iGEM 2022 — Team hopkins

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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## What should this page contain?

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* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Timelines

###### April 2019

— The launch of Aryacoin; AYA ICO, resulting in over 30BTC collected

###### December 2019

— The launch of AYA Pay

###### April 2020

— The successful Hamedan Hardfork, supported by all AYA exchanges, aimed at integrating the dPoW algorithm, improving the security of the AYA blockchain.

###### June 2020

— Aryna Exchange goes live, opening more trading opportunities globally

###### July 2020

— The enabling of our Coin Exchanger

###### November 2020

— The implementation of Smart Contracts into the Aryacoin Ecosystem

###### Q1 2021

— Alef B goes live (more details coming soon)

## Inspirations

---

* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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{% block title %}Engineering Success{% endblock %}
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Below we describe how we accomplished engineering success by going through the Design -> Build -> Test -> Learn cycle while assembling our genetic constructs. More details about our wetlab work can be found in our [lab notebook](https://static.igem.wiki/teams/4452/wiki/entries.pdf).

## 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](https://www.uniprot.org/uniprotkb/Q8GXF8/feature-viewer).

##### 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](https://www.uniprot.org/uniprotkb/Q0WVX5/feature-viewer). 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](https://www.uniprot.org/uniprotkb/Q9MAQ0/feature-viewer).

##### 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](https://www.uniprot.org/uniprotkb/Q9LV91/feature-viewer).

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.

![Gene design for composite part BBa_K4452011.](https://static.igem.wiki/teams/4452/wiki/engg-success-main-gene-construct.png)

BBa\_K4452011: 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

| Part | Description | BsaI Site |
| --- | --- | --- |
| [BBa\_K4452000](http://parts.igem.org/Part%3ABBa_K4452000) PLT2 promoter | GGAG / TACT | |
| [BBa\_K4452001](http://parts.igem.org/Part%3ABBa_K4452001%20), [BBa\_K4452002](http://parts.igem.org/Part%3ABBa_K4452002), [BBa\_K4452003](http://parts.igem.org/Part%3ABBa_K4452003%20), [BBa\_K4452004](http://parts.igem.org/Part%3ABBa_K4452004), [BBa\_K4452005](http://parts.igem.org/Part%3ABBa_K4452005%20), or [BBa\_K4452006](http://parts.igem.org/Part%3ABBa_K4452006) Transit Peptide | TACT / AATG | |
| [BBa\_K4452007](http://parts.igem.org/Part%3ABBa_K4452007) Pyrococcus furiosus ferritin (PFt) | AATG / TTCG | |
| [BBa\_K4452008](http://parts.igem.org/Part%3ABBa_K4452008) GFP with 5xGS linker | TTCG / GCTT | |
| [BBa\_J428082](http://parts.igem.org/Part%3ABBa_J428082) Plant threeUTRs Tnos | GCTT / CGCT | |
| [BBa\_J428341](http://parts.igem.org/Part%3ABBa_J428341) pJUMP29-1A | GGAG / CGCT | |

##### Gene 2: RUBY Reporter

| Part | Description | BsaI Site |
| --- | --- | --- |
| [BBa\_J428074](http://parts.igem.org/Part%3ABBa_J428074) Plant promoter CaMV35S | GGAG / TACT | |
| [BBa\_J428088](http://parts.igem.org/Part%3ABBa_J428088) Plant fiveUTRs AtRbcS2B 5UTR | TACT / AATG | |
| [BBa\_K3900028](http://parts.igem.org/Part%3ABBa_K3900028) RUBY | AATG / GCTT | |
| [BBa\_J428082](http://parts.igem.org/Part%3ABBa_J428082%20) Plant threeUTRs Tnos | GCTT / CGCT | |
| [BBa\_J428342](http://parts.igem.org/Part%3ABBa_J428342) pJUMP29-1B | GGAG / CGCT | |

##### Gene 3: nptII Antibiotic Resistance

| Part | Description | BsaI Site |
| --- | --- | --- |
| [BBa\_J428074](http://parts.igem.org/Part%3ABBa_J428074) Plant promoter CaMV35S | GGAG / TACT | |
| [BBa\_J428088](http://parts.igem.org/Part%3ABBa_J428088) Plant fiveUTRs AtRbcS2B 5UTR | TACT / AATG | |
| [BBa\_J428077](http://parts.igem.org/Part%3ABBa_J428077) nptII | AATG / GCTT | |
| [BBa\_J428082](http://parts.igem.org/Part%3ABBa_J428082) Plant threeUTRs Tnos | GCTT / CGCT | |
| [BBa\_J428343](http://parts.igem.org/Part%3ABBa_J428343) pJUMP29-1C | GGAG / CGCT | |

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.

| Part | Description | BsmBI Site |
| --- | --- | --- |
| [BBa\_K4452011](http://parts.igem.org/Part%3ABBa_K4452011), [BBa\_K4452012](http://parts.igem.org/Part%3ABBa_K4452012), [BBa\_K4452013](http://parts.igem.org/Part%3ABBa_K4452013), [BBa\_K4452014](http://parts.igem.org/Part%3ABBa_K4452014), [BBa\_K4452015](http://parts.igem.org/Part%3ABBa_K4452015%20), or [BBa\_K4452016](http://parts.igem.org/Part%3ABBa_K4452016) Gene 1: Ferritin | GGAG / AATG | |
| [BBa\_K4452017](http://parts.igem.org/Part%3ABBa_K4452017) Gene 2: RUBY Reporter | AATG / AGCC | |
| [BBa\_K4452018](http://parts.igem.org/Part%3ABBa_K4452018) Gene 3: nptII Antibiotic Resistance | AGCC / TTCG | |
| [BBa\_K4452009](http://parts.igem.org/Part%3ABBa_K4452009) Gene 4: M13R primer | TTCG / CGCT | |
| [BBa\_K4452010](http://parts.igem.org/Part%3ABBa_K4452010) pLX-B3(omega)1 | GGAG / CGCT | |

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.

| Part | Description |
| --- | --- |
| [BBa\_K4452021](http://parts.igem.org/Part%3ABBa_K4452021) Ferritin with prFB transit peptide + RUBY + nptII | |
| [BBa\_K4452022](http://parts.igem.org/Part%3ABBa_K4452022) Ferritin with prcpHsc70-1 transit peptide + RUBY + nptII | |
| [BBa\_K4452023](http://parts.igem.org/Part%3ABBa_K4452023) Ferritin with prSGR9 transit peptide + RUBY + nptII | |
| [BBa\_K4452024](http://parts.igem.org/Part%3ABBa_K4452024) Ferritin with prSS4 transit peptide + RUBY + nptII | |
| [BBa\_K4452025](http://parts.igem.org/Part%3ABBa_K4452025) Ferritin with prSSG1 transit peptide + RUBY + nptII | |
| [BBa\_K4452026](http://parts.igem.org/Part%3ABBa_K4452026) Ferritin with prDPE1 transit peptide + RUBY + nptII | |

## Build

---

The first step of assembling our genetic constructs was to generate enough DNA of the basic parts. For the parts from the 2022 iGEM Distribution Kit, we located their well locations in the distribution plates, resuspending the DNA and transformed some of the DNA into E. coli. The transformed cells were plated on LB agar plates with the appropriate antibiotic resistance according to the part’s plasmid vector. Following growth on plates, bacterial colonies were transferred to liquid cultures which were miniprepped to get purified plasmids for assembly.

The table below shows the location of parts in the iGEM distribution kit plate 1 and the antibiotic resistance conferred by the parts’ plasmid backbone.

| Part Number | Description | Well | Plasmid | Antibiotic Resistance |
| --- | --- | --- | --- | --- |
| [BBa\_BBa\_J428341](http://parts.igem.org/Part%3ABBa_J428341) pJUMP29-1A | 2A | BBa\_J428326 | Kanamycin | |
| [BBa\_J428342](http://parts.igem.org/Part%3ABBa_J428342) pJUMP29-1B | 2C | BBa\_J428326 | Kanamycin | |
| [BBa\_J428343](http://parts.igem.org/Part%3ABBa_J428343) pJUMP29-1C | 2E | BBa\_J428326 | Kanamycin | |
| [BBa\_K3900028](http://parts.igem.org/Part%3ABBa_K3900028) RUBY | 19I | pSB1C5C | Chloramphenicol | |
| [BBa\_J428074](http://parts.igem.org/Part%3ABBa_J428074) 35S | 19K | pSB1C5A | Chloramphenicol | |
| [BBa\_J428088](http://parts.igem.org/Part%3ABBa_J428088) AtRbcS2B | 20E | pSB1C3SB | Chloramphenicol | |
| [BBa\_J428077](http://parts.igem.org/Part%3ABBa_J428077%20) nptII | 21C | pSB1C5C | Chloramphenicol | |
| [BBa\_J428082](http://parts.igem.org/Part%3ABBa_J428082%20) Tnos | 21O | pSB1C5SD | Chloramphenicol | |

Basic parts that were synthesized by Twist were resuspended to be used directly in assembly reaction.

### Level Alpha Assembly

---

Using the NEBridge® Golden Gate Assembly Kit (BsaI-HF®v2), we set up the eight assembly reactions in the table below for level alpha assembly with the following protocol:
 **(37°C, 1.5 min → 16°C, 3 min) x 30 cycles → 60°C, 5 min → 80°C, 20 min**

| Reaction # | Inserts | Vector | Resulting Part | Description |
| --- | --- | --- | --- | --- |
| 1 | BBa\_K4452000  BBa\_K4452001  BBa\_K4452007  BBa\_K4452008  BBa\_J428082 | BBa\_J428341 | BBa\_K4452011 | Gene 1: Ferritin with prFB transit peptide |
| 2 | BBa\_K4452000  BBa\_K4452002  BBa\_K4452007  BBa\_K4452008  BBa\_J428082 | BBa\_J428341 | BBa\_K4452012 | Gene 1: Ferritin with prcpHsc70-1 transit peptide |
| 3 | BBa\_K4452000  BBa\_K4452003  BBa\_K4452007  BBa\_K4452008  BBa\_J428082 | BBa\_J428341 | BBa\_K4452013 | Gene 1: Ferritin with prSGR9 transit peptide |
| 4 | BBa\_K4452000  BBa\_K4452004  BBa\_K4452007  BBa\_K4452008  BBa\_J428082 | BBa\_J428341 | BBa\_K4452014 | Gene 1: Ferritin with prSS4 transit peptide |
| 5 | BBa\_K4452000  BBa\_K4452005  BBa\_K4452007  BBa\_K4452008  BBa\_J428082 | BBa\_J428341 | BBa\_K4452015 | Gene 1: Ferritin with prSSG1 transit peptide |
| 6 | BBa\_K4452000  BBa\_K4452006  BBa\_K4452007  BBa\_K4452008  BBa\_J428082 | BBa\_J428341 | BBa\_K4452016 | Gene 1: Ferritin with prDPE1 transit peptide |
| 7 | BBa\_J428074  BBa\_J428088  BBa\_K3900028  BBa\_J428082 | BBa\_J428342 | BBa\_K4452017 | Gene 2: RUBY Reporter |
| 8 | BBa\_J428074  BBa\_J428088  BBa\_J428077  BBa\_J428082 | BBa\_J428343 | BBa\_K4452018 | Gene 3: nptII Antibiotic Resistance |

The level alpha assembly products were transformed into competent E. coli cells and grown on kanamycin plates. Two colonies from each plate were grown in LB liquid culture overnight and miniprepped.

To test the success of level alpha assembly, we designed primers that would bind to the ends of the pJUMP backbone so we could amplify our gene inserts sequences with PCR.
We ran the PCR products on a gel looking for the appropriate band sizes (Gene 1: 2855 bp, Gene 2: 5083 bp, Gene 3: 1927 bp)

![Photo of gel for diagnostic PCR.](https://static.igem.wiki/teams/4452/wiki/engg-success-gel-image-1.png)

Gel for diagnostic PCR for level alpha assembly.

We also sent our miniprep product with the forward primer for Sanger sequencing. For reactions #1 to #6 we sent miniprep product #1 and for reactions #7 and #8 we sent miniprep product #2.

With the results of sanger sequencing, we confirmed that Gene 2 (BBa\_K4452017) included the CaMV35S promoter, the AtRbcS2B 5UTR, and the beginning of RUBY. We also confirmed that Gene 3 (BBa\_K4452018) included the CaMV35S promoter, the AtRbcS2B 5UTR, and the beginning of nptII. For each of the versions of Gene 1 (BBa\_K4452011 - BBa\_K4452016), we could confirm the PLT2 promoter and the beginning of the transit peptide.

### Level Omega Assembly

---

Because the miniprep yields of the parts needed for level omega assembly were too low, we needed to amplify the products using PCR before attempting the assembly. We cleaned up the amplicons with Qiagen QIAquick PCR Purification Kit.

For the level omega assembly, we used the NEBridge® Golden Gate Assembly Kit (BsmBI-v2) and we set up the six assembly reactions in the table below with the following protocol:
**(42°C, 1.5 min → 16°C, 3 min) x 30 cycles → 60°C, 5 min → 80°C, 20 min**

| Reaction # | Inserts | Vector | Resulting Part | Description |
| --- | --- | --- | --- | --- |
| 1 | BBa\_K4452011  BBa\_K4452017  BBa\_K4452018  BBa\_K4452009 | BBa\_K4452010 | BBa\_K4452021 | Ferritin with prFB transit peptide + RUBY reporter + nptII |
| 2 | BBa\_K4452012  BBa\_K4452017  BBa\_K4452018  BBa\_K4452009 | BBa\_K4452010 | BBa\_K4452022 | Ferritin with prcpHsc70-1 transit peptide + RUBY reporter + nptII |
| 3 | BBa\_K4452013  BBa\_K4452017  BBa\_K4452018  BBa\_K4452009 | BBa\_K4452010 | BBa\_K4452023 | Ferritin with prSGR9 transit peptide + RUBY reporter + nptII |
| 4 | BBa\_K4452014  BBa\_K4452017  BBa\_K4452018  BBa\_K4452009 | BBa\_K4452010 | BBa\_K4452024 | Ferritin with prSS4 transit peptide + RUBY reporter + nptII |
| 5 | BBa\_K4452015  BBa\_K4452017  BBa\_K4452018  BBa\_K4452009 | BBa\_K4452010 | BBa\_K4452025 | Ferritin with prSSG1 transit peptide + RUBY reporter + nptII |
| 6 | BBa\_K4452016  BBa\_K4452017  BBa\_K4452018  BBa\_K4452009 | BBa\_K4452010 | BBa\_K4452026 | Ferritin with prDPE1 transit peptide + RUBY reporter + nptII |

## Test

---

### Sanger Sequencing

Following transformation of our level omega products, we picked two colonies from each plate and cultured them in LB overnight. We then directly miniprepped them and got a high yield for one colony from each plate. These were sent for sanger sequencing with the M13 reverse primer, which should produce a ~1kbp sequence for part of our nptII. Puzzlingly, sequencing results were only returned for the prFB (BBa\_K4452021) and prSGR9 (BBa\_K4452023) assemblies, for which we received near-identical sequences of our ferritin and nos terminator (0 mismatches and 8 mismatches respectively). We hypothesized that the other plasmids did not return results because the M13 spacer did not make it in, whereas for the two that did, the M13 spacer was somehow next to the PFt transcriptional unit, and RUBY and nptII might not be present.

![Plasmid map of expected plasmid sequence for BBa_K4452021.](https://static.igem.wiki/teams/4452/wiki/engg-success-plasmid.png)

Expected plasmid sequence for BBa\_K4452021.

## 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.

![Photo of Miniprep digest gel 1](https://static.igem.wiki/teams/4452/wiki/engg-success-gel-image-2.png)

Miniprep digest gel 1 (\* = returned sequencing results).

![Photo of Miniprep digest gel 2](https://static.igem.wiki/teams/4452/wiki/engg-success-gel-image-3.png)

Miniprep digest gel 2 (redo)

### 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.

![Photo of Colony PCR gel](https://static.igem.wiki/teams/4452/wiki/engg-success-gel-image-4.png)
![Photo of Colony PCR gel](https://static.igem.wiki/teams/4452/wiki/engg-success-gel-image-5.png)

PFt colony PCR screen

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.

![Photo of Colony PCR gel](https://static.igem.wiki/teams/4452/wiki/engg-success-gel-image-6.png)

PFt colony PCR screen

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

![Photo of Colony PCR gel](https://static.igem.wiki/teams/4452/wiki/engg-success-gel-image-7.png)

NptII colony PCR screen / PFt colony PCR screen

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.

![Photo of Colony PCR gel](https://static.igem.wiki/teams/4452/wiki/engg-success-gel-image-8.png)

RUBY colony PCR screen

### 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.

## Success

---

We did have one successful assembly with the correct orientation. Full sequencing results revealed that assembly for colony A of the SSG1 construct (BBa\_K4452025) worked perfectly.

### References

---

[1] Matsumoto, Yuri, et al. "Engineering intracellular biomineralization and biosensing by a magnetic protein." Nature communications 6.1 (2015): 1-10. <https://doi.org/10.1038/ncomms9721>

[2] Singh, Manjul et al. "Striking the right chord: signaling enigma during root gravitropism." Frontiers in Plant Science 8 (2017): 1304. <https://doi.org/10.3389/fpls.2017.01304>

[3] Li, Huchen et al. “Plant-Specific Histone Deacetylases HDT1/2 Regulate GIBBERELLIN 2-OXIDASE2 Expression to Control Arabidopsis Root Meristem Cell Number.” The Plant cell vol. 29,9 (2017): 2183-2196. <https://doi.org/10.1105/tpc.17.00366>

[4] Marquès-Bueno, Maria Del Mar et al. “A versatile Multisite Gateway-compatible promoter and transgenic line collection for cell type-specific functional genomics in Arabidopsis.” The Plant journal : for cell and molecular biology vol. 85,2 (2016): 320-333. <https://doi.org/10.1111/tpj.13099>

[5] Santuari, Luca et al. “The PLETHORA Gene Regulatory Network Guides Growth and Cell Differentiation in Arabidopsis Roots.” The Plant cell vol. 28,12 (2016): 2937-2951. <https://doi.org/10.1105/tpc.16.00656>

[6] Galinha, C., Hofhuis, H., Luijten, M. et al. “PLETHORA proteins as dose-dependent master regulators of Arabidopsis root development”. Nature 449, 1053–1057 (2007). <https://doi.org/10.1038/nature06206>

[7] Aida, Mitsuhiro et al. “The PLETHORA Genes Mediate Patterning of the Arabidopsis Root Stem Cell Niche.” Cell vol. 119, 1 (2004): 109-120. <https://doi.org/10.1016/j.cell.2004.09.018>

[8] Chiung-Chih Chu, Krishna Swamy, Hsou-min Li, Tissue-Specific Regulation of Plastid Protein Import via Transit-Peptide Motifs, The Plant Cell, Volume 32, Issue 4, April 2020, Pages 1204–1217, <https://doi.org/10.1105/tpc.19.00702>

[9] Moritaka Nakamura, Masatsugu Toyota, Masao Tasaka, Miyo Terao Morita, An Arabidopsis E3 Ligase, SHOOT GRAVITROPISM9, Modulates the Interaction between Statoliths and F-Actin in Gravity Sensing , The Plant Cell, Volume 23, Issue 5, May 2011, Pages 1830–1848, <https://doi.org/10.1105/tpc.110.079442>

[10] Roldán, I., Wattebled, F., Mercedes Lucas, M., Delvallé, D., Planchot, V., Jiménez, S., Pérez, R., Ball, S., D'Hulst, C. and Mérida, Á. (2007), The phenotype of soluble starch synthase IV defective mutants of Arabidopsis thaliana suggests a novel function of elongation enzymes in the control of starch granule formation. The Plant Journal, 49: 492-504. <https://doi.org/10.1111/j.1365-313X.2006.02968.x>

[11] Seung D, Soyk S, Coiro M, Maier BA, Eicke S, et al. (2015) PROTEIN TARGETING TO STARCH Is Required for Localising GRANULE-BOUND STARCH SYNTHASE to Starch Granules and for Normal Amylose Synthesis in Arabidopsis. PLOS Biology 13(2): e1002080. <https://doi.org/10.1371/journal.pbio.1002080>

[12] Tenorio, G., Orea, A., Romero, J.M. et al. Oscillation of mRNA level and activity of granule-bound starch synthase I in Arabidopsis leaves during the day/night cycle. Plant Mol Biol 51, 949–958 (2003). [https://doi.org/10.1023/A:1023053420632](https://doi.org/10.1023/A%3A1023053420632)

[13] Steven M. Smith, Daniel C. Fulton, et al. Diurnal Changes in the Transcriptome Encoding Enzymes of Starch Metabolism Provide Evidence for Both Transcriptional and Posttranscriptional Regulation of Starch Metabolism in Arabidopsis Leaves, Plant Physiology, Volume 136, Issue 1, September 2004, Pages 2687–2699, <https://doi.org/10.1104/pp.104.044347>

[14] Lin YC, Chang SC, Juang RH (2017) Plastidial α-glucan phosphorylase 1 complexes with disproportionating enzyme 1 in Ipomoea batatas storage roots for elevating malto-oligosaccharide metabolism. PLOS ONE 12(5): e0177115. <https://doi.org/10.1371/journal.pone.0177115>

[15] Wang, Q., Mercogliano, C. P., & Löwe, J. (2011). A ferritin-based label for Cellular Electron Cryotomography. Structure, 19(2), 147–154.
<https://doi.org/10.1016/j.str.2010.12.002>

[16] Kim, S.-E., Ahn, K.-Y., Park, J.-S., Kim, K. R., Lee, K. E., Han, S.-S., & Lee, J. (2011). Fluorescent ferritin nanoparticles and application to the Aptamer Sensor. Analytical Chemistry, 83(15), 5834–5843. <https://doi.org/10.1021/ac200657s>

[17] Sarrion-Perdigones A, Falconi EE, Zandalinas SI, Juárez P, Fernández-del-Carmen A, et al. (2011) GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules. PLOS ONE 6(7): e21622. <https://doi.org/10.1371/journal.pone.0021622>

[18] Pasin, Fabio et al. “Multiple T-DNA Delivery to Plants Using Novel Mini Binary Vectors with Compatible Replication Origins.” ACS synthetic biology vol. 6,10 (2017): 1962-1968. <https://doi.org/10.1021/acssynbio.6b00354>

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}