Team:BGU Israel/Bricks

Overview

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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	Lambda 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

3. Creating new parts

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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)

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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]

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.

BBa_K1223001 - P.A.S.E 1 cassette:

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.

BBa_K1223005 - cI Translational Unit:

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.

BBa_K1223002 - P.A.S.E 2 cassette:

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.

Creating the world’s first UAA logic AND gate

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.

Improving existing parts

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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.

BBa_K1223007 - cI translational unit with HisTag

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

Genetic code expansion using stop codon (Amber) suppression in bacteria

Natural translation process is achieved via conservative mRNA-tRNA codon-anticodon specific base pairing; the meaning of each codon is interpreted mainly through stringent substrate specificity of Aminoacyl tRNA synthetases (AARS) in the aminoacylation reaction. This reaction is an interpretation level in the context of the flow of genetic information transmission.[1]
In order to incorporate the #21 man made, synthetic Unnatural amino acid (UAA) one must first find a way to expand the genetic code to add a translational sense codon for that amino acid. We used a method called "Stop codon suppression"[2] developed by Prof. Peter Schultz and co-workers. This method uses orthogonal tRNAcua and Aminoacyl-tRNA synthetase from archaea. Orthogonal means that these components do not interact with the different host organism’s cellular pathways.

Orthogonality:
Since the PylRS originating from archea must be orthogonal in the host cell, our BioBricks could and should be used only in bacteria [2]. There is no orthogonality between archea and mammalian cells for example.

Use in our project:
The following parts were used by us to incorporate the unnatural amino acid proparagyl-L-lysine (as a model UAA) into our PASE 2 essential protein – TyrRS. When one of the following parts or the UAA itself are missing, one of the logical AND gate (Link to PASE2 project description) conditions are not met and essential protein translation is disabled.
Pyrolysyl tRNA synthetase (PylRS) from Methanosarcina barkeri str. Fusaro (Archea):
In well-studied model organisms such as E.coli , yeast and in mammalian cells, the natural AARS catalyse via the aminoacylation reaction the amino acid activation and accurate biosynthesis of aminoacyl-tRNA, the immediate precursors for encoded proteins. Note that each AARS should select its "own" (i.e cognate) amino acid (AA) which is subsequently covalently linked to the cognate tRNA isoacceptor "Fished" from the cellular pool. Immediately after their dissociation from AARS, the aminoacyl tRNA are shuttled or channeled to the ribosome where the anticodon is matched to the mRNA codon and the tRNA is deacylated , with the amino acid being added as the next residue of the a nascent protein chain.[1]
In our project we used a class 2 AARS that is capable of charging Pyrolysine (Pyl – a natural, but rare, amino acid) and several unnatural Amino acids (Fig.1) onto the tRNA_cua^pyl.

PylRS substrates:
The PylRS can use different derivatives of Pyrolysine with high specificity and fidelity, The substrates of the PylRS are [3]:



As can be seen in Fig. 2 the aminoacylation of the tRNA by the PylRS is specific and selective to the latter substrates[3].



In Fig.2 tRNA were incubated with various lysine derivatives and PylRS, Next all samples were subject to acidic PAGE and stained with methylene blue. All samples that have 2 bends have been aminoacylated with the relevant lysine derivative.
In our study proparagyl-lysine (figure 1, (8)) was used as a model amino acid.

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.

Additionally, we intend to characterize and develop the Lambda red incorporation machinery (pKD Bba_K1223012) used in our project to a working and well defined biobrick .

Continue the journey: read about Our achievements.