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The BUGSS team has been working to cultivate a strain of E. Coli that produces metallothionein and RFP in the presence of heavy metals like lead. Adequate production of metallothionein protein can sequester lead in order to make the metal non-bioavailable. Again, the BUGSS team is building on earlier work on lead sequestration,utilizing some parts from the iGEM registry while producing and submitting new and composite parts. Sequences were found and edited for metallothionein production (BmtA), and the PbrR regulator. PpbrA promoters were connected to RFP and Bmta genes in order to regulate metallothienein and RFP production, inducing their generation in the presence of lead. In order for this new construct to work, the E. Coli's natural heavy metal resistance gene, Znta, must be deactivated. The team is utilizing a one step gene inactivation protocol using phage lambda red recombinase to recombine the gene with a kanamycin resistance insert.
March 20, 2015
Gel run on March 14th suggested that the reactions had too much template. Ran the following controls with less template:
Plan for next week: PCR of znta knockout using pkd20 linear plasmid.
Ran gel on colony PCR. Results inconclusive. Lacked a positive control.
Performed Colony PCR on Part 69 Transformants
Each tube used 5μL of DNA in 100μL of cells.
To do next: Check transformants and colony PCR screening.
To do: Transformations!
Filename: PSB1C3_Verifications_2_02_15 (in Maurice folder)
January 28, 2015
Past: #71 – verified by PCR for size. We have vector with #71
Vector PSB1C3 25ug/ml (green 1.5ml eppendorf, IGEM box)
1h at 37C. Then frozen in Maurice's box (-20C).
To do: Verify PSB1C3 with electrophoresis. Ligate 3 remaining parts cut with Xba1 and Spe1.
PCR done with Positive (F2620 1299bp) and Negative Control (Template DNA)
Performed plasmid miniprep on cmr transformant. A260: 0.072 = 3.6Ξμg/mL A260/280=1.6
Used 3 plates of commercial competent cells and 3 plates of lab-prepared (from CAT119) competent cells.
November 5, 2014
BB-RBS-BmtA-tt-BB, Labeled 68
All reactions left overnight.
Using igem protocol: http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones
To do: Grow cells with PSB1C3 positive control and cut out RFP fragment by digestion and gel isolation.