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Team:BostonU/Results
From 2012.igem.org
Results Summary
- 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.
Converting Biobrick to Moclo Parts:
- 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.
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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.
Creating Level 1 Moclo Parts
- We combined level 0 promoter, RBS, gene and terminator in a one pot reaction. Which then we transformed, then screened for white colonies on IPTG/X-Gal plates.
- We checked the level 1s through a double digest and checking it on a gel. The gel below shows bands at 2 kb to be the destination vector and the smaller bands at 75~800 bp indicating the presence of the transcriptional unit.
- Lastly, we sequenced the level 1 parts, but the results yielded sequences of only the destination vector and not the transcriptional unit. We are currently trouble shooting and we plan to present the results at the Jamboree.
- We combined level 0 promoter, RBS, gene and terminator in a one pot reaction. Which then we transformed, then screened for white colonies on IPTG/X-Gal plates.
Creating Moclo Level 0 Parts
Basic Genetic Circuits
Things to include: SBOL figures of circuits, gels, sequence data, plate pictures, characterization data summary
Below are the step by step results we obtained in making all of our destination vectors:
