Our vectors were linearized through digestion by restriction enzymes and our inserts were PCR-generated.
Restriction enzyme-treated vectors can have 5´ overhangs, 3´ overhangs or blunt ends.

One of the unique features of this Assembly Master Mix is its ability to remove both 3´ and 5´ end flap sequences upon fragment assembly.

T5 Exonuclease removes the bases from the 5-prime end, generating a 3-prime overhang. After that, single-stranded 3-prime ends can anneal, the DNA polymerase fills in the occurring gap and DNA ligase joins the adjacent fragments.

In that way, the 3´- and 5´-end flap sequences occurring after a particular restriction enzyme digestion are removed. This allows fragments generated by restriction enzyme digestion to assemble while eliminating the remaining restriction site sequences on both the 5´ and 3´ ends in the fragment junction.

Primer Design

The first part of HiFi DNA Assembly is the Primer design. The basic rules we followed to design our primers are as follows:

The entire overlap sequence must originate from the vector sequence and must be added to primers that will be used to amplify the insert.

The overlap region of the forward primer for the gene of interest (orange) should line up with the 3´ end of the overhang on the vector’s left arm. This primer should also include a gene-specific sequence at its 3´-end (gray).

Primer design - HiFi DNA Assembly

IRIS’s chromosomal DNA is also synthetically engineered. Clonetegration is a commonly used method for editing bacterial chromosomal DNA and incorporating gene inserts into it.

Clonetegration is a two-plasmid system designed to integrate a gene into a specific site within the bacterial chromosome. This approach combines DNA Cloning/assembly and chromosomal integration into a single procedure.

Clonetegration relies on bacteriophage-derived integrases, which are naturally involved in recombination processes between specific DNA sequences. These integrases typically mediate recombination between attB sequences in the bacterial chromosome and an attP sequence found on bacteriophage.

These attP sequences are incorporated into a plasmid, so they direct the integration of that plasmid into the chromosomal attB sequence. Clonetegration comprises two key plasmids:

OSIP plasmid
E-FLP plasmid

OSIP Plasmid:

OSIP is practically the plasmid responsible for the integration of the gene of interest in the bacterial chromosomal DNA. It is composed of two modules:

Integration/Propagation Module

This module includes the following components:

Site-specific Recombinase Gene: This gene is controlled by the lamda Cl ts repressor facilitating precise site-specific recombination between attP and attB sequences.

Attp Attachment Sequence: This sequence guides the integration of the plasmid into the bacterial chromosome

Antibiotic Selection Marker: The marker is essential for the selection of the transformed cells

R6Kγ Origin of Replication: This origin functions exclusively in strains that carry the pir gene. It permits the propagation of the plasmid in pir+ stains and allows the stable integration of the plasmid into the chromosome of the strain that lacks the pir gene.

FRT Sites: These strains flank the integration/propagation module serving as recognition sites for the pirFillipase (FLP) recombinase enzyme which facilitates the excision of this module from the bacterial chromosome.

Cloning Module

This module includes the following components: Site-specific Recombinase Gene: This gene is controlled by the lamda Cl ts repressor facilitating precise site-specific recombination between attP and attB sequences. Split Multiple Cloning Site (MCS) this site accommodate BioBricks element BBa_I52002. Mini pUC Origin: it enables the production of high DNA yields of the unmodified plasmid ccdB gene: It functions as a negative selection marker because it expresses a toxin that binds to the DNA gyrase enzyme. DNA gyrase manages the DNA supercoiling by temporarily introducing double-stranded breaks in the DNA backbone, and the interference by the ccdB toxin leads to cell death. Genes integrated: The genes Rluc8 and T7 RNA pol are inserted to the cloning module in order to be integrated to the bacterial chromosome.

E-FLP plasmid:

The role of this vector is to express the Flp recombinase which recognize the Flippase Recognition Sites (FRT) sites and catalyze the recombination between these sites. This recombination results in the excision of the integration module from the bacterial chromosome.

To enable IRIS to perform its diverse functions, it underwent a transformation from a typical E. coli bacterium into a significantly altered engineered machine. To achieve this, our team incorporated three different plasmids housing the required genes, as well as integrated two genes into IRIS's chromosomal DNA.

All the engineering was based on four distinct constructs:

Construct no. 1

Construct 1 - pTU2-A-RFP (p15A origin)/ hemD Vector: pTU2-A-RFP
Gene inserted: hemD


To achieve a moderate expression of hemD gene, we employed pTU2-A-RFP, a low-copy-number plasmid, as the vector for this particular gene.

This vector features a p15A origin of replication and it is chloramphenicol resistant.



pTU2-A-RFP (p15A origin) vector

It lacks any T7 promoter/terminator or RBS sequences. To address this, we incorporated a T7 promoter, a T7 terminator, and an RBS, all originated from pETDuet-1, in the final insert to serve as regulatory elements for controlling the expression of the hemD gene.

The vector was linearized through digestion by SacI restriction enzyme. We didn’t take into account any overhangs left by the enzyme when designing primers and the actual insert, since HiFi DNA assembly removes both 3´ and 5´ end flap sequences in any case.



Construct 1 overview

Construct no. 2

Construct 2 - pET-29c(+) / hemL, hemAs, hemF Vector: pET-29c(+)
Gene inserted: hemL, hemA^s, hemF


In order to achieve high-level overexpression of hemL, hemA^s and hemL we employed pET-29c(+), a high-copy-number plasmid, as the vector for these three genes.

This vector features a ColE1/pMB1/pBR322/pUC origin of replication and it is kanamycin resistant.



pET-29c(+) vector
In order to achieve the best results in 5-ALA accumulation, hemL and hemA^s are expressed under the same T7 promoter, while hemF is expressed under a separate promoter. [x] This means that two different transcription units, both containing the necessary regulatory elements, must be integrated in one construct.

This vector already includes one T7 promoter, one RBS and one t7 terminator, so in order to regulate the second unit of our insert, we integrated a T7 promoter, a T7 terminator and an RBS sequence, all originated from pETDuet-1.

The vector was linearized through digestion by two different restriction enzymes, BglII and SacI. These enzymes were specifically utilized to cut off a thrombin site. Once again, we didn’t have to think about complementary one-stranded edges left by the enzymes, as blunt edges are always left during the HiFi DNA Assembly procedure.

To ensure their proper expression, each gene had to include a tag for its detection using Western blotting. Consequently, we incorporated an HA-tag and a Myc-tag in our hemA and hemF inserts respectively. The hemL gene utilizes the S-tag provided by the vector.

Construct overview:



Figure 10. Construct 2 overview

Construct no. 3

Figure 11. Construct 3 - pETDuet-1 / Catalase, rhtA Vector: pETDuet-1
Gene inserted: Catalase, rhtA exporter


In order to achieve the overexpression of catalase and rhtA exporter, we employed pETDuet-1 as our carry out vector.

This vector features a ColE1/pMB1/pBR322/pUC origin of replication and it is ampicillin resistant.



Figure 12. pETDuet-1 vector


pETDuet-1 is a high copy number plasmid that features two T7 promoters and two RBS sequences. However, it contains only one T7 terminator. As a result, each gene is expressed under its dedicated T7 promoter, but one of the inserted genes requires the inclusion of a T7 terminator for proper regulation. The rhtA insert is the one carrying the extra T7 terminator.

In order to assemble our constructs, two cloning steps needed to be performed. Firstly, the vector was linearized through digestion with the BglII restriction enzyme to allow the insertion of the catalase gene. Subsequently, another digestion with SacI was necessary to clone the rhtA exporter gene into our vector.

In order to confirm our gene expression, an S-tag was incorporated in the catalase insert making it possible to detect through Western-Blotting. The rhtA exporter utilizes a 6xHis-tag which is already an integral part of the plasmid.

Construct overview:



Figure 13. Construct 3 overview

Construct no. 4

Figure 18. Construct 4 Vector: pOSIP-TT (P21)
Gene inserted: Rluc8


pOSIP is the vector mediating the insertion of Rluc8 in the bacterial chromosomal DNA as outlined in the clonetegration protocol.

Among the various plasmids within the OSIP family capable of performing this task, we chose pOSIP-TT (P21) due to its tetracycline resistance, which was useful for simultaneous transformation with other plasmids.

This vector features a ColE1/pMB1/pBR322/pUC ori as well as a gamma replication origin from E. coli plasmid R6K and it is tetracycline resistant.



Figure 19. pOSIP-TT (P21) vector
pOSIP-TT (P21) lacks a T7 promoter, an RBS and a T7 terminator. To ensure the expression of our Rluc8 gene after its integration into the chromosomal DNA, it's essential to incorporate these regulatory elements into our insert.

The vector was linearized through digestion with two different restriction enzymes, XhoI and EcorI. These enzymes were responsible for removing the ccdB toxin and ColE1/pMB1/pBR322/pUC origin of replication in order to make the detection of the cloned vectors easier. Within this region, the Rluc8 gene was inserted, complete with its regulatory elements.

It's unnecessary to incorporate a tag into our insert to verify the proper expression of the Rluc8 gene because Rluc8 is an enzyme that interacts with its substrate, yielding a visible outcome.

Construct overview:



Figure 20. Construct 4 overview

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