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Team:BGU Israel/Achievements
From 2013.igem.org
Biobricks
The following biobricks were designed, sent to the registry and characterized:
- Bba_K1223002 – P.A.S.E 2 cassette
- Bba_K1223003 - KanR (promoter+CDS)
- Bba_K1223005 - cI translational unit
- Bba_K1223006 - HisTag + stop codon
- Bba_K1223007 - cI translational unit with His-tag
- Bba_K1223011 - ampR translational unit (ampicilin resistance CDS+promoter)
The following biobricks were designed, characterized, and we currently work on sending them to the registry:
- Bba_K1223001 – P.A.S.E 1 cassette
- Bba_K1223013 - Pyrolysyl-tRNA synthetase CDS
- Bba_K1223014 - tRNA-Pyl (pylT) gene from Methanosarcina barkeri str. Fusaro
In addition, we work on removing illegal restriction sites from pkd46 functional unit (Bba_K1223014). The biobrick itself works and was characterized.
P.A.S.E 1
- The system’s parts have been designed and synthesized.
- 2 out of 3 of the system’s parts have been assembled in e. coli – pkd78 (recombination system) and pUC57 cI (protective element)
- Since we haven’t succeeded yet in proving the recombination system works, an alternative design was conceived for purposes of proof of concept, relying on the same principles as the original design. Instead of using a linear DNA with recombination sites for the toxin cassette, we used a plasmid as a cloning vector. The completed system was assembled and it is comprised of an e. coli transformed with pUC57 cI and pUC57 Toxin (toxin regulated by cI binding promoter). Characterizing the system is undergoing.
- Characterization of P.A.S.E 1 cassette and collection of evidence regarding its lethal activity in the cells.
- Characterizing the expression of cI repressor protein under the regulation of Lac/Ara-1 IPTG inducible promoter.
- Construction of cI + his tag BioBrick.
P.A.S.E 2
- The system’s parts have been designed and synthesized.
- A recombination system (pkd78) with different antibiotic resistance have been constructed (we replaced the chloramphenicol resistance of pkd78 with carbenicillin resistance).
- Site specific incorporation of Unnatural amino acids (UAA).
- pKD + UAA machinery Transformed into BL-21 E. coli.
- Creation of models of expected protein concentrations.
Attributions
All of the experimental work described here was performed only by the team, so as all the designing and assembly of the Bio Bricks. Prof. Lital Alfonta from the Avram and Stella Goldstein-Goren Department of Biotechnology Engineering of Ben Gurion University provided the laboratories we worked in this summer.
Collaboration
Paris_Bettencourt Paris_Bettencourt team has sent us one of their parts for characterization: BBa_K1137008. This part codes for the enzyme TDMH, which degrades mycobacterial cell wall. The enzyme has a his tag, so we would use Western blot to characterize its expression in BL21 over time – after 1, 2, 3 and 6 hours from induction with IPTG. Additionally, a negative control (an uninduced culture) will also be used for the blotting. We sent their team our part Bba_K1223005, so they could characterize the lac/ara-1 promoter we use in this construct.
Marburg The sunlight-driven microalgae Phaeodactylum tricornutum has been established for the production of complex proteins. Marburg team from Germany are developing a system in the P. tricornutum which secretes the proteins to the medium after they are produced. This system provides a much more efficient and simpler way to purify recombinant proteins. They tested their system with the production and secretion of Hepatitis B antibody which was placed under the control of a nitrate inducible promoter (BBa_K1071004). Since our instructors have experience with yeast we offered to check if the Nitrate Reductase (NR) promoter works in yeast. If so, we would characterize it by checking how much nitrate and ammonia must exist in the medium to work properly. This collaboration was offered in order to examine the potential of their unique system in yeasts. We received a GFP reporter gene under the expression of the NR promoter. We intended to grow the yeast (post plasmid transformation and under selection conditions) in the presence and in the absence of different concentrations of nitrate compounds. The reporter gene would then be examined by using flow cytometry (FACS).
Tuebingen Excreted progestin are not eliminated from purified water in sewage treatment plants and therefore the progestin-contaminated purified water mixes with river-water and adversely affects the fish. Tuebingen team from Germany are developing a progestin measurement system based on yeast to function as a biosensor to detect the concentrations of progestin in the river. Since we had access to yeast and we take environmental issues very seriously, we offered our help to characterize their regulated FET3 promoter. The FET3 promoter is an iron regulated promoter of S. cerevisiae which is repressed by vertebrate progesterone receptors. It was sent to us on the pTUM100 backbone (BBa_K801000) with the URA gene for selection in yeast and mOrange as the reporter gene. We intended to transform the plasmid into our EDY100 yeast and grow them in standard SC-URA medium. FET3 should be usually repressed in the presence of iron and becomes active in a low iron environment . Therefore are intention was to grow them in different mediums containing various concentrations of iron and examine the activity of the promoter using FACS at 550nm which detects the mOrange expression.
