Engineering Success

Design

To restore directional root growth in microgravity, we propose that the existing gravitropic mechanisms can be engineered to respond to an artificial cue. We set out to engineer roots to grow in the direction of magnetic field gradients: magnetotropism.

Plants sense gravity via statoliths—starch-laden organelles in root tip columella cells—which sediment due to their weight. Statolith sedimentation triggers changes in the efflux of auxin, a universal plant hormone that induces plant cell elongation. Polarized auxin accumulation along the upper and lower sides of roots causes differential elongation of cells, guiding root growth in the direction of gravity.

We predicted that filling statoliths with iron-loading proteins, like ferritin, would allow the statoliths to move in response to a magnetic gradient. For our project we designed a genetic construct that allows for ferritin to be expressed in Arabidopsis and imported into statoliths.

Main Basic Parts

Pyrococcus furiosus Ferritin (PFt): BBa_K4452007

Our engineered magnetotropism depends on efficiently loading the ferritin with iron. We decided to use a Pyrococcus furiosus ferritin mutant (PFt) which included one point mutation (L55P), engineered by Matsumoto et al. to have an expanded iron core [1].

PLT2 Promoter: BBa_K4452000

We wanted the ferritin to be expressed specifically in the columella cells of the root cap as those cells contain the statoliths that sense gravity and translate that signal into directional root growth [2].

We researched root-specific Arabidopsis promoters looking for ones that have specificity to columella cells. Candidates included:

• PYK10-1457 promoter (BBa_K2286005), a seedling and root specific promoter which was engineered from Pyk10 to increase strength by several times
• RCH1 Promoter, ROOT CLAVATA HOMOLOG1 (RCH1) promoter is generally highly active in the root tip, but not specific to the columella cells [3]
• PIN7 Promoter, active in the columella with off-target activity in stele, but has less activity in stele when root is differentiated [4]
• FEZ Promoter, preferentially expressed in columella cells of root, but sometimes not active in the columella cells but instead active in the lateral root cap [4]
• PLT2 Promoter, expresses the PLT2 gene of the PLETHORA (PLT) protein family in the columella and meristem sections of roots [5, 6, 7]

We did not pursue PYK10-1457 promoter and RCH1 Promoter because they were not specific enough to columella cells. We noted that PIN genes are responsive to changes in the auxin maximum. Since statolith sedimentation is supposed to impact the auxin maximum, using PIN7 promoter may inadvertently make a positive feedback loop between statolith sedimentation and ferritin expression, and we are unsure how this would affect magnetotropism. Therefore we eliminated PIN7 as a promoter candidate.

We decided to use the PLT-2 promoter, specifically the shorter 1.3 kb version (instead of the longer 5.8kb version) because the sequence’s shorter length makes it easier to assemble.

Transit Peptides

In addition to overexpressing ferritin in the correct cells, we need to import the ferritin into the correct intracellular compartments, i.e the statoliths.

Statoliths are starch-laden organelles in plant cells. The class of organelles in plants that are involved in food synthesis and storage are called plastids. The plastids that are responsible for food synthesis usually contain pigments. Chloroplasts are an example of plastids that are involved in food synthesis and they contain the green pigment chlorophyll.

Plastids without pigments are called leucoplasts. Leucoplasts are responsible for food storage and are renamed for the type of food they store. Leucoplasts that store starch are called amyloplasts, and thus, statoliths are a type of amyloplast.

Proteins that originate from nuclear transcripts must have a transit peptide (TP) sequence on their N terminus, which will bind to translocons on the plastid membrane after its initial translation. The rest of the protein will then be translated directly into the plastid. No validated statolith import sequences are known, so we selected a couple sequences to test. We searched for papers that mentioned root amyloplast TPs, and searched the Gene Ontology (GO) Resource for proteins known to be in the statolith that had annotated TPs. Upon advice from Dr. Hsou-min Li, the principal investigator of the Chu et al. paper cited below, we used the first 60 residues of the preproteins pulled from GO instead of the sequence that was annotated, as Dr. Li reported that TPs are not typically shorter than 50 residues.

Our 6 candidate TPs included:

prFB Transit Peptide: BBa_K4452001

Chu et al. identified transit-peptide motifs that enhanced import of proteins into root leucoplasts. They found that the preprotein Fibrillin 1B (prFB) exhibited 50% import efficiency into leucoplasts in pea (Pisum sativum) roots, whereas for its closely related homolog prPGL35 (80% identical amino acid sequence; 52% identity transit-peptide sequences) import into leucoplasts was very limited.

Furthermore, to test if the prFB transit peptide contains motifs that can specifically confer high leucoplast import efficiency, Chu et al. engineered mutants in which they replaced regions of the prPGL35 transit peptide that differ from prFB with the corresponding prFB sequence. From these experiments, Chu et al. found two motifs of the prFB transit peptide that are sufficient to increase the leucoplast import efficiency of prPGL35 up to more than threefold [8].

prcpHsc70-1 Transit Peptide: BBa_K4452002

Chu et al. also found that Arabidopsis (Arabidopsis thaliana) preprotein prcpHsc70-1 imported very well into leucoplasts, with a leucoplast import efficiency of 45%. Additionally, knockout mutants of Arabidopsis cpHsc70-1, one of a family of plastid Hsp70 proteins, have small size and slow root growth. This suggests that cpHsc70-1 may not only be localized to leucoplasts but statoliths in particular. [8]

prSGR9 Transit Peptide: BBa_K4452003

RING-type E3 ligase SHOOT GRAVITROPISM9 (SGR9) SGR9 has been shown to be localized to amyloplasts within gravity-sensing cells in Arabidopsis thaliana. Also, SGR9 has been reported to modulate the interaction between these statoliths and actin filaments. In the SGR9 mutant, there is reduced gravitropism and amyloplasts do not sediment but instead jump around [9]. The transit sequence of prSGR9 is annotated on UniProt ID:Q8GXF8.

prSS4 Transit Peptide: BBa_K4452004

Starch synthase IV (SS4) is one of the five classes of starch synthases necessary for the synthesis of starch in plastids. From studies of a Arabidopsis mutant defective in SS4, it is speculated that SS4 is involved in the priming of starch granule formation [10]. The transit sequence of prSS4 is annotated on UniProt ID:Q0WVX5. The transit peptide is annotated as a chloroplast import sequence, but due to SS4’s function in starch synthesis, the sequence could target amyloplasts as well.

prSSG1 Transit Peptide: BBa_K4452005

Granule-bound starch synthase 1 (SSG1) is an enzyme involved in the starch biosynthesis pathway, specifically in the synthesis of amylose, one of the two distinct polymers within statolith starch granules. SSG1 is expressed in roots, but is more highly expressed in leaves, where it exhibits circadian up-regulation during the day [11, 12, 13]. The transit sequence of prSSG1 is annotated on UniProt ID:Q9MAQ0.

prDPE1 Transit Peptide: BBa_K4452006

Studies suggest that disproportionating enzyme 1 (Dpe1) is involved in starch biosynthesis. Also, it has been demonstrated that Dpe1 is localized to amyloplasts in sweet potato (Ipomoea batatas) storage roots [14]. The transit sequence of prDE1 is annotated on UniProt ID:Q9LV91.

We decided to experimentally validate which of the six candidate transit peptides could efficiently import into statoliths, and set out to assemble six different variations of our construct.

GFP with 5xGS linker: BBa_K4452008

To identify the localization of ferritin, we wanted to include a fluorescence reporter that would be fused to the ferritin. Both N-terminus and C-terminus GFP-tagged ferritins have been successfully used in past studies [15,16]. From discussions with APL scientist Jeff Shipp, we decided that GFP was a sufficient choice for observing localization in Arabidopsis roots. Autofluorescence from chlorophyll could normally pose a challenge for fluorescence microscopy, but roots do not have chlorophyll. We added a flexible linker with five Glycine-Serine units (5xGS) to the beginning of GFP so that the folding of GFP would not interfere with the folding of the ferritin.

Plant threeUTRs Tnos: BBa_J428082

We utilized the plant terminator (composed of the nopaline synthase 3' untranslated region) provided in the 2022 iGEM Distribution Kit.

Main Gene Construct

Using the above basic parts, we designed six different gene variations which allow for testing of the six transit peptide candidates. The genes correspond to the following composite parts: BBa_K4452011, BBa_K4452012, BBa_K4452013, BBa_K4452014, BBa_K4452015, and BBa_K4452016.

Looking at BBa_K4452011 as an example, the standard design is as follows: PLT2 promoter + transit peptide + ferritin + 5xGS-GFP + Tnos.

Other Genes

In order to select for Arabidopsis plants that express the above gene of interest, our plasmid also needs to include two other genes: a visual marker for seed selection and an antibiotic resistance gene for seedling selection.

RUBY reporter under 35S promoter: BBa_K4452017

For visual selection of positively transformed seed, we utilized the RUBY reporter construct which will make the seeds red from the expression of betalain pigments. The composite part to express RUBY in Arabidopsis was designed using only basic parts provided in the 2022 iGEM Distribution Kit. We selected the constitutive CMV35S plant specific promoter, a 5’ UTR from the AtRbcS2B gene, and a 3’ UTR plant terminator.

nptII Antibiotic Resistance Gene for Plant Selection: BBa_K4452018

For selection of positively transformed seedling on agar plates, we utilized nptII which confers resistance to neomycin/kanamycin. The composite part to nptII in Arabidopsis was designed using only basic parts provided in the 2022 iGEM Distribution Kit. We selected the constitutive CMV35S plant specific promoter, a 5’ UTR from the AtRbcS2B gene, and a 3’UTR plant terminator.

Assembly Plan

Background

Since our genetic constructs are composed of new basic parts, which we had synthesized with Twist, and existing basic parts that we could resuspend from the 2022 iGEM distribution kit, we needed a plan to assemble the basic parts into genes. Furthermore we needed a plan to assemble our three genes (Ferritin, RUBY, nptII) onto one plasmid.

Initially, we considered a variety of assembly strategies including BioBrick Standard Assembly, Gibson Assembly, and Type IIS assembly methods such as GoldenBraid, MoClo, and ProClo. Due to lack of success in the previous year with BioBrick assembly and concerns that Gibson Assembly would not work for the large plasmid we intended to assemble, we preferred to utilize a Type IIS assembly method.

GoldenBraid is a standardized assembly system based on type IIS restriction enzymes “that allows the indefinite growth of composite parts through the succession of iterative assembling steps.” This criteria is important for us because our cloning plan requires assembling basic parts into three distinct genes and then assembling those genes together. Additionally, GoldenBraid was designed with the intention of being an assembly standard for plant synthetic biology [17], so it would be an appropriate method for our project.

Implementation of GoldenBraid requires (1) specific type IIS restriction sites on basic parts and (2) specific destination plasmids with type IIS restriction sites positioned inside the vectors to allow for “braiding” parts binarily in indefinite successive iterations.

Basic parts are flanked with BsaI recognition-cleavage sites using distinct 4 bp cleavage sequences for neighboring basic parts such that the parts can be assembled in a specified sequence. When the parts are ligated with the correct destination plasmid that is flanked by BsaI sites in divergent orientation, all BsaI recognition sites disappear from the resulting expression plasmid. This process of putting the parts into a destination plasmid with BsaI digestion is referred to as level alpha assembly.

To assemble parts from level alpha plasmids into another destination plasmid requires BsmBI digestion, this is referred to as level omega assembly. The level alpha plasmids and the level omega destination plasmid will be flanked by complementary 4 bp BsmBI cleavage sites.

Implementation

While we only require BsaI and BsmBI for our assembly plans, we checked the interior of the sequences of each basic part when designing them to remove the following restriction sites: BsaI, SapI, BsmBI, BtgZI, BbsI, AarI, EcoRI, NotI, XbaI, SpeI, PstI.

For our level alpha assembly, we chose to use the Joint Universal Modular Plasmids (JUMP) collection which were included in the 2022 iGEM Distribution Kit. Each gene uses a different pJUMP backbone. For the ferritin gene, we use pJUMP29-1A (BBa_J428341). For the RUBY reporter gene, we use pJUMP29-1B (BBa_J428342). For the nptII antibiotic resistance gene, we use pJUMP29-1C (BBa_J428343).

The following tables show the 4 bp BsaI recognition-cleavage sites that flank the basic parts and the pJUMP backbones.

Gene 1: Ferritin

Gene 2: RUBY Reporter

Gene 3: nptII Antibiotic Resistance

For our level omega assembly, we chose pLX-B3(omega)1, a vector from the pLX vector collection. These vectors are designed for expression in Agrobacterium tumefaciens for Agrobacterium-mediated transformation of plants [18].

The following table shows the 4 bp BsmBI recognition-cleavage sites that flank the composite parts in the pJUMP backbones and the pLX-B3(omega)1 destination plasmid.

Note that the level omega assembly into pLX-B3(omega)1 requires four genes to be ligated into the plasmid backbone. We only had three genes (ferritin, RUBY, and nptII) in mind for a functional construct. For the fourth gene position, we had to choose a spacer. We decided to have the spacer be the M13R primer to permit Sanger sequencing of our final constructs to test for successful assembly.

After level omega assembly, we have six final genetic constructs.

Learn

Following the unexpected sequencing results, we decided to verify if assembly occurred correctly by running a diagnostic restriction digest and diagnostic colony PCR.

Diagnostic Restriction Digest

For a diagnostic restriction digest we used BsaI. Two BsaI sites should be present in the level omega pLX backbone, flanking our construct. We expected two bands: a 3kb band for the pLX backbone and 9kb band for our full genetic construct. We could also get a 12kb band if the plasmid isn’t completely digested. However, none of our miniprep products showed this. Instead, they had a doublet or singlet band at around 4 kb.

Interpreting the gel was difficult since when we tried to run the bands apart further, they disappeared, presumably because of the low amount of miniprep product. In case EtBr had diffused out of the gel, we soaked the gels in a 0.5 µg/ml EtBr solution for 30 minutes, then overnight, but this did not improve our bands.

Diagnostic Colony PCR

We then ordered primers for each transcriptional unit (PFt, RUBY, and nptII) and ran colony PCR on 4 colonies picked from each plate, as well as for the FB and SGR9 miniprep products that has returned sequencing results (out of curiosity). We decided to screen using two rounds of PCR; first for PFt as it was considered our most valuable gene, and second for RUBY and nptII, out of those that passed the first round.

4 of the newly picked colonies (SSG1 A, SSG1 C, SSG1 D, FB C) had successful PFt bands.

We repeated colony PCR on 3 colonies picked from each of the plates that failed (Hscp, DPE1, SGR9, SS4) and found one successful (SGR9 F). However, because we started running out of gels, and we already had a Pft positive SGR9 plasmid (SGR9 MP), we did not decide to continue screening this one.

Five colonies proceeded to the nptII PCR, and all of them had successful bands.

The same five proceeded to the RUBY PCR. Ultimately, the fully positive plasmids were FB C, SSG1 A, SSG1 C, and FB MP, all of which we sent for full-plasmid sequencing through Plasmidsaurus.

Redo level omega assembly

While waiting for sequencing results, we redid level omega assembly for the four transit peptides for which we had no good colonies - Hscp, SS4, SGR9, and DPE1 - and then performed the same 2-round colony PCR screen. This time, we got at least one colony from each plate that passed the PFt and RUBY screens, but none passed the nptII screen. Nonetheless, considering that RUBY would likely be sufficient to select successfully transformed seeds, we continued with agrobacterium transformation.

Agrobacterium transformation was performed at Jeff Shipp’s lab at APL. Unfortunately, because the transformation window for our first batch of Arabidopsis had passed, we were not able to transform them. We planned to perform a floral dip transformation of Wisconsin FastPlants (a fast-cycling variety of Brassica rapa) on 10/10. Because RUBY expression will be immediately obvious, turning the seeds red even within the seed pod, we hope that the plants will produce harvestable seeds and can grow for a few days in the clinostat before the Jamboree.

Full-Plasmid Sequencing

Full plasmid sequencing showed that for colony C of the FB construct (BBa_K4452021) level omega assembly worked, but level alpha assembly had failed, since the PFt transcriptional unit was missing the nos terminator.

The FB MP construct (BBa_K4452021) that had been sequenced with M13 reverse primer and showed an unexpectedly placed PFt was also sent to be fully sequenced. The results showed that the plasmid had ligated to double the expected size at 23kb and was rotationally symmetrical!

Results of sequencing for SSG1 C (BBa_K4452025) showed that PFt, RUBY, and nptII resistance genes were all included, albeit backwards.