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Team:BGU Israel/Bricks
From 2013.igem.org
1. Introduction
Here is a collection of all the parts we have designed and constructed over the course of our project. Not only did we assemble existing parts in order to create more complex Bio bricks, but we created new parts and improved existing ones. Details of each part can be found in the Registry by a click on the Bio Brick in the following table.
| Part Number | Part Name | Type |
|---|---|---|
| Bba_K1223001 | P.A.S.E1 cassette | Project |
| Bba_K1223002 | P.A.S.E2 cassette | Project |
| Bba_K1223003 | KanR (promoter+CDS) | Coding |
| Bba_K1223004 | Lambd cI CDS | Coding |
| Bba_K1223005 | cI translational unit | Translational Unit |
| Bba_K1223006 | HisTag + stop codon | Tag |
| Bba_K1223007 | cI translational unit with His-tag | Reporter |
| Bba_K1223008 | pUC57-P.A.S.E.2 | Plasmid |
| Bba_K1223009 | pUC57 backbone (REVERSED) | Plasmid Backbone |
| Bba_K1223010 | pUC-57-P.A.S.E.1 | Plasmid |
| Bba_K1223011 | ampR translational unit (ampicilin resistance CDS+promoter) | Translational Unit |
| Bba_K1223012 | pKD46 functional unit | Device |
| Bba_K1223013 | Pyrolysyl-tRNA synthetase CDS | Coding |
| Bba_K1223014 | tRNA-Pyl (pylT) gene from Methanosarcina barkeri str. Fusaro | Coding |
2. Assembly of parts
We mixed and matched different parts from the registry with our finishing touch to build our state of the art P.A.S.E machinery.
This part was designed to fulfill the self destruct system of P.A.S.E 1. It contains a toxin system based on phage lysis system of holin (BBa_K112805) and lysozyme (BBa_K112301). Holin protein causes "pores" in the inner membrane, which allows lysozyme to access and break down the peptidoglycan of the cell wall, causing lysis and eventually death. The toxins are regulated by cI regulated promoter (BBa_R0051). This part is designed to integrate into the cell's genome via homologous recombination and therefore it contains homologous regions at its ends. Kanamycin resistance was added for selectivity. Therefore, when transforming in bacteria only the cells that have gone through double recombination with the insert will survive.
The part was characterized through sequencing and restriction digest with BamHI and EcorRV.
This part was designed to function as a biological timer for our P.A.S.E 1 system. It includes an assembly of two existing parts from the Registry, Lac/Ara-1 IPTG Inducible promoter (BBa_K354000) and LVA-tagged cI repressor protein (BBa_K327018). In order to extend the half life of the cI protein, we removed the LVA tail.
The part was characterized through sequencing and restriction digest with Pvull and HindIII. In addition, the promoter’s performance under the presence of IPTG and Arabinose was analyzed via comassie staining. The cI protein is 29 KDa.
This part is the heart of the P.A.S.E. 2 system. It is intended to be incorporated into E.coli BL21 genome by recombineering, and therefore has a homologous region in each side that direct the recombination into the right place in the genome. This part is used to replace the native promoter and regulatory sequences upstream of the CDS of the TyrS gene that encodes for the enzyme Tyrosine Synthetase. The native promoter is replaced with an IPTG/Arabinose induced promoter (BBa_k354000) and BBa_b0034 RBS. In addition, it has a kanamycin resistance gene (KanR) to aid in selection of the desired transformants.
The sixth amino acid in the TyrS sequence was replaced with an amber stop codon (TAG). The TAG stop codon is used for the incorporation of unnatural amino acids (UAAs) into the protein sequence. This system gives us better control of the translation process and prevents expression of TyrS when the unnatural amino acid is not present in the medium.
The fail proof aspect of this system is achieved by creating a logic AND gate which is depended on IPTG/Arabinose AND UAA in order to synthesize an active TyrS protein.
The part was characterized through sequencing and restriction digest with BamHI and XbaI.
3. Creating new parts
We added two new biobricks that can be used for the incorporation of Unnatural amino acids:
BBa_K1223013 - Pyrolysyl-tRNA synthetase CDS
This part is the coding sequence for the Pyrolysyl-tRNA synthetase enzyme from the archaea Methanosarcina barkeri str. fusaro. The enzyme 'load' the amino acid pyrolysine onto its dedicated tRNA for subsequent incorporation into proteins during the translation process. The tRNA synthetase is part of the unnatural amino acid (UAA) incorporation machinery along with the dedicated tRNA molecule (BBa_K1223014).
Application:This part was used by us to incorporate the unnatural amino acid propargyl-L-lysine into various proteins. In the fluorescent gel below we show incorporation of the unnatural amino acid into copper oxidase of E.coli in various loactions H117,N262,D411,M412 - position and amino acid that was replaced with the UAA. CueO - native protein without UAA incorporated.
we used a reactive fluorescent dye to identify the incorporated UAA in the gel.
BBa_K1223014 - tRNA-Pyl (pylT) gene from Methanosarcina barkeri str. Fusaro
This is the DNA coding sequence for the tRNA of pyrrolysine from the Archaea Methanosarcina barkeri str. Fusaro.
This tRNA is a part of the machinery that is used to incorporate pyrolysine into proteins in E.coli.
The tRNA synthetase that charges the tRNA with pyrrolysine can be found in biobrick BBa_k1223013.
The tRNA has the anticodon CUA - which means that the sense codon needed to incorporate pyrrolysine is TAG - the amber stop codon.
We added another two new biobricks: Assembling parts to create a new DNA strand always requires taking into account all the restriction sites that exists on each part. Not always is it possible to assemble specific parts for they may contain overlapping restriction sites which do not enable to "cut and paste" between them. In order to overcome this obstacle, there is a need for expanding the selection of options for each part. We contributed to this issue by adding two new parts, though with similar activity to existing ones but with a different sequence. Both parts are a common antibiotic resistance:
1. BBa_K1223003 - kanamycin resistance gene (promoter + CDS)
2. BBa_K1223011 - ampR translational unit (ampicillin resistance CDS + promoter)
4. Improving existing parts
Purifying a specific protein from the cell and analyzing its expression requires precise and often expensive tools and ability. Attaching a certain tag to the protein usually simplifies this process and makes it much easier. However, some tags usually have an effect on the function and activity of the protein such as shorting its half life or disrupting its function.
This part is an improvement of the existing BBa_K327018 LVA tagged cI repressor protein. We added a his tag instead of the LVA tag in order to offer a convenient way to study the protein without damaging it. The his tag is located at its C-terminal, therefore having no effect on its function [1]. The part was characterized through comassie staining and western blotting with His probe anti-histag antibody.
comassie staining western blotting
5. In development
We are currently working on inserting the Lac/Ara-1 IPTG inducible promoter (BBa_K354000) into a pGFPuv plasmid through side-directed mutagenesis. This is done to improve the BBa_K354000 BioBrick by attaching to it a GFP reporter gene and by improving our ability to characterize it. After a few unsuccessful tries, iGEM team Paris_Bettencourt kindly offered us their help. They are currently helping us characterizing this part by putting a GFP gene under the expression of this promoter. We are currently developing our UAA machinery as a BioBrick compatible which will contain the neccessary components for the incorporation of unnatural amino acids into a protein during translation. These components are an orthogonal tRNAcua which loads the UAA to be incorporated into the protein in addition to the Acetyl-lysyl tRNA synthetase, regulated by Lacl promoter which recognizes both the orthogonal tRNA and the UAA. This future BioBrick will contain a constituently expressed resistance for Chloramphenicol (for selection).
References
[1] M. Pedersen, M. Ligowska, K. Hammer, Characterization of the CI repressor protein encoded by the temperate lactococcal phage, Journal of Bacteriology 29, [2010]
