Team:Marburg/Toolbox Design

Toolbox Design

Standardization in Synbio

The standardization of components in common engineering disciplines, from screw threads to printed circuit boards, is one of the key elements to facilitate both the speed of innovation and the economy of production processes. The foundation of synthetic biology lies in applying engineering principles to biology and one of the key aspects is the concept of standardization, which is of utmost importance when building biological parts with defined functions. From the collection of over 20.000 Biobricks included in the iGEM registry only 18 are intended to be used in the chloroplast. Therefore the aim of our project was to design, build and test novel parts for engineering chloroplasts.

Golden-Gate and Modular Cloning

Before starting the design of our chloroplast toolbox, we had to decide on a cloning standard we would use. Our choice for this was the Modular Cloning (MoClo) standard. First introduced by Weber et al., the MoClo Assembly is based on the Golden-Gate cloning system [2] which can be used to build plasmids in a one step, one pot reaction. It relies on the ability of type IIs enzymes to cut outside of their recognition site to create a 4bp staggered cut.

Figure 1: Functionality of Type IIs restriction enzymes
Type IIs restriction enzymes (BsaI shown here) can cut DNA outside their recognition site, allowing for the standardization of the resulting 4bp overhang

These overhangs, in turn, dictate the position of each part in the plasmid. This standard includes unique overhangs for promoters, 5’UTRs, CDS and more; the only three enzymes used are BsaI, Esp3I and BpiI [1].

Phytobrick Standard

As there are several modular cloning standards that hugely differ from each other(e.g. the selection marker, type IIs enzyme, etc.), we wanted to select a standard which is as compatible as possible with other phototroph iGEM Teams.
In order to facilitate the easy exchange of standardized genetic parts in plant and phototrophic chassis, the international plant science community established a common genetic syntax for DNA parts [3]. Prior to the agreement of the Phytobrick standard, many labs had started their own standardisation efforts by converting in house plasmids to the Golden Gate standard. The GoldenBraid2.0 (GB2.0) [4] and Golden Gate Modular Cloning (MoClo)[1] assembly standards have been adopted by the plant research communities and are partly but (unfortunately) not entirely compatible. Even small cariations in the different standards prevent the creation of a collection of standardized, highly characterized parts and therefore hinder their exchange between labs. The syntax of the Phytobrick standard addresses exactly that; By establishing a common syntax for the plant science community - while also being compatible with most widely adopted Type IIS based standards - Phytobrick strongly contributed the progress of plant synthetic biology.

Marburg Collection: Recap

In addition to making our Collection Phytobricks-compatible, we also made use of the plasmid backbones provided by the Marburg collection [5], a Golden Gate based toolbox introduced by the iGEM Team Marburg 2018. The collection was originally built for the fast-growing bacterial chassis Vibrio natriegens, though its modular design makes it suitable for any organism.
Compared to other bacterial toolboxes, the Marburg Collection shines with superior flexibility and modularity. The collection overcame the rigid paradigm of plasmid construction - thinking in fixed backbone and insert categories - by achieving complete de novo assembly of plasmids. And The introduction of novel 5’ and 3’ connectors allows for the creation of up to 5 transcriptional units facing in either direction to be assembled in one plasmid. If you would like to read a detailed explanation of the Marburg collection visit this page

Over the years, the repertoire of genetic parts in the Marburg collection has grown far beyond the size of traditional toolboxes. In 2018, genetic parts for the genetic engineering of Gammaproteobacteria along with the cloning systems framework were introduced (connectors, syntax). During 2019, the collection was extended with the addition of genetic modules suitable for usage in cyanobacteria and state of the art dropout sequences that allow for high-throughput characterization of genetic parts (BBa_K3228060, BBa_K3228061 and BBa_K3228063).

This year we wanted to expand the Marburg collection even further by introducing genetic parts needed for chloroplast synthetic biology. These parts include regulatory sequences like promoters, 5’ and 3’ untranslated regions from a diverse group of plants, several coding sequences as well as two integration sites for Nicotiana tabacum chloroplasts. Prior to our contribution, only 18 parts in the iGEM registry were available for usage in chloroplasts, sadly none of them were ever characterized.

Figure 2: Overview of our Part Collection
Positions in the MoClo syntax are described by the 4bp overhangs that are can be seen below the different part types. The numbering system was introduced in order to better refer to the positions

Chloroplast Gene Expression

In order to develop chloroplast synthetic biology, standardization has to be established for characterized genetic parts, construct designs and for part measurements. A typical genetic device for plastid expression consists of a promoter, a 5’ untranslated region (UTR), a coding part and a 3’ UTR.

Figure 3: SBOL scheme of an exemplary transcriptional unit of a chloroplast gene
A chloroplast gene consists of a promoter, 5'UTR, coding sequence and a 3'UTR. The figure includes also a representation of the connectors we use for cloning

Promoters

Transcription in the chloroplast is mainly driven by two different RNA Polymerases: plastid-encoded polymerase (PEP) and nuclear-encoded polymerase (NEP).

The PEP Promoter

The PEP is a bacterial-like polymerase that is a remnant of the chloroplast’s cyanobacterial ancestor and is only capable of promoting gene expression in the plastid. These polymerases are able to interact with nuclear-encoded sigma factors and therefore are able to recognize bacterial promoter motives such as the -35 (TTGACA) and the Pribnow (TATAAT) box. Similar to bacteria, there are different sigma factors promoting gene expression under different growth conditions. As the PEP is structurally more sophisticated, there are even more peptides involved in DNA transcription that are not fully understood yet.

The NEP Promoter

The NEP is a T3/T7 phage-like polymerase that is encoded in the nucleus and is imported into the chloroplast. It was proposed that this polymerase is a remnant of a horizontal gene transfer from a bacterium to an eubacterial ancestor of today's plant cells [6, 7]. This type of polymerase mainly promotes gene expression in the early developmental stages of the chloroplast. In mature chloroplasts, it continues to transcribe housekeeping genes, like the subunits of the plastid-encoded polymerase (rpoA, rpoB, rpoC1, and rpoC2) and proteins involved in fatty acid biosynthesis such as acetyl-CoA carboxylase (accD). In contrast, the PEP is rather active in mature chloroplasts and is primarily involved in the expression of photosynthetic genes. For other non-photosynthetic genes, motives of both polymerases can be found, and it has been shown that both can promote transcription using deletion studies of important promoter sequences [8, 9, 10].

If you want to learn more about the design of promoters in the chloroplast visit our Best Basic Part page.

PPR proteins

The chloroplast is embedded in an eukaryotic background and, consequently, is heavily regulated by the nucleus of the cell. The regulation of gene expression in the chloroplast is primarily of translational nature. By importing nuclear-encoded RNA binding proteins pentatricopeptide repeat (PPR) proteins the nucleus can respond to a variety of different environmental cues and in turn react with upregulation or downregulation of the activity of certain genes. These environmental stimuli include changes in temperature [11, 12], light intensity [13], water availability [14] and redox regulation [15]. PPR proteins recognize specific RNA binding motives and the code of this mRNA recognition has been extensively studied and completely elucidated [16]. PPR proteins can bind specific RNA sequences and for example trim primary transcripts. By specific binding and trimming of the primary transcripts [17], the PPRs protects the maturing RNA from degradation via endonucleases found in the chloroplast.

Figure 3: Schematic for PPR protein RNA interaction in the chloroplast
PPR protein interaction with RNA in the chloroplast enabling the expression of transgenes by protecting the mRNA from exoribonucleases (Figure adapted from Rojas et al [18])

5'UTR

The 5’ untranslated region is a major decisive factor for initiation and regulation of translation in the chloroplast. Although plastids originate from cyanobacteria and their translation machinery being similar to most bacteria, their translation initiation mechanism is not exclusevely regulated by the availability of a Shine Dalgarno sequence directly upstream of the start codon. Translation initiation is oftentimes influenced by specific RNA binding proteins that promote or repress translation. In addition, it has been shown that gene expression in the chloroplast can also be regulated by specific secondary structure folding [19]. Another special mechanism by which the plant controls the expression of genes in the chloroplast is the utilization of specific pentatricopeptide repeats (PPR) proteins, which bind to specific sequence motifs within the 5’UTR. With this mechanism, they shield maturing RNA. In the past years there have been many studies investigating individual PPR proteins and their interactions with RNA in the chloroplast. Knockout mutants helped elucidate the specific function of these PPR proteins.

3'UTR

3’ untranslated regions do not function as efficient transcription terminators. They rather form stem-loop RNA secondary structures that are thought to protect the mature RNA from 3’ exoribonucleases [20]. While 3’UTRs play a strong role in the accumulation of RNA transcripts [21], their effect on protein accumulation is in most cases rather limited [22]. Similarly to the 5’UTR, some 3’UTRs are trimmed by specific pentatricopeptide repeat (PPR) proteins that are imported from the nucleus of the plant [23].

One difference of engineering plastids to commonly used chassis is the importance of translational control and protein stability rather than transcriptional regulation. Chloroplast transcripts are processed and regulated, by intercistronic cleavage [24], intron splicing [25] and mRNA editing [26]. Furthermore, mRNA levels and processing are regulated by PPR proteins (Figure 3), that bind to a specific mRNA sequence [27]. They also have been used for inducible gene expression in the chloroplast[18].

Existing Chloroplast Parts

Currently, the collection of genetic parts suitable for the chloroplast is fairly limited. In Literature, a handful of regulatory sequences are present, but the range of readily available inducible parts is limited [28, 29]. In the context of the iGEM competition there was already one team working with chloroplasts, namely iGEM Cambridge 2016. They were working on the chloroplast of the model algae Chlamydomonas reinhardtii. It was thanks to this team, 18 parts for chloroplasts have already been added to the iGEM registry. In 2019, a vast collection named the “MoChlo collection” was introduced to the field of chloroplast synthetic biology. The toolbox included in total 128 parts: 47 promoters, 38 5ʹ UTRs, 9 promoter:5ʹUTR fusions, 10 3ʹUTRs, 14 genes of interest, and 10 chloroplast-specific destination vectors. The paper demonstrated the usefulness of the collection by a proof of concept where they introduced two versions of the lux operon fused to an Emerald reporter gene. Although the collection included a neat variety of well-described chloroplast parts from Nicotiana tabacum, it is not compatible with the PhytoBrick standard described above, and a huge portion of the parts remained uncharacterized. As the collection decided to take Kanamycin as their lvl0 antibiotic marker, the parts were not compatible with our lvl2 destination vector. In order to minimize problems in the future, we decided to exchange the antibiotic for as many of the parts as we could in order to include them in a standardized manner in the Marburg collection. Besides these two resources of parts we just mentioned, there is still a lack of characterized parts in the field of chloroplast synbio.

Transcriptomics

A huge portion of the parts we created this year were created by combining two software tools. First, we used existing Transcriptomic datasets from several plant species that were deposited in the sequence read archive of the National Center for Biotechnology Information (NCBI). Datasets for almost all plant species we were working on this year could be found in this archive. Although most of the reads found in these datasets are mainly of nuclear origin, a small fraction coming from the chloroplast can also be extracted from these RNA-seq runs. The raw Illumina sequencing runs were downloaded and then converted into fastq files using the fastq-dump function of the SRA-toolkit. The fastq files could subsequently be further processed using a Transcriptomic pipeline: Spliced Transcripts Alignment to a Reference (STAR) [30]. By creating a genome index from the sequence in fasta file format and the corresponding gene annotations using gff3 file format, the fastq dataset can be aligned to the genome index in order to create a sequence alignment map (SAM) format file that can be displayed using a genome viewer software like Benchling or Geneious Prime. The information we gained from these data sets differed a lot between the different RNA-seq data. While some runs were performed in order to identify smRNA, they were particularly helpful in identifying PPR-binding sites in Nicotiana tabacum. These binding motives were later implemented in synthetic 5’UTRs. Other data sets were particularly interesting, because they contained a large portion of primary transcripts. With this, it was possible to accurately define full-lengths of 5’ and 3’ untranslated regions of individual genes as well as defined transcription start sites (TSS). But we did not stop here.

Figure 4: Coverage plot of the rbcL region of Spinach
Coverage plot in the region of the rbcL gene of Spinach. The y axis is displayed in logarithmic form in order to better visualize the graph

Chloroplast Database Based Part Design

The second software tool that immensely helped us during the design process of our basic parts was our Chloroplast Database. Chloroplast genomes of different plant species are highly conserved across the phylogenetic tree. Very early during our project we realized this conservation is particularly clear when comparing regulatory sequences of genes like rbcL (large subunit of RuBisCo), psbA (Photosystem II protein D1) or the PEP promoter region of the 16S ribosomal RNA. This became even more evident after seeing that some 16S promoters of certain organisms only deviate in 1 bp across the whole core region, it gave us a good indication that some genetic parts might be better transferable between organisms than others. Our chloroplast database currently consists of 6065 chloroplast genomes, which we used for nucleotide sequence homology analyses. This way, the mapping of promoters, 5’ and 3’UTR became easier than ever. The combination of this technique together with the comparison of the transcriptomic data set we had produced helped the indentification of new genetic parts. With the combined forces of these two software tools, we were able to design our parts based on established data. This is something we wanted to highlight, because normally chloroplast parts in literature are rather identified by taking random length chunks upstream or downstream of certain genes and then experimentally validating their behaviour, without considering any standardization efforts.

Workflow - Natural Part Creation

After the identification of our desired regulatory sequences, we set out to domesticate these parts into our modular cloning standard. DNA synthesis does not work for most chloroplast parts, as a lot of the regulatory sequences include stem loop structures, AT-rich regions or a multitude of inverted repeat motives. Most of the possible sequences we input into the online tools of IDT and Twist were denied due to their high complexity score. As a consequence, we were dependent on PCR amplification from chloroplast DNA and subsequent golden gate reaction in order to obtain them. To do this, we made use of a genomic DNA extraction method that is routinely used for isolation of high quality DNA from Chlamydomonas reinhardtii [DNA Extraction method]. We tried to transfer this protocol to even more plant species, but the isolated DNA from higher plants was always of poor quality and not usable for downstream applications. After literature research, we found a protocol suitable for the extraction of high quality DNA from higher plants, which worked significantly better. The usage of CTAB allowed for higher quality isolations [31] through binding unwanted polysaccharides and phenolic compounds that otherwise inhibited our PCR reactions. After we were able to isolate high-quality DNA samples of Chlamydomonas, Tobacco, Spinach, Wheat and Rice, we designed the primers needed to amplify the regulatory sequences from the chloroplast genomes. After purification of the PCR products, the fragments were cloned into a Universal acceptor vector[BBa_K2560002] for following modular cloning assemblies.

Figure 5: CTAB DNA Extraction method
Described here ist the process of genomic DNA purification from plant tissue. CTAB is used in order to wash Polysaccharides and phenolic compounds out of the DNA solution

Construct Design

The design of our devices were divided into two distinct projects.

(1) The first aim was to create a vector that could be used to detect activity in any cell-free system that is created by the cell-free extract subgroup, regardless of the organism. For this construct we made use of Decoupling, a method used in order to divide bigger problems into smaller ones, which can be worked on separately [32]. This part was named T7 Universal Test Construct and went through seven rounds of iterations of the design-build-test-learn cycle in order to optimize the vector as much as possible. We introduced this part as our best composite part and all of the optimization efforts can be found here.

Figure 6: SBOL scheme of our best composite part
Our best composite part consisting of a T7 promoter, gene10 5'UTR from T7, NanoLuc coding sequence and TMV 3'UTR. This plasmid was used to screen activity in our cell-free extracts

(2) For our second project we wanted to produce measurement vectors that can be used to efficiently characterize regulatory parts in a high-throughput manner. We therefore decided to make use of GFP placeholder parts introduced by the iGEM Marburg team of 2019. These placeholders harbour outwards facing BsaI cutting sites and are located in different positions of the MoClo syntax. This allows us to build preassembled lvl2 acceptor vectors, which in turn enables us to rapidly exchange parts of a certain position in shorter time frames. This way lvl0 parts can be directly cloned in lvl2 construct without intermediate cloning steps. These measurement vectors were built using the T7 promoter and also included a second inverse cassette that is used to ratiometrically normalize data to. If you want to learn more about this normalization approach click here.

Figure 7: SBOL scheme of our measurement vector design
The general structure of our lvl2 measurement vectors including a Firefly normalization cassette and a NanoLuc luciferase expression cassette with placeholder sequences in the positions of the promoter, 5' and 3'UTR enabling high-throughput characterization capabilities

During our project, we noticed we were not necessarily bound to using plasmid DNA as templates, therefore we developed a novel method for high-throughput screening of genetic designs using PCR prototyping.

Workflow - Golden-Gate -> PCR -> Prototype

Inspired by our successful approach of using Linear DNA templates (Results), we were asking ourselves if we could come up with an even faster workflow of testing and prototyping genetic designs in a high throughput manner. DNA synthesis is not yet an option for larger libraries of constructs due to cost limitations. With that in mind, we developed a workflow which combines our Golden-Gate based modular cloning toolbox with our linear DNA template approach by skipping all of the in vivo part of the cloning process. Firstly, a PCR is performed directly after the Golden-Gate reaction with standardized primer pairs. A similar approach has already been reported for different assembly types, which uses a PCR amplification after the Golden-Gate to directly use it for a Gibson Assembly, in order to build high-level constructs within one cloning cycle. We aim to make use of that approach by directly using the PCR product from the Golden-Gate assembly and setting up measurements on the same day. This workflow allows for high-throughput testing of chloroplast parts within one day! Starting with ideas for genetic design in the morning, followed by the Golden-Gate reaction and PCR and finally acquiring data from our cell-free systems in the evening.

Figure 8: Workflow of our improved characterization method
The figure describes the workflow coming from individual lvl0 parts to a cell-free measurement in the end. This is possible due to PCR amplification performed in the Golden-Gate reaction mix.

Synthetic 5'UTRs

In Literature, several studies from various plant species have elucidated the effect of specific PPR proteins on gene regulation. We applied this knowledge and implemented it in the design of de novo synthetic UTR modules.

As a basis we used well described PPR binding motives present in the 5’UTRs of prominent genes in the chloroplast, like atpH, rbcL and psbA. Ultimately, we combined a synthetic ribosome binding site from the MoChlo collection with different combinations of PPR Protein motifs and synthesized them via IDT as full transcriptional units, which can be directly tested in our cell-free systems.
In total, we built 4 synthetic 5’UTR sequences including PPR binding motives of the following genes:


  1. atpH PPR10 binding site [33]
  2. a combination of aptH PPR10 + rbcL MRL1 binding site
  3. rbcL MRL1 binding site [34]
  4. psbA HCF173 binding site [35]



Using our approach to rapidly prototype synthetic 5' UTRs in our cell-free extracts, we demonstrate the strength of our cell-free systems. These synthetic parts are of particular interest to chloroplast engineering, as using, and especially reusing, endogenous sequences will result in unwanted homologous recombination and excise any introduced transgene. In literature, it has been shown that a bare minimum of 51bp can suffice to enable recombination events in vivo in Chlamydomonas reinhardtii chloroplasts [36]. With our contribution of synthetic UTRs, we present fully orthogonal UTRs that bypass the problem of unwanted recombination in vivo.

Outlook

Our focus this year was mainly directed to the creation of regulatory parts from a wide variety of chloroplasts from different plant species. With this toolkit we want to lay the foundation for future, more elaborate, engineering projects. As an outlook to the future of the project, we propose the creation and testing of inducible elements for the chloroplast, as these tools are particularly rare in the field of chloroplast engineering. The importance for inducible parts becomes especially clear, since high expression in the chloroplast can lead to severe phenotypic effects and growth defects.
Furthermore, we noticed throughout our project that viral sequences from various origins can be used in the chloroplast in order to drive gene expression. We were able to characterize the 5’UTR of the T7 phage and 3’UTR sequences from the Tobacco mosaic virus, Brome mosaic virus and the Turnip yellow mosaic virus. The analysis of these plant pathogen 3’UTRs showed that their effectiveness most likely stems from their particularly strong stem loop structures. After doing more research on these viruses, we noticed that there is a huge sequence space available for further 5’ and 3’UTRs of different plant pathogens that are very likely to work in a similar fashion.
Our chloroplast database has provided the basis for the creation of endogenous parts, however sequence alignments can be performed to evaluate the score of individual base pair probabilities of different regulatory parts. With this information, whole collections of promoters can be designed that differ in their transcription strength. This enables more precise control over gene expression, much like the Anderson promoter collection introduced in 2006. In other experiments we were able to show that the community RBS (much like the 5’UTR of the T7 phage and the Synthetic RBS sequence) can successfully be used in our cell-free systems. These parts are all orthogonal to the chloroplast genomes and only include distinct ribosome binding sites that are the reason for their efficiency. By applying the design principles of the Community RBS collection, RBS collections specifically tailored for the chloroplast could be designed and tested using our cell-free systems.
Another major engineering problem in plastids is the abundant read-through transcription caused by inefficient termination [37, 38, 39, 40]. It has been shown that 3’UTRs do not function as transcription terminators, and are mainly responsible for transcript stabilization. This leads to a poorly insulated part behaviour and the genetic context can highly affect the expression levels, especially as it has been shown that chloroplasts need a mechanism to cope with antisense transcription readthrough from downstream antisense promoters. However, it has been demonstrated that tRNA genes trnS and trnH or the heterologous E. coli Thr attenuator terminate plastid transcription with at least 85% efficiency. These functioning sequences could be used as the basis to build functioning insulators or can be the blueprint for new synthetic terminators, which potentially enhance the robustness of the genetic designs.
All in all, we strongly believe that our chloroplast cell-free extracts offer a great opportunity to catch up the backlog to common synbio chassis by enabling the development of the missing tools and characterized parts. With this we want to lay the foundation for future more complex engineering endeavours in plant synbio.

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