Team:MADRID UCM/Engineering

Engineering - 4C_FUELS

During our project, we have developed genetic engineering strategies that might be useful for the synthetic biology community. In this page you will find how we have designed this gene edition tools and applied them to adress our own needs during labwork.

I-SceI Unmarking System

Industry demands markerless recombinant strains

Common laboratory practices handle genetically modified organisms that carry one or more antibiotic resistance. This is a common procedure to force mutant strains to maintain the foreing construction we transformed them with. Antibiotic resistance cassettes are left in the modified organisms. They could be seen as a marker left by the cloning and genetic modification procedures.
However, industrial demands are far from this traditional approach. When bioprocesses scale up, dependency on external substances like antibiotics or inducers turns into high operational costs. Moreover, if genetically modified organisms are going to be used for some specific purpose like feedstock or cosmetics they aren’t allowed to be cultured with antibiotics.

Current strategies

Because of the need to move on towards this antibiotic resistance-free scenario, some techniques and approaches have been developed to remove antibiotic resistance from mutant strains. One of the most popular for prokaryotic microorganisms is based on the introduction of the sacB gene from Bacillus subtilis, which codes for the levansucrase protein. sacB is constitutively expressed, and when bacterial cells are cultured in the presence of sucrose, sacB converts this sugar into a toxic compound. This acts as a counter-selection strategy. Once the mutants have been modified and selected using the antibiotic resistance, they are plated on sucrose to force the survival of only those colonies who have lost the antibiotic resistance-sacB cassette.

SacB unmarking strategy already works on prokaryotes such as cyanobacteria. We wanted to expand the repertoire of engineering tools available for phototrophs, focusing on unmarking strategies. That is why we looked for other systems that had not been tested on photosynthetic chassis. We found an interesting tool based on recombineering and I-SceI restriction enzyme. We aimed to adapt it to cyanobacteria, as well as improving it for the synthetic biology community.

Our Unmarking system

Our aim is to adapt current I-SceI mediated recombination system for the generation of markerless mutants in cyanobacteria. Following our design could be possible to quickly obtain a markeless recombinants after two transformation and selection rounds. The key players of our system are a integration by single recombination and a rare restriction enzyme: I-SceI.

I-SceI: A 18bp rare cutting endonuclease

Unlike traditional restriction enzymes used in Molecular Biology, I-SceI is a rare cutting restriction enzyme that recognizes a 18 bp long sequence. This specific target has a chance to occur almost once in every 7*10^10 base pairs. This fact makes I-SceI suitable for genetic engineering approaches that take place inside the cells. The chance of encountering its target sequence in our host’s genome is barely none. We can safely express it within our cells without disrupting the genome of the chassis.
I-SceI expression together with recombineering approaches can achieve high efficiency markerless genomic integrations or deletions. Here we present an overview of the procedure as well as our improvements and applications.

Single Recombination Integration

Genome integration techniques usually rely on double recombination events between synthetic constructs and the host’s genome. However, these events can be quite rare depending on the organism, and are definitely less common than single recombination events. I-SceI unmarking strategy relies on single recombination events.
If a plasmid carries an homology region and a single recombination event takes place, the whole plasmid gets inserted within the genome. I-SceI cargo plasmids carry two I-SceI target sequences flanking the construct we want to integrate. This plasmid also carries an antibiotic resistance cassette, so recombinants could be selected.

How does the system work?

The proposed design is a 2-plasmid system, first an integrative plasmid for the selection of market mutants and secondly an I-SceI expression plasmid, which will drive the removal process of undesired marker genes. Eventually, this former plasmid must be curatated from the strain, a process which could become tedious. To overcome this limitation we have considered two simultaneous strategies to reduce procedure times and ease the selection of unmarked mutants.

A global perspective of the proposed system at work is depicted below

ISceI overview

Briefly, the wild type is transformed by cosingle recombination with the integrative plasmid which harbors two homology arms and a a resistance cassete in the plasmid backbone. After that, transformants could be selected by resistance to this antibiotic. Then, the selected mutants would be transformed with the I-SceI replicative plasmid, and selected by the Chloramphenicol resistance of the I-SceI expression plasmid. Once transformants are obtained, I-SceI expression is induced in liquid culture. After a short time, I-SceI will generate double strand breaks in the DNA, forcing the DNA recombination, regenerating either the unmarked Wild-Type or mutant pehnotype. Eventually, I-SceI expression plasmid will be self-cured by the action of I-SceI on the I-SceI site present in expression plasmid backbone. Curated unmarked transformants could be then easily selected by plating in Agar-Sucrose. This is becaue the introduction of a SacB cassete within I-SceI expression plasmids impedes the non-curated cells to grow on sucrose.

System Components

Adapted Acceptor Plasmids

Our aim is to make this system compatible with our assembly standard, then we have adapted the Marburg Collection Lv.1 and Lv.2 acceptor plasmids to serve as the single-recombination integrative vector. To do so, we have designed modified Lv. 1 and Lv. 2 Modular Cloning acceptor plasmids plasmids that harbor two I-SceI restriction sequences flanking the genetic device to integrate.

Since both of this plasmids are only required for co-integration, the standard Lv1 (AmpR/ColE1) / Lv2 (KanR/ColE1) Marburg Collection acceptor plasmids has been used as template for the introduction of I-SceI recognition sites.

I-SceI Expression Plasmid

In order to express I-SceI we have considered to adapt the system already build in pSEVA328S. This plasmid contains an I-SceI expression casete under the control of Pm/XylS system, which is induced by the pressence of benzoate and its derivates. Pm/XylS system has been reported to achieve fine regulation and show reduced leakiness in standard laboratory chassis. In addition, the plasmid harbors a Chloramphenicol resistance cassete (compatible with the MoClo Lv.1 and Lv.2 selection markers).

Our design contemplates to replace by cloning with AscI and FseI the origin of replication by the pANs Ori, documented in part BBa_K3228069. Likewise, a sacB cassete can be included by cloning with SanDI and SpeI in the gadget position of pSEVA328S

The I-SceI Unmarking system in detail

An step by step description of how does our system behaves is shown below. The presented procedure corresponds with our theoretical design of I-SceI unmarking system, which could be virtually employed for almost any prokaryotic organism capable of homologous recombination.

1. Marked Mutants Generation

First step of the process is the transformation of the wild type strain with the integrative plasmid harboring the desired expression construct. During this transformation, the aim is to allow the cells to co-integrate the plasmid within their genetic material, acquiring the whole plasmid.
Because of this, selection can be then performed by plating antibiotic containing media.

2. I-SceI expression, DSB and recombination

Once recombinants are selected, we proceed by transforming them with I-SceI expression plasmid. This plasmid carries an I-SceI expression cassette under the control of a tightly regulated inducible promoter, and a second antibiotic resistance. By exposing cells to both antibiotics we can select those colonies that harbor both I-SceI expression plasmid and I-SceI cargo plasmid integrated in the genome.
The I-SceI cassette is then induced and the enzyme is expressed. I-SceI will cut the two target sequences that were flanking the construct to be integrated, causing Double Strand Breaks (DSBs) in the host’s genome.

At this point the cell must repair the DSB in order to survive. Using the Homology-Directed Repair (HDR) machinery, cells can overcome this problem. Due to how the I-SceI cargo plasmid is designed, there are two ways cells can repair the DSB: one of them reverts the integration to a wild-type version, and the other leaves our desired construct markerlessly integrated in the genome. This process depends on chance and in theory it should render a 50% chance of success.

In our case I-SceI expression plasmid has been designed in a double origin of replication plasmid: pRO1600/ColE1 for replication in E. coli cloning strains, and pANS origin of replication. The latter has been demonstrated to be a functional origin of replication in Synechococcus. In this way we aim to express I-SceI enzyme within cyanobacteria.

3. I-SceI expression plasmid curation

One of the main problems of I-SceI systems is the strain curation process. After the counterselection I-SceI expression plasmid remains within the cell. Curation is usually done by passing cells several times without antibiotics to encourage them to lose the I-SceI expression plasmid. However, this relies on pure chance and can be quite a long process. For common heterotrophs like E. coli or P. putida this can be achieved in a week, approximately. But when working with organisms like cyanobacteria the whole process could take much longer because of their long replication times.

This is why we decided to incorporate some novel features in our design such as a I-SceI target sequence within I-SceI expression plasmid to achieve self-curation. Also, we implemented a SacB expression cassette within I-SceI expression plasmid to force plasmid curation by plating mutants in the presence of sucrose at the end of the process.

However, the introduction of this features implies that I-SceI expression must be tightly controlled, since excessive expression or a leaky promoter baseline expression could drive the plasmid curation even before the double strand break event takes place.

Design Build Test & Learn Cycle: Our Iteration

In our project we have embraced the Design Build Test and Learn (DTBL) principles for all of our laboratory work. Our aim is to iteratively develop our technology, starting with the construction of a purely theoretical design and refining the design according to the observed outcomes.

Particularly we have applied DTBL philosophy for the experimental design for the validation of newly identified neutral sites (read more about it in our Software page) and the evaluation of I-SceI Unmarking System performance. In this way, our aim was to combine simultaneously both systems, co-integrating multiple plasmids harboring the identified neutral sites homology regions, as well as a control plasmid with the well defined Neutral Integration site I for Synechococcus e. PCC11801.

However, issued during cloning and delays in strain acces limited our possibility to perform a full iteration to DBTL cycle. On the one hand, digestion-ligation cloning of Lv.1/Lv.2 entry vectors didn't yielded any transformants harboring the desired I-SceI flanked dropout. On the other hand, we succesfully assembled the I-SceI expression plasmid with pANs/ColE1 origin of replication and an internal I-SceI recognition site. However the Pm/XylS system demonstrated to be leaky enough in different cloning E. Coli strains to compromise cell survival. This is because the baseline "leaky" expression of I-SceI was enough to self-destreoy the expression plasmid, which is ultimately responsible for cell survival during selection with chloramphenicol.

We observed how strains transformed with I-SceI expression plasmid harboring an internal I-SceI site did not grown properly on solid agar.

After designing and building the expression plasmid, we have observed how the streaks grow remarkably slow and they did not survive when inoculated in liquid media containing chloramphenicol. This results encouraged us to re-design the expresion plasmid in two different ways:

First, replacing the Pm/XylS system, screening for more tightly regulated promoters either in E. Coli and cyanobacteria. A reduced expression background is a requirement for succesful transformation with the plasmid.

Second, removing the internal I-SceI site of the expression plasmid, and relying solely on SacB cassete, which allows to select colonies by its sensitivity/insensitivity to sucrose. In this case, after I-SceI induction, bacteria should be cultured longer in liquid media without antibiotics, in order to ease the plasmid curation. Then, only those strains which have lost the plasmid will survive when plated.

Sun, T., Li, S., Song, X., Diao, J., Chen, L., Zhang, W., 2018. Toolboxes for cyanobacteria: Recent advances and future direction. Biotechnol. Adv. 36, 1293–1307.

Jones, C.M., Parrish, S., Nielsen, D.R., 2021. Exploiting Polyploidy for Markerless and Plasmid-Free Genome Engineering in Cyanobacteria. ACS Synth. Biol. 10, 2371–2382.

Chen, Z., Ling, W., Shang, G., 2016. Recombineering and I-SceI-mediated Pseudomonas putida KT2440 scarless gene deletion. FEMS Microbiol. Lett. 363.

Gawin, A., Valla, S., Brautaset, T., 2017. The XylS/Pm regulator/promoter system and its use in fundamental studies of bacterial gene expression, recombinant protein production and metabolic engineering. Microb. Biotechnol. 10, 702–718.

Egger, E., Tauer, C., Cserjan-Puschmann, M., Grabherr, R., Striedner, G., 2020. Fast and antibiotic free genome integration into Escherichia coli chromosome. Sci. Reports 2020 101 10, 1–10.

Lea-Smith, D.J., Vasudevan, R., Howe, C.J., 2016. Generation of Marked and Markerless Mutants in Model Cyanobacterial Species. JoVE (Journal Vis. Exp. 2016, e54001.

Dempwolff, F., Wischhusen, H.M., Specht, M., Graumann, P.L., 2012. The deletion of bacterial dynamin and flotillin genes results in pleiotrophic effects on cell division, cell growth and in cell shape maintenance. BMC Microbiol. 12, 298.

Elnitski, L., Hardison, R.C., Li, J., Yang, S., Kolbe, D., Eswara, P., O’Connor, M.J., Schwartz, S., Miller, W., Chiaromonte, F., 2003. Distinguishing Regulatory DNA From Neutral Sites. Genome Res. 13, 64–72.

Chaves, J.E., Wilton, R., Gao, Y., Munoz, N.M., Burnet, M.C., Schmitz, Z., Rowan, J., Burdick, L.H., Elmore, J., Guss, A., Close, D., Magnuson, J.K., Burnum-Johnson, K.E., Michener, J.K., 2020. Evaluation of chromosomal insertion loci in the Pseudomonas putida KT2440 genome for predictable biosystems design. Metab. Eng. Commun. 11, e00139.