Team:MADRID UCM/Synbio

SynBio – bueno – iGem Madrid

SynBio

"Synthetic biology is a) the design and construction of new biological parts, devices and systems and b) the re-design of existing natural biological systems for useful purposes"

This page includes the information related to the Synthetic Biology present in our project, from the design to the results obtained, and the future steps to take.

The first section is dedicated to the assembly system employed to join our parts and constructs, as well as the vectors used for that purpose and their functioning. We have chosen to work with Golden Gate cloning technology, through which multiple inserts can be assembled into a vector backbone simultaneously, using a single Type IIS restriction enzyme and T4 DNA ligase [1]. The rest of the page is divided into two parts that represent the creation of the two synthetic platforms, which solve two of the problems we faced during our project:

The first part relates to the creation of E. cholira, our biological platform which enables us to perform the separation and selection of aptamers. It includes the development of the whole idea from the very start, and the DNA parts involved on it.

The second part is related to the design, synthesis and purification of streptavidin - cellulose binding domain fusion proteins, used in LFA assays (if you want to know more, all the information can be foundhere).

SynBio has been one of the most challenging parts of our project, as we have had to face completely unexpected problems and difficulties. However, even if we have not been able to clone and characterize all of the intended parts and constructs, we are proud of our work. We truly believe that the parts we have created will be useful tools for future synthetic biology projects.

1 MoClo standard assembly

First of all, why Golden Gate?

Golden Gate is a molecular cloning method which allows us to assemble several DNA fragments into a single vector in a single reaction. The particularity of this method relates to the use of type IIS restriction enzymes. These enzymes have the ability to cleave double-stranded DNA several bases apart from their recognition sequence, leaving non-specific overhangs. This has several advantages [2]:

1

We can design the sequence of overhangs once we find the restriction sites of the enzyme.

2

By combining different overhang sequences, including those of the digested vector, we can assemble multiple fragments directionally. In the past, this could only be achieved by using one different endonuclease for each overhang combination, but now we only need one enzyme to obtain as many different overhang sequences as we need.

3

If the recognition sites are correctly designed in both the fragments and the destination plasmid, with opposite orientations, that sequence will be lost after the cleavage and ligation, avoiding a new cut and making the reaction irreversible. This allows us to perform the reaction in a single tube including all the necessary enzymes (restriction enzyme and ligase) within a suitable buffer.

4

The reaction, if the overhangs are carefully designed, is typically scarless, as they fit together perfectly.

But there is more. We did not only clone our parts using Golden Gate, but we also needed to combine them inside specific vectors. And we chose MoClo to achieve this goal.

But, what is MoClo?

MoClo is a Modular Cloning System for Standardized Assembly of Multigene Constructs. This cloning system is based on the Golden Gate cloning technology, and the main advantage that it offers is the ability to assemble complex DNA molecules in various predefined arrangements, in a hierarchical and standardized way [3]. Therefore, an otherwise complex and tedious cloning can be made just by pre-designing the genetic modules according to the MoClo standard.

Finally, we can combine our new constructs using binary steps: we can switch between two levels of plasmid, alpha and omega, which alternate the order of two different and opposed restriction sites for two different types of IIS enzyme. Thus, we can assemble as many fragment combinations as we want in one single plasmid.

For this binary-step method, we relied on 2018_Valencia_UPV team’s kindly donated vectors pARK1 and pARK2 as level 1 (alpha plasmids), and pRMSO1 as level 2 (omega plasmid) [4]. For level 0, our inserts are basic parts or level 0 parts themselves, as they are designed and were ordered already carrying the correct restriction sites. However, they were cloned into pSEVA182 plasmids (kindly lent by Victor de Lorenzo’s laboratory) to create a stock library of basic parts.

Our plasmids

Level 0

Level 1

Level 2

Antibiotic resistance genes:

Insert carrying the resistance to the indicated antibiotic. This part will allow the selection of transformants: as the cell strain is sensible to this substance, only bacteria harbouring the plasmid may survive.

pUC - Ori:

Origin of replication is the point where plasmid replication is initiated. In the case of pUC origin, it produces a very high number of copies of the plasmid.

mrfp1:

This is a basic (constitutively fluorescent) red fluorescent protein. It is the marker protein used for recombinant selection: if the insert is correctly cloned, the insert will disappear and colonies will grow as usual colis, with a white colour. But if not, colonies will keep a reddish colour, allowing us the detection of religates.

LacZ:

Gene coding for minor subunit of B-galactosidase. It is another kind of recombinant selection system: when cells are in the presence of IPTG inductor and XGal substrate, a blue colored product is released to the media, and therefore colonies appear blue-ish. But when our construct is correctly cloned, the gene is interrupted and colonies appear white.

SmaI restriction site:

SmaI is a type II endonuclease which recognices and cuts the sequence CCCˇGGG leaving blunt ends. It was used to clone our level 0 parts into pSEVA182 vector to allow subsequent amplification, as our fragments were dsDNA with blunt ends too.

BsaI restriction site:

BsaI is a type IIS endonuclease which recognices and cuts the sequence GGTCTC(N)1ˇ, leaving pre-defined cohesive ends. It was used to clone our parts into pARK1 vector assembled as a construct, by cleavage of both insert and plasmid. After ligation, restriction site is lost.

BsmBI restriction site:

BsmBI is a type IIS endonuclease which recognices and cuts the sequence CGTCTC(N)1ˇ, leaving pre-defined cohesive ends. It was used to clone our level 1 constructs into pRMSO1 vector by cleavage of both insert and plasmid. After ligation, restriction site is lost.

Now, by alternating BsaI and BsmBI enzymes, we can extract constructs of one plasmid alpha and integrate them together into an omega one, then the other way around, and so on. This is the basis of MoClo.

2 Escherichia cholira

Context

The problem

As we explain in our Description page, the way to develop our aptamers is through the Cell-SELEX (Systematic Evolution of Ligands by EXponential enrichment). This method relies on the availability of target cells.

As we aim to develop aptamers for the specific detection of a pathogen, the selection mechanism consists in isolating those which bind best to specific membrane proteins, while the cell is still intact. This constraint is due to the nature of the pathogen in the real samples; alive and with the entire membrane untouched. The reason is that when bacteria die, they expose several different molecules to the extracellular medium compared to when they were alive and pathogenic. Therefore, if we used non-viable cells, our aptamer could bind to these non naturally exposed domains, and our results would be distorted. As such, the best candidate to perform this selection and reach the highest specificity is V. cholerae itself.

At this point we face a considerable drawback. Vibrio cholerae is a gram-negative enteric pathogen, and the causative agent of the diarrheal disease cholera. Its toxicity guarantees our bacteria a level 2 of biosafety (BSL-2) [5]. This means that working with V. cholerae in the lab would be hard and expensive, due to the strict protocols we would need to follow and the equipment required, not to mention the high cost of any error.

Our solution: Synthetic Biology

In our case, we are using SynBio to create a harmless, non-toxic and human-friendly recombinant microorganism expressing those specific V. cholerae membrane proteins: a microorganism that ‘looks like’ the pathogen.

The chosen microorganism is Escherichia coli, the laboratory bacteria par excellence, and one of the most studied organisms in the world. The protein from V. cholerae which we are expressing is the porin-like, outer membrane protein T, and the chosen technology to design our biological part is the efficient and seamless Golden Gate Assembly, the latest-generation technique for assembling DNA fragments.

Target protein choice: why OmpT

We chose the Outer Membrane Protein T after exhaustive research into V. cholerae specific proteins.

First of all, the chosen protein had to fulfill several requirements:

1

It had to be an external membrane protein. This is important for detection, since the aptamer will bind to it only as long as it is on the surface of the bacteria.

2

It should have been common in gram negatives, as both bacteria belong to this group, to ensure its correct expression, folding and transport to the membrane.

3

Its structure should have been resolved to allow us to study its interactions with aptamers, and to compare it with other proteins.

We started searching for the target protein based on the third point, as it was the crux of the choice. Among the different outer membrane proteins of V. cholerae found in the Protein Data Bank database, OmpT was chosen due to the high number of related structures described there (showing that it has been extensively studied) [7].

OmpT is described as a member of the family of outer membrane proteases called omptins, involved in the virulence of pathogenic gram-negative bacteria [8]. Moreover, other studies show a correlation between this protein and the regulation of cholera toxins, such as ToxR, and the presence of OmpT is directly connected to the sensitivity of the pathogen to host bile, hindering the colonization by the pathogen [9]. In conclusion, these facts proved that OmpT is present in pathogenic V. cholerae strains and performs an important role in them, and could therefore be used as the target protein of our study.

Outer-membrane expressed OmpT of Vibrio cholerae (Source: PDB)

Before confirming the decision to work with OmpT, some further aspects had to be checked:

Extracellular locations. Since it has a beta barrel structure, the protein has extracellular and intracellular domains. Therefore, extracellular domains had to be determined and one of them identified as the target for the aptamer interaction. We decided to work on the most exposed loop, marked in the right-hand figure in red colour.

Immunogenicity study of the chosen regions. This loop should also have been an immunogenic region to favour binding of the aptamer. Immunogenicity depends on size, exposition and charge. In our case, the chosen loop was big enough and the most exposed part of the protein to the extracellular medium, and the composition consisted of polar amino acids [10]. Thus, it fulfilled all the conditions.

Alignment. Outer membrane proteins are common among gram negatives, and so our protein had to be different enough from E. coli’s ones for aptamers to discriminate between them. Structural alignments were made using Pymol software to compare the structures of both OmpT found in PDB database. Results show that the chosen loops differ enough to make a significant difference.

Finally, codon optimization and restriction-site clean-up of OmpT gene had to be carried out to ensure optimal expression in E. coli and to adapt it to the biobrick standard.

Building E. cholira

Expression system: OmpT

Once the target protein is chosen, the goal is to build the microorganism. The first step is to decide the display system of the target protein.

There are different ways in which we can express the protein in the membrane. At this point, collaboration with CNB provided us with new approaches to this step: after several meetings with Víctor de Lorenzo, instructor of our team, we evaluated three final possibilities:

Cloning directly the entire protein, trusting that coli would synthesize, fold, transport and expose it properly.

Cloning only the part of interest of the protein, which was the loop we had chosen, in a “support” protein such as LamB. This protein casts a permissive extracellular loop at amino acid 153 that allows insertion of short peptides and its subsequent display [11].

Using a display system whose support is a beta barrel protein. This approach would expose much more the area of interest than the others, and a couple of tags would be placed before and after the fragment to verify its correct cloning and allow purification.

The second and third approaches had already been tested by De Lorenzo´s laboratory with other tags, so they appeared to be great possibilities. However, in our case they did not ensure the correct protein loop folding either. In fact, it is less probable that a single loop will fold as expected than a whole protein will. Furthermore, the first approach is more similar to the natural state of the protein. For these reasons, we chose to express the whole OmpT in the outer membrane of Escherichia coli.

In addition, an epitope tag may be added in a periplasmic location of the protein to allow its further detection. These tags are highly immunoreactive, being useful to detect recombinant proteins to which they are fused in western blot assays. In this case, the chosen tag was c-Myc.

Separation system: LamB

This section began by mentioning SELEX, which is the method that allows us to develop aptamers against a specific target. For this to happen we need to be able to retain target cells both while an aptamer library is being presented to them and during washes, and to achieve this goal a retention system is needed.

We decided to express a six-unit histidine tag in the permissive loop of LamB. The peculiarity of this tag is that histidines bind strongly to nickel beads subjected to a magnetic field. Since histidine is a polar amino acid, negatively charged (due to its multiple amine groups) in a wide range of pH, it can easily interact with ions of metallic particles [12]. Using this approach, we suggest that by expressing enough histidine tags in the membrane of our target cells we would be able to induce conjugation between magnetic beads and cells when incubated together in the magnetic module. For further information, visit our Robo-SELEX page [link to SELEX].

Adaptation to MoClo Assembly

Our Level 0 genes

Finally, having decided the structure of both the expression of the target protein and the separation from the cell of interest systems, their genetic constructs need to be created. The origin, adaptations and registry sites of each gene are described in the final part of this section, “Parts”.

These genes were designed to have a BsaI type IIS restriction enzyme recognition site at each of their ends, oriented to the core of the gene. Therefore, after cleavage, the recognition site will be lost, leave pre-designed cohesive ends that fit the MoClo standard.

All the new genes needed to create E. cholira were ordered to Doulix, and the existing ones were donated by 2018_Valencia_UPV iGEM team (see our Collaborations page here).

Our Level 1 constructs

These genes are combined as follows in a Level 1 pARK1 or pARK2 vectors:

Promoter - RBS - LamB - Terminator in pARK 1

Promoter - RBS - LamB - 6xHis - Terminator in pARK 1

Promoter - RBS - OmpT - cMyc - Terminator in pARK 2

Our Level 2 product: the heart of E. cholira

The last piece of the puzzle is to combine the constructs Promoter - RBS - LamB - 6xHis - Terminator (in pARK 1) with the Promoter - RBS - OmpT - cMyc - Terminator (in pARK 2) in a Level 2 plasmid, which is in this case pRMSO1.

Transforming a pop6510 Escherichia coli strain (mutant for lamB) with this final plasmid would result in our E. cholira bacteria. This is how, by using just a single plasmid, we can express all of the constructs we need (LamB, OmpT, Histidines…) in a chosen cell line. Thus, this is the final microorganism that will be used for the Cell-SELEX performance to develop a library of specific aptamers against OmpT protein from Vibrio cholerae.

Parts

Part	Type	Original Biobrick	Description
BBa_K3122000	Coding	None	AEGIS's lamB display system for Gram-negative bacteria
BBa_K3122001	Tag	BBa_K1223006	6xHis Tag (Adapted to AEGIS' lamB display system)
BBa_K3122002	Composite	BBa_K3122000 BBa_K3122001	AEGIS' lamB display system exposing 6xHis Tag
BBa_K3122003	Regulatory	BBa_J23107	Constitutive promoter J23107 (TypeIIS adapted)
BBa_K3122004	Composite	BBa_K3122003 BBa_K2656009 BBa_K3122002 BBa_K2656026	Constitutive medium promoter + strong RBS + AEGIS's lamB display system + 6xHis Tag + Terminator
BBa_K3122005	Composite	BBa_K3122003 BBa_K2656009 BBa_K3122000 BBa_K2656026	Constitutive medium promoter + strong RBS + AEGIS's lamB display system + Terminator
BBa_K3122007	Coding	None	Outer membrane protein T ( Vibrio cholerae ) (TypeIIS adapted)
BBa_K3122008	Tag	BBa_K823036	cMyc-Tag (TypeIIS adapted)
BBa_K3122013	Composite	BBa_K3122003 BBa_K2656009 BBa_K3122007 BBa_K3122008 BBa_K2656026	Constitutive medium promoter + strong RBS + Outer membrane protein T + cMyc Tag + Terminator