with the added fusion sites
The second picture is the result of ligation PCR of the J series promoters.
Afterwards we transformed and plated cells with level 0 Moclo parts on to IPTG/X-Gal plates for blue white screening where we look for white colonies. In the picture we see white colonies, indicating the LacZ was cut out of the destination vector and replaced by parts with the Moclo fusion sites.
We confirmed the Moclo level 0 parts through sequencing. Below there is an example of the sequence file we received for a promoter, RBS, gene and terminator. The files were edited with highlights to indicate the correct locations of the part, fusion sites and the restriction sites.
This is a work in progress. We are currently working on the MoClo Levels 1 reactions and unfortunately we couldn`t get them ready before the Wiki freeze.
