iGEM 2023 — Team eastcoastbiocrew

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# Notebook

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March

**25th March**- First East Coast BioCrew meetup. Introduced members and researched previous iGEM projects to learn more about the competition.

April

**1st April**- Split into subgroups to brainstorm ideas for our project

**15th April**- Narrowed down idea: combat phosphorus pollution and air pollution-- amplifying or modifying plant organism to absorb air pollution and phosphorus

**22nd April**- Attended "Future of syntehtic Biology Conference"

May

**20th May**- Researched literature regarding rhizobium. Found glucose dehydrogenase (GDH) with cofactor Pyrroloquinline quinone (Pqq) had the ability to solubilize mineral phosphate.

June

**3rd June**- Started compiling information about Rhizobium tropici CIAT 899. Researched various medias R. tropici could grow in.

**10th June**- Planned for a two part system: one making the phosphate soluble and the other stimulating phosphate uptake. Researched systems for increasing phosphate uptake and found pstSCAB operon, which was used by previous iGEM teams.

**17th June**- Engineered gene constructs on Benchling and ordered them.

**24th June**- Spoke with Dr. Reid through zoom. Decided to make the project for private use after going through pros and cons. Began drafting emails to stakeholder groups.

July

**1st July**- Overview of media: ATCC 111 (soil), yeast mannitol (high Ca), yeast mannitol (low Ca). Began blue white screening with E. coli and empty plasmid.

**8th July**- Measured optical densities of R. tropici growing in three medias and found no significat growth rate differences. Blue white screening did not work as planned. Discussed future plans and modification to protocol.

**15th July**- Had succesful blue white screening. Made plans for plant experiments with root nodulation.

**22nd July**- GenSpace received R. tropici and empty plasmids.

**29th July**- Soybean plants grew, and we began the innoculation process. GenSpace performed PCR screening and discovered the genes were not inserted properly due to primer issues.

August

**5th August**- Set up Q5 PCR doubling this protocol (PCR using Q5) with tubes 1 and 2 as pBBR1 with PpqC primers and tubes 3 and 4 as pBBR1 with pstSCAB primers. We repeated the PCR and transformation.

**11th August**- Two team members attended the iGEM Mid-Atlantic Regional meetup and presented project, receiving feedback from other iGEMers.

**12th August**- Set up Q5 colony PCR. Sets 1 and 2 are pBBR2 that contain PqqC and gcd. Ran 20 clones from set 1 (1-1 through 1-20). Ran 8 clones from set 2 (2-1 through 2-8). Sets 3 and 4 are pBBR2 that contain pstSCAB. Ran 13 clones from set 3 (3-1 through 3-13). Ran 10 clones from set 4 (4-1 through 4-10). Positive clones appear to be 1-1, 1-2, 2-1, 3-1(?), 3-3 (looks small), 3-4 (looks small), 3-13, 4-1, and 4-7. Grew cultures overnight in LB+kanamycin (except for #3-4).

**19th August**- Ran PCR and gel electrophoresis with positive clones from last week. Plasmid Concetration; DNA recordings. Measured plasmid concentration with nanodropper. Set up PCR using 1 ul of each DNA as template using Q5 2X Master mix with primers M13F and M13R.

September

**2nd September**- Used electroporation to transofmr our R. tropici with Pqqc+gcd and pstSCAB genes.

**4th September**- GenSpace made 10% glycerol solution for bacterial washing before electroporation and innoculated transformed E. coli from BUGGS on to agar plates.****

**5th September**- GenSpace isolated plasmid DNA from E. coli bacterial cultures with MiniPrep and pelleted R. tropici inEppendorf tubes for easier storage.

**9th September**- Began working on promotional video and judging form. Began work on parts registry.

**10th September**- GenSpace prepared for transforming electrocompetent R. tropici by making SOC medium, TSB Yeast medium plates, and preparing R. tropici for electroporation (competent cells stored in -80)

**16th September**- PCR of R. tropici in plasmid has been started. Plants have been growing quicker, temperature has increased since the plants were put in the incubator, the next measurement may not be ready in time for the wiki freeze. Genspace has made their competent cells, have made their first transformation.

**23rd September**- Began phosphate uptake plate assays. Met with the Chesapeake Bay Foundation-- will be writing a summary about what we learned: phosphate solubility relies on pH, when testing test in a range of pH conditions.

**24th September**- GenSpace removed the bacteria after two days of growth at 30 C and placed in minifridge for storage. Results - looking like some kind of contamination on plates 1 and 3. GenSpace Replated remainder of transformed Rhizobium on fresh TSB + Kanamycin plates, grew at 30C for 48 hrs, stored in minifridge.

**30th September**- Bacterial ODs are being taken again today for the model. Phosphate plate assays were not viable-- retesting with different bacterial dilutions and added step of drying plates under laminar flow hood. Phosphate colorimetric kit has arrived at Genspace.

October

**1st October**- GenSpace made 500 mL TSB Broth according to protocol and aliquoted 5 mL into 15 mL snap cap tubes. Inoculated: 3 colonies of PqqC + gcd, 3 empty pBBR2, and 3 colonies of pstSCAB.

**4th October**- GenSpace received package sent from BUGGS containing calcium phosphate tribasic, R. tropici, mannitol, P. fluorescens. Made 250 mL of tricalcium phosphate broth + kanamycin according to protocol. Measured OD values of GenSpace-grown Rhizobium. Inoculated GenSpace-grown R. tropici into tricalcium phosphate broth and incubated at 30 C. Inoculated BUGGS-grown R. tropici into TSB and incubated at 30 C.

**7th October**- GenSpace took OD of BUGGS R. tropici. Added amount from tryptic soy broth (TSB) to tricalcium phosphate broth (TCP). Added varying amounts to ensure that equal OD (0.881) was added. Found value by dividing lowest OD (0.881) by each sample's respective OD value. BUGGS ran protein gel on 8 colonies: 5 contained PqqC + gcd genes and 3 contained pstSCAB genes. Protein gel did not provide conclusive results.

**10th October**- Protein gel was run again, and results were...

**11th October**- GenSpace ran phosphate colorimetric assays, and results were....

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# Engineering Success

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## Design

## Selecting Chassis

When we were designing our project, our goal was to genetically modify an organism to uptake phosphate and solubilize it to utilize unavailable phosphate and prevent unwanted runoff. Originally, we wanted to engineer a plant to be able to solubilize phosphate, but we were worried that engineering a eukaryotic organism would not fit the scope of this project. However, we wanted to prioritize the use of plants since our team is based in two coastal cities at risk of flooding due to the lack of greenery since encouraging our city governments and people to plant more greenery will help protect our communities from flooding. While researching, we encountered the bacteria rhizobia, which has a symbiotic relationship with plants, solubilizing phosphate so that it can be available to plants. Because of this pre-existing relationship, we decided to engineer rhizobia to increase its uptake and conversion of phosphate. This allowed us to engineer a prokaryotic organism, which would fit the scope of our project, while also encouraging the use of plants in our coastal cities.

## Choosing Soil Media ![](https://static.igem.wiki/teams/4649/wiki/od-graph.png) When researching prior projects and experiments related to *Rhizobium tropici*, we encountered three different types of media in which the bacteria were grown by the ([2019 Tacoma team](https://static.igem.org/mediawiki/2019/a/a1/T--Tacoma_RAINmakers--otsAOverexpression_Notes.pdf)). In order to determine which media we should use for our experiments, we conducted a trial in each media. The optical densities of the different samples at different times are recorded in the table above as well as in the graph below. A higher optical density indicates that there is a higher concentration, and therefore quantity, of bacteria. The negative readings for samples 3 and 4 are likely machinery errors due to high concentrations of Ca causing opacity, and due to those readings it was not possible to generate an equation for the OD in the soil media. Overall the yeast mannitol with low calcium showed an exponential growth of y= 0.0479e0.445x with an r2 value of 0.99. The yeast mannitol with high calcium showed an exponential growth of y=0.0343e0.4582x with an r2 value of 0.94. A t-test paired two samples for means performed between the ATCC 111 media, which was the most commonly used one in literature, and the yeast mannitol extract with low Ca concentrations yielded a p-value of 0.11 (greater than the threshold of 0.05) showing no significant statistical difference found in growth. A t-test of two samples for the means between the low Ca concentration yeast mannitol and high Ca concentration yeast mannitol yielded a p-value of 0.11. Therefore, since there was no statistical difference in growth, it was decided to use the yeast mannitol low Ca soil media since a low Ca was preferred for increased accuracy of measurements and lower cost and there was no evidence to suggest that the yeast mannitol with higher concentrations of Ca would yield higher rhizobium ODs.

## Design of Plasmid

Our research and guidance from our mentors led us to discover two key pathways in Rhizobacteria for mineral phosphate solubilization: the Pqq(FABCDEG) genes, which are believed to play a role in Pyrroloquinoline quinone (Pqq) biosynthesis as a redox cofactor to glucose dehydrogenase (GDH) enzyme, and the pst(SCAB) genes, which have been identified as being involved in phosphate transportation. We chose to use the pBBR1MCS vector in the pBBR1MCS-2 plasmid from addgene ( that the [2019 Tacoma team](https://2019.igem.org/Team%3ATacoma_RAINmakers/Design) used because this vector contained the lac operon, a well documented promoter, and it contains a multiple cloning site with blue white selection which would allow us to easily see if our plasmid was transforming successfully. Since this lac promoter is a derepressed promoter in E. coli, we chose it because it may be constitutive in R. tropici.

![](https://static.igem.wiki/teams/4649/wiki/screenshot-2023-10-11-220831.png)

We decided to use these chosen genes in a coupled approach to solving the issue of phosphate contamination: solubilizing phosphate to make it available to plants (with the Pqq and GDH) and to increase the uptake of the mineral phosphates (with the pst(SCAB) transporter) by rhizobia. We chose the lac operon promoter for our codon optimized genes because we desired constitutive promoter for our construct and the lac operon promoter is both constitutive and very well-documented. We codon optimized these genes with Benchling and had them synthesized by Twist Bioscience.
![](https://static.igem.wiki/teams/4649/wiki/ptscab-figure1.png)
![](https://static.igem.wiki/teams/4649/wiki/ptscab-figure2.png)

## Build

## Build Plasmid

To figure out how best to transform our plasmid into rhizobia, we practiced transforming the plasmid into rhizobia before we had cloned our genes into the plasmid. For our first attempt, we followed a blue-white screening procedure from the 2014 Hannover team for rhizobium electroporation.
To test that our plasmid was functional, we performed a blue-white screening. The intact plasmid contains a functioning LacZ gene, so when we add Xgal and IPTG to the plasmid, it should turn blue. However, when the genes have been inserted into the plasmid and the LacZ gene has been disrupted, the plasmid should turn white.

We performed the blue-white screening on the plasmid before cloning our genes to ensure that the plasmid was operational. The results of this procedure were not successful; no bacterial colonies turned blue and mold had formed on the plates, compromising the validity of the results.
In our next attempt, we changed the concentration of DNA while following the same procedure and holding all other variables constant and ensured that the lids of the petri dishes were tighter than before to prevent any mold from getting in and disturbing the samples. This time the results showed blue colonies, showing that our plasmids had an operating LacZ gene.

## Building Our Inoculated Plants

To test compatibility of different plants native to our area with rhizobia, we grew and inoculated four species of plants (showy tick trefoil, the common bean, American senna, and soybeans) in African violet soil at maturation. Once a week we measured the plants growth and looked for nodules. We only observed nodulation in one showy tick trefoil plant, so we did not consider these experiments a success. After discussing with Dr. Sharon Long (more information can be found here: ) we changed the temperature of the plants' environment from 20 degrees celsius to 30 degrees celsius and began measuring the pH of the soil to see if that was affecting the plants' growth.

## Test

## Results of Cloning

The plasmid that we are using for cloning, pBBR1MCS-2, has a LacZ gene in it, so we can do blue-white screening in E. coli to see if our genes were inserted. Our blue-white screening showed that our plasmids were successfully assembled in E. coli. From there, we screened the plasmids by PCR, isolated the plasmids from E. coli, and then transformed them into Rhizobia. We then screened our Rhizobium by PCR, and our PCR and gel electrophoresis experiments showed that our transference to rhizobia was successful. We were able to confirm the length of inserted fragments. We expected a fragment length of around 3700 base pairs for pstSCAB and a fragment length of around 3200 for PqqC + gcd. Colonies that begin with the numbers 1 & 2 contain pstSCAB (~3700), and colonies that begin with the numbers 3 & 4 contain PqqC+gcd (~3200) From the image below, samples 1-1, 1-2, and 2-1 showed successful gene insertion of pstSCAB genes; samples 3-3, 3-4, 4-2, 4-3, 4-4, 4-5, and 4-6 showed successful gene insertion of PqqC+gcd genes.

![E. coli Transformation 8/5/23](https://static.igem.wiki/teams/4649/wiki/20230807-133107.jpg)
![E. coli Transformation 8/5/23](https://static.igem.wiki/teams/4649/wiki/copy-of-20230807-133114.jpg)
![R. tropici Transformation 8/5/23](https://static.igem.wiki/teams/4649/wiki/20230807-133121.jpg)
![R. tropici Transformation 8/5/23](https://static.igem.wiki/teams/4649/wiki/20230807-133128.jpg)
![Gel 8/12/23](https://static.igem.wiki/teams/4649/wiki/picture1.png)

We then transformed our plasmid into e.coli. Once we had successfully transformed them into e.coli, we transformed them into rhizobium. Our PCR test showed that our Rhizobium contained our genes, but we wanted to see if the proteins were actually produced from the inserted genes. Our first protein gel did not provide conclusive results. The ladder we used, (SMOBIO ExcelBand™ 3-color Regular Range Protein Marker, PM2500), allowed us to see that most of our proteins were under 25000 Da, which is not expected because the masses of our proteins are greater than 25000Da. In addition, there appeared to be a lot of debris in the gel-- either from cell lysis or proteins congregating. We ran another protein gel, ensuring cell debris is not inside the gel. This yielded more success as we saw promising results for gcd protein synthesis. We expected 87 kDa for GDH enzyme and saw a corresponding band for one of our PqqC+gcd samples- the arrow marks the band in the image below. It is worth noting that we were expecting to see the proteins running at their individual sizes, but because pstA, pstB, and pstC form the pstABC transporter and, therefore, form strong associations, we were also aware that we might see them still associating in the gel and a protein complex at a larger size. This would correspond to around 95 kDa.

![Expected Protein Masses](https://static.igem.wiki/teams/4649/wiki/screenshot-2023-10-08-164332.png)
![Protein Gel 2 10/10/23](https://static.igem.wiki/teams/4649/wiki/screenshot-2023-10-11-170801.png)

For the future, we will continue phosphate uptake and solubilization assays.

## Learn

We learned from the phosphate plate assays that the plate phosphate assays do not work with our bacteria. This may be because we used media that was not mentioned in literature. We would want to test the phosphate plate assays with other media recipes used in published phosphate solubilization studies to see if this is the case.

We learned from the protein gel that the genes in the transformed plasmid may not have effectively expressed. To produce successful expression, we may change the promoter or the vector. It could be that the lac promoter or the vector we used is not compatible with R. tropici or that a different origin of replication may be required than that of the vector we used. It is important to note that the Tacoma 2019 team did not get to assemble their vector, so this may support our hypothesis that this vector is not compatible with R. tropici.

Furthermore, when it comes to our plant experiments we want to experiment to find the optimal CFU count for rhizobium inoculation of different plant species since we believe that is the reason we did not observe nodulation since the pH we measured and the temperature we held constant should have been ideal. We could also do some experiments seeing if it is better to inoculate the plants at germination rather than maturation.

More information about our specific procedures can be found in our  [Notebook](https://2023.igem.wiki/eastcoastbiocrew/notebook)  and our  [Experiments and Parts](https://2023.igem.wiki/eastcoastbiocrew/experiments)  pages.

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## Overview

Our project focused on two different pathways that can be used to increase phosphate uptake. One of them used glucose dehydrogenase to remove the heavy metals in mineral phosphates, thereby increasing the ability of the plants to uptake the phosphate. The other amplified expression of the pstSCAB operon, which is already present in Rhizobacteria, with the intent of increasing the number of ion channels in the membrane of the Rhizobium. All of the coding sequences for both of these pathways will be included below, as well as the promoters, ribosome binding sites, and terminators used in the creation of the composite parts.

## Pqq/gcd Pathway

### Basic Parts

| Gene name | Description | Species of origin | Registry number |
| --- | --- | --- | --- |
| PqqC | Creates pyrroloquinoline-quinone which acts as a cofactor to glucose dehydrogenase. | Agrobacterium radiobacter | [BBa\_K4649000](http://parts.igem.org/Part%3ABBa_K4649000) |
| GCD | Creates glucose dehydrogenase which converts glucose into gluconic acid | Escherichia coli Strain K12 | [BBa\_K4649001](http://parts.igem.org/Part%3ABBa_K4649001) |

### Composite Parts

| Construct name | Description | Registry number |
| --- | --- | --- |
| PqqC + gcd | These two genes produce gluconic acid, which can separate phosphate ions from heavy metals to increase bioavailability. | [BBa\_K4649010](http://parts.igem.org/Part%3ABBa_K4649010) |
![Parts](https://static.igem.wiki/teams/4649/wiki/screenshot-2023-10-11-221057.png)

## pstSCAB Pathway

### Basic Parts

| Gene name | Description | Species of origin | Registry number |
| --- | --- | --- | --- |
| pstS | Works with other genes in its operon to transport phosphate into the cell. Specifically binds to inorganic phosphate and allows it to enter the ion channel. | Escherichia coli Strain K12 | [BBa\_K4649002](http://parts.igem.org/Part%3ABBa_K4649002) |
| pstA | Works with other genes in its operon to transport phosphate into the cell. Specifically forms half of the ion channel used by phosphate. | Escherichia coli Strain K12 | [BBa\_K4649003](http://parts.igem.org/Part%3ABBa_K4649003) |
| pstB | Works with other genes in its operon to transport phosphate into the cell. Specifically hydrolyzes ATP to extract the energy needed to move phosphate through the ion channel. | Escherichia coli Strain K12 | [BBa\_K4649004](http://parts.igem.org/Part%3ABBa_K4649004) |
| pstC | Works with other genes in its operon to transport phosphate into the cell. Specifically forms half of the ion channel used by phosphate. | Escherichia coli Strain K12 | [BBa\_K4649005](http://parts.igem.org/Part%3ABBa_K4649005) |

### Composite Parts

| Construct name | Description | Registry number |
| --- | --- | --- |
| pstSCAB | With these four genes combined in an operon, the complete phosphate ion channel can be made. pstA and pstC create the ion channel imbedded in the membrane, pstS binds to the phosphate, and pstB gathers the energy to allow it into the cell. | [BBa\_K4649009](http://parts.igem.org/Part%3ABBa_K4649009) |
![Parts](https://static.igem.wiki/teams/4649/wiki/screenshot-2023-10-11-221036.png)

## Other Parts

In addition to these coding sequences, we also cataloged 3 different promoters and 2 primer sequences

| Gene name | Description | Species of origin | Registry number |
| --- | --- | --- | --- |
| T7 Promoter | A strong promoter for E. coli | T7 Bacteriophage | [BBa\_K4649006](http://parts.igem.org/Part%3ABBa_K4649006) |
| T3 Promoter | A promoter for various bacteria. Unknown strength. | Recombinant E. coli | [BBa\_K4649007](http://parts.igem.org/Part%3ABBa_K4649007) |
| Lac Promoter | A constituitively activated promoter that can be activated by lactose or IPTG. | Escherichia coli Strain K12 | [BBa\_K4649008](http://parts.igem.org/Part%3ABBa_K4649008) |
| SK Primer | Designed primer specific to genes inserted at multiple cloning site. Used pBluescript vector pBBR1MCS-2, a mobilizable shuttle and expression vector that can replicate in many Gram-negative bacteria such as E. coli and R. tropici. | pBBR1MCS-2 | [BBa\_K4649011](http://parts.igem.org/Part%3ABBa_K4649011) |
| KS Primer | Designed primer specific to genes inserted at multiple cloning site. Used pBluescript vector pBBR1MCS-2, a mobilizable shuttle and expression vector that can replicate in many Gram-negative bacteria such as E. coli and R. tropici. | pBBR1MCS-2 | [BBa\_K4649012](http://parts.igem.org/Part%3ABBa_K4649012) |

## Experiments & Results

## Testing media growth rates (July 2023)

For this experiment, we explored the growth rates of Rhizobium tropici in three different media: ATCC medium 111, high calcium yeast mannitol, and low calcium yeast mannitol.

Introduction: ATCC Medium 111, also known as Rhizobium X Medium, is a type of soil extract medium used in microbiology. It's typically used for the growth and propagation of certain types of bacteria, such as Rhizobium tropici. The specific composition of the medium can support the survival, proliferation, and biological function of these organisms. We choose to test this medium because our bacteria dwells in the rhizosphere, and we believed this media would act as a close representation to the real environment.

Results: A t-test of two samples for the means between the low Ca concentration yeast mannitol and high Ca concentration yeast mannitol yielded a p-value of 0.11. Therefore, since there was no statistical difference in growth; it was decided to use the low Ca yeast mannitol media since a low Ca was preferred for increased accuracy of measurements and lower cost and there was no evidence to suggest that the yeast mannitol with higher concentrations of Ca would yield higher rhizobium ODs.

Procedures for making media:

ATCC Medium 111 (Rhizobium X Medium):

Introduction: ATCC Medium 111, also known as Rhizobium X Medium, is a type of soil extract medium used in microbiology. It's typically used for the growth and propagation of certain types of bacteria, such as Rhizobium tropici. The specific composition of the medium can support the survival, proliferation, and biological function of these organisms. We choose to test this medium because our bacteria dwells in the rhizosphere, and we believed this media would act as a close representation to the real environment.

Materials:

* Yeast extract 1.0g
* Mannitol 10.0g
* Agar 15.0g
* Distilled water 800.0mL
* Soil extract 200.0mL
* African violet soil 77.0g
* Na2CO3 0.2g
* Distilled water 200.0mL

Procedure:

* Combine all materials
* Adjust pH to 7.2 and autoclave with 15lbs. pressure at 121 C for one hour to sterilize
* Filter medium through paper to remove soil before using

The final product should be translucent and tan in color. There may be a slight precipitate. It is recommended to store the medium at a temperature between 2-8 C.

Yeast Mannitol:

Introduction: Yeast Mannitol Broth is used for the cultivation and enumeration of soil microorganisms like Rhizobium species. Yeast extract provides a source of readily available amino acids, vitamin B complex, and accessory growth factors for Rhizobia. Mannitol is the carbohydrate source. The pH of the medium is adjusted to 6.8±0.2 at 25 C. In addition to Rhizobium, this medium can also be used for other symbiotic nitrogen-fixing soil microorganisms.

Materials:

* Yeast extract 1.0g
* Mannitol 10.0g
* Dipotassium phosphate 0.5g
* Magnesium sulfate 0.2g
* Sodium chloride 0.1g
* Calcium carbonate (use 1.0g for high calcium media and 15mg for low calcium media)
* Distilled water 1.0L

Procedure:

* Suspend 12.8 g of Yeast Mannitol Broth in 1000 ml of distilled water.
* Boil to dissolve the medium completely
* Sterilize by autoclaving at 15 lb. pressure at 121 C for 15 minutes

## Transformation Test (July 2023):

For this experiment, we tested whether our protocol for plasmid insertion was effective. Our empty plasmid contained Kanamycin antibiotic resistance and lacZ genes with the inducible lac promoter. We used blue-white screening to confirm integration.

X-gal, or 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside, is a chromogenic substrate for the enzyme β-galactosidase. This means it changes color when it is cleaved by the enzyme. In molecular biology, X-gal is used in a technique known as blue-white screening, which is a rapid and efficient method for the identification of recombinant bacteria.

A plasmid vector is engineered to contain a segment of the lacZ gene, which codes for β-galactosidase. This plasmid is taken up by R. tropici cells through a transformation of electro-competent cells(https://2014.igem.org/Team:Hannover/Protocols/Rhizobium\_Electroporation). When X-gal is added to the growth medium, it is cleaved by any β-galactosidase that is present, producing an insoluble blue pigment. If a foreign DNA sequence is inserted into the lacZ gene on the plasmid (where we designed our multiple cloning site to be located), it disrupts the gene and prevents it from producing β-galactosidase.

Therefore, colonies of bacteria that have taken up the plasmid but have not had foreign DNA inserted into the lacZ gene (non-recombinant) will turn blue. In contrast, colonies that have taken up the plasmid and have had foreign DNA inserted into the lacZ gene (recombinant) will remain white because they cannot cleave X-gal. This allows for easy visual identification of recombinant bacteria.

Results: We were able to confirm the insertion of an empty plasmid with blue-white screening; our colonies were blue.

## Ligation (August 2023):

Introduction: Ligation is a process in molecular biology that involves the joining of two nucleic acid fragments through the action of an enzyme. It is an essential laboratory procedure in the molecular cloning of DNA, whereby DNA fragments are joined to create recombinant DNA molecules. This process can occur when a foreign DNA fragment is inserted into a plasmid. For our project, we needed to join two gcd gene fragments with a PqqC gene fragment.

We performed ligation using T4 DNA ligase. This enzyme is broadly used in vitro as a molecular biology research tool due to its capability of joining both sticky and blunt DNA ends. The ends of DNA fragments are joined by the formation of phosphodiester bonds between the 3’-hydroxyl of one DNA terminus with the 5’-phosphoryl of another. This process is catalyzed by an enzyme called DNA ligase.

Results: Successful ligation is proved by PCR performed after gene insertion.

Protocol:

Set up the following reaction in a microcentrifuge tube on ice. (T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.)

![Ligation Table](https://static.igem.wiki/teams/4649/wiki/screenshot-2023-10-08-171619.png)

Procedure:

* Gently mix the reaction by pipetting up and down and microfuge briefly.
* For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
* For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours.
* Heat inactivate at 65 C for 10 minutes.
* Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.

## Plasmid Gene Insertion (August-September):

Introduction: The NEBuilder® HiFi DNA Assembly Cloning Kit is a tool developed to improve the efficiency and accuracy of DNA assembly. It allows for seamless assembly of multiple DNA fragments, regardless of fragment length or end compatibility. It can be used to assemble either single-stranded oligonucleotides or different sizes of DNA fragments with varied overlaps (15–30 bp). The kit is useful for the synthetic biology community, as well as those interested in one-step cloning of multiple fragments due to its ease of use, flexibility, and simple master-mix format. It can join DNA fragments together more efficiently, even with larger fragments or low DNA inputs. The kit can be used in successive rounds of assembly, as it removes 5´ and 3´ end mismatches.

Preparation Protocol:

* pstSCAB contains 4 genes (pstS, pstC, pstA, & pstB), add 1μL of each
* PqqC + gcd contains 3 genes (PqqC, gcd 1, & gcd2), add 1μL of each
* 300 ng of back bone (2 μL)
* 10μL of master mix
* add water to end of with 20 μL total
* Incubate for 1 hour @ 50 C

Assembly Protocol:

* Thaw and vortex master mix thoroughly and keep on ice
* Incubate samples in a thermocycler at 50 C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4 - 6 fragments are being assembled. Following incubation, store samples on the ice at - 20 C for subsequent transformation
* Transform competent cells with 2 μL of the assembly reaction, using the following the transformation protocol

Chemically Competent Cells Transformation Protocol:

* Thaw chemically competent cells on ice
* Add 2 μL of the chilled assembled product to the competent cells. Mix gently by pipetting up and down by flicking the tube 4 - 5 times. Do not vortex
* Place the mixture on ice for 30 minutes. Do not mix.
* Heat shock at 42 C for 30 seconds. Do not mix.
* Transfer tubes to the ice for 2 minutes
* Add 950 μL of room-temperature SOC media to the tube
* Incubate the tube at 37 C for 60 minutes. Shake vigorously (250 rpm) or rotate
* Warm selection plates to 37 C
* Spread 100 μL of the cells onto the selection plates. Use Amp plates for the positive control sample
* Incubate overnight at 37 C

## Gene Confirmation with Polymerase Chain Reaction (PCR) and Gel Electrophoresis (September 2023):

For this experiment, we tested whether our genes were integrated into our plasmids. We performed a colony PCR with Q5 and a gel electrophoresis. Colony PCR is a high-throughput method for determining the presence or absence of insert DNA in plasmid constructs. Specifically, we wanted to confirm our genes by confirming the insert length (from the colony PCR) with gel electrophoresis.

Results: As seen in the images below, we were able to confirm the length of insertion fragments. We expected a fragment length of around 3700 base pairs for pstSCAB and a fragment length of around 3200 for PqqC + gcd.

Colony PCR Protocol:

Materials:

* 10uM primer VF2
* 10uM primer VR
* 2X Q5 HotStart Master Mix
* Distilled water
* Agar plate with bacterial colonies
* PCR tubes
* Toothpicks

Collecting Bacterial Cultures + Reaction Setup:

Be sure that you save each bacterial colony so that if the PCR shows what you want, you can go back to the original bacterial cells. You can do this by dipping the toothpick in step 5 below into liquid media or using to swab an agar plate before adding the DNA into the PCR tube.

* Add 1.3 ul of primer VF2 to PCR tube
* Add 1.3 ul of primer VR to PCR tube
* Add 12.5 ul 2x Master Mix to PCR tube
* Add 10 ul of water to PCR tube
* Using toothpick, take 1 colony of the bacteria from the agar plate and add it to the PCR tube (step 5)
* Put PCR tube into thermocycler and run using the following parameters:

PCR Cycle:

* Initial denaturation @ 98 C for 30 seconds

25-30 Cycles of the following:

* 98 C for 5-10 seconds
* 50-72 C for 10-30 seconds (variable depending on primers used; select temperature based on primer annealing)
* 72 C for 20-30 seconds per kilobase
* Final extension @ 72 C for 2 minutes
* Hold @ 4-10 C

Gel Electrophoresis Protocol:

Preparing the gel:

* Weigh out 0.5g of the agarose on a piece of weigh paper. Transfer to an Erlenmeyer flask. Add 50 ml of 1X TAE.
* Place the flask in the microwave for 30 seconds to 1 minute. Heat until the agarose and buffer mixture is fully dissolved and remove with cooking mittens. Periodically swirl mixture to ensure the mixture is melting evenly.
* Remove the flask of clear agarose and allow it to cool. This will take about 10 minutes. If you are able to grasp the flask comfortably it is cooled enough to pour.
* While the agarose is cooling, place the gel tray into the gel box and add blocker and a comb.
* When the agarose is cooler, add 2 ul of Apex Safe DNA Gel Stain to the melted agarose.
* Swirl the agarose to incorporate the dye and pour the agarose into the gel tray—your gel should be about 1 cm thick.
* Allow at least 20 minutes for the gel to solidify. Once solid, carefully remove the comb and blockers, and place the solidified gel (still on the tray) oriented on the negative side.
* Add enough 1X TAE buffer to completely cover the gel by 1 cm.

Preparing the Sample:

On a piece of parafilm, spot 1 ul of 6X DNA loading dye with a p20 pipette. You should have one spot for each of your PCR products. Wait until your gel has started to solidify before beginning the step since the spots will dry out after about 10 minutes. You can also do this step in a tube.

* Add 5ul of water to each spot of dye.
* Add 5ul of the PCR product.

Running a Gel:

* Into the first lane of the gel load 5ul of the 2-log DNA ladder.
* Into lane 2-5 load 10ul of each of your PCR products including the water and dye.
* Place gel lid with electrodes on the gel box.
* Set the voltage to 100 volts. You should see bubbles coming up from the wire electrode.
* After a few minutes, check to make sure everything is going smoothly. Run the gel for approximately 30 minutes or until the dye is 2/3 the way down the gel.
* Take a picture of the gel.
* Use the DNA ladder to approximate the size of each of your DNA bands. Make sure you check that your DNA band is the correct size.

## Phosphate Uptake Assays (August-September):

For this experiment, we wanted to confirm whether our engineered Rhizobium would more effectively uptake an inorganic phosphate. During our initial control tests, we found that our strain of R. tropici was unable to uptake inorganic phosphate, making it a negative control. Therefore, we used Pseudomonas fluorescens as a positive control because it was present in our lab and has been shown to uptake inorganic phosphate in numerous literature.

To do this, we made agar plates with a modified low calcium yeast mannitol that replaced the dipotassium phosphate with tricalcium phosphate. Next, we put bacterial spot dilutions and let them incubate for 7 days. After the incubation period, we observed the “halo” – a clearing made around the bacterial spot – and compared the area.

We also performed liquid assays to account for the high motility of our positive control, P. fluorescens. We made 22 liquid media samples with the protocol for the tricalcium phosphate yeast mannitol. We also made 9 agar plates with tricalcium phosphate media. All bacterial solutions were 10uL in 5mL of tricalcium phosphate yeast mannitol liquid media. Samples 1-6 did not contain kanamycin or IPTG. Samples 7-22 contained 25uL kanamycin and 50uL IPTG per sample.
Plates 1 and 2 did not contain kanamycin. 1x = 200uL of bacterial solution spun down with 190uL of supernatant removed. 2x = 400uL of bacterial solution spun down with 385uL of supernatant removed. P. fluorescens 1x = 500uL of bacterial solution spun down with 490uL of supernatant removed. P. fluorescens 2x = 1000uL of bacterial solution spun down with 985uL of supernatant removed.

Protocol:

Materials:

* Yeast extract 0.5g
* Mannitol 5.0g
* Magnesium sulfate 0.1g
* Sodium chloride 0.05g
* Calcium carbonate 0.005g
* Tricalcium phosphate 2.5g
* Agar 7.5g
* Distilled water 500mL

Procedure:

* Swirl to combine all materials in a 1L glass
* Sterilize by autoclaving at 15lb pressure at 121 C for 15 minutes
* Let it cool to 50 C (should be able to touch with bare hand without burning)
* Add 50ng/uL kanamycin; swirl to combine
* Pour plates

Testing Phosphate Assays:

* Spread 100uL 100mM IPTG before adding bacterial spots
* Let plates dry under laminar flow hood for about 1 hour
* Wrap with parafilm to ensure moisture is not lost and incubate at 32 C for 7 to 10 days

## Phosphate Solubilization Assays (September-October):

The colorimetric phosphate assay is a method used to measure the concentration of phosphate in a sample.

In a dilute orthophosphate solution, ammonium molybdate reacts under acid conditions to form the heteropoly acid, molybdophosphoric acid. In the presence of vanadium, yellow vanadomolybdo-phosphoric acid is formed. The intensity of the yellow color is proportional to the phosphate concentration. The Phosphate Colorimetric Assay Kit provides a simple and direct procedure for measuring phosphate levels (ranging from 1–5 nmole/well) in a variety of samples. Phosphate reacts with a chromogenic complex, which results in a colorimetric (650 nm) product proportional to the amount of phosphate present.

The sample is mixed with the vanadate-molybdate reagent and the absorbance is measured. The phosphate concentration in the sample can then be determined by comparing its absorbance with that of the calibration standards.

Procedure

* All samples and standards should be run in duplicate.

Phosphate Standards for Colorimetric Detection

* Dilute 10 mL of the 10 mM Phosphate Standard with 990 mL of water to prepare a 0.1 mM Phosphate
  Standard Solution.
* Add 0, 10, 20, 30, 40, and 50 mL of the 0.1 mM Phosphate Standard Solution into a 96 well plate, generating 0 (blank), 1, 2, 3, 4, and 5 nmole/well standards.
* Add water to each well to bring the volume to 200 mL.

Sample Preparation

Samples can be measured directly.

* Add 1–200 mL of sample to wells.
* Bring samples to a final volume of 200 mL with water.

Note: For unknown samples, it is suggested to test several sample dilutions to ensure the readings are within the linear range of the standard curve.

Assay Reaction

* Add 30 mL of the Phosphate Reagent to each of the wells.
* Mix well using a horizontal shaker or by pipetting, and incubate the reaction for 30 minutes at room temperature.
* Cover the plate and protect from light during the incubation.
* Measure the absorbance at 650 nm (A650).

Note: When using 1.0 mL cuvettes, increase the volume of all reaction components 5-fold. The 1 mL total reaction volume will contain 1–500 mL of sample, 150 mL of Phosphate Reagent, and bring to a final volume of 1 mL with water.

* Incubate at room temperature for 30 minutes.
* Measure the absorbance at 650 nm (A650).

## Protein Gel (October):

The main purpose of a protein gel, also known as protein gel electrophoresis, is to separate proteins based on their size. We ran a protein gel to confirm the synthesis of our various parts. The fragment lengths we were looking for are:

gcd: GDH enzyme- 86,747 mass (Da)

PqqC: Pqq synthase- 47,425 mass (Da)

pstS: pstS Pi binding protein- 37,024 mass (Da)

pstC: pstC membrane transport protein- 34,121 mass (Da)

pstA: pstA membrane transport protein- 32,322 mass (Da)

pstB: pstB ATP-binding protein- 29,027 mass (Da)

Results: Our first protein gel was unsuccessful as the bars were unclear and under 25000Da.

Protocol:

Cell Lysis:

* Discard the medium in culture dishes with cells and wash the cells using ice-cold Phosphate-buffered saline (PBS).
* Discard the PBS, add ice-cold lysis buffer.
* Scrape the cells using a cold plastic cell scraper.
* Collect the cells in microcentrifuge tubes.
* Agitate the contents in microcentrifuge tubes for 30 min at 4 °C.
* Centrifuge the tubes at 16,000 x g for 20 min at 4 °C.
* Collect the supernatant in a fresh tube and place on ice. Discard the pellet.

Sample Preparation:

* To a volume of protein sample (cell lysate), add an equal volume of loading buffer.
* Boil the above mixture at 95 °C for 5 min.
* Centrifuge at 16,000 x g for 5 min.
* These samples can be stored at -20 °C or may be used to proceed with gel electrophoresis.

Gel Electrophoresis:

* Load your samples into wells created in the stacking gel.
* Apply an electric field across the gel. The proteins will migrate through the gel based on their charge and size.
* Once electrophoresis is complete, stain the gel to visualize the proteins. This can be done using various methods such as Coomassie dye staining or silver staining.
* Remove excess dye from the gel matrix background.

Results: Our second protein gel showed promising results for gcd protein synthesis. We expected 87 kDa and saw a corresponding band for one of our PqqC+gcd samples.

## Plant Experiments:

Our ultimate goal is to measure the micro symbiosis between our Rhizobium and our various plant species (Common Bean, Soybean, Showy Pink Trefoil, American Senna.). We need to do this, because we want to know if we can implement the project using native plants as our micro symbionts.

We are also trying to measure if our Rhizobium is actually beneficial for the plant's growth/if it experiences phosphate poisoning. If we find negligible differences in growth overtime, if we see no differences, then we will know that either the Rhizobium never took root (hehe) or that the Rhizobium doesn’t help the plant that much. We need to do this in order to see if our “built in killswitch” theory holds true.

The first thing we do is acquire a wet paper towel, and drape it on top of a ziploc bag. Then, we place the chosen amount of seeds on one half of the paper towel. Fold in half, and place the paper towel inside of the ziplock bag.
We do this so that we can track germination. Rhizobium has a limited window of 3 days after germination for inoculations to be successful.

Once germinated, transfer GERMINATED SEEDS EXCLUSIVELY into a small planter. Inoculate a portion of the seeds in dirt with Rhizobium.(One portion will be a control). Once the plant outgrows the planter, gently transfer to a pot. We do this to prevent our plant from getting root bound. Let the plant grow ⅔ of the way until maturity, and then that is when you begin nodulation checks.

In the beginning, we planned a series of experiments to test the optimum conditions for our Rhizobium to survive. This included assessing the best microsymbiont, and assessing what the best age for inoculating is.

However, we have unfortunately been pressured to annul the age experiments. Our first Rhizobium check proved fruitless aside from a single set of modules on a showy pink trefoil.

As a result, we want to maximize our chances with inoculations, so we have been removing innoculatory controls from experiments. Since we have measured all previous growth rates, but they proved to be invalid, we have deemed them to be considered as controls.

For the past week or two we have been studying a hypothesis that it may be temperature that caused our lack of success. As a result, we are now incubating our plants at 29° C. We will then contrast the growth rates with our previous rates.

Important dates:

* Began plant experiments 7/15/2023
* First inoculation 7/27/2023
* First nodulation check 9/2/2023
* First nodules found 9/9/2023
* Temperature Hypothesis Transfer 9/23/2023
* Final Nodule Check 10/7/2023

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# Results

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### Soil Media

When researching prior projects and experiments related to Rhizobium, we encountered three different types of media in which the bacteria were grown. We obtained our strain from ATCC, which recommended ATCC#111 media, which contains African violet soil in it. This was a difficult media to make, so we wanted to see if we could instead use yeast mannitol media, which we found in the literature to work with R. tropici (del Cerro P, Rolla-Santos AA, Gomes DF, Marks BB, del Rosario Espuny M, Rodríguez-Carvajal MÁ, Soria-Díaz ME, Nakatani AS, Hungria M, Ollero FJ, Megías M. Opening the "black box" of nodD3, nodD4 and nodD5 genes of Rhizobium tropici strain CIAT 899. BMC Genomics. 2015 Oct 26;16:864. doi: 10.1186/s12864-015-2033-z. PMID: 26502986; PMCID: PMC4624370.) The mannitol media had a high concentration of calcium and was cloudy, which made it hard to use to measure bacterial growth. So, we also tried lowering the calcium concentration to its solubility level in yeast mannitol media (15 mM).

In order to determine which media we should use for our experiments, we conducted a trial in each media. The optical densities of the different samples at different times are recorded in the graph below. A higher optical density indicates that there is a higher concentration, and therefore quantity, of bacteria. The negative readings for samples 3 and 4 are likely errors due to high concentrations of Ca which made the values of our blanks very high. Overall, the yeast mannitol with low calcium showed an exponential growth of y= 0.0479e0.445x with an r2 value of 0.99. The yeast mannitol with high calcium showed an exponential growth of y=0.0343e0.4582x with an r2 value of 0.94. A t-test paired two samples for means performed between the ATCC 111 media, which was the most commonly used one in literature, and the yeast mannitol extract with low Ca concentrations yielded a p-value of 0.11 (greater than the threshold of 0.05) showing no significant statistical difference found in growth. Therefore, since there was no statistical difference in growth; it was decided to use the low Ca yeast mannitol media since a low Ca was preferred for increased accuracy of measurements and there was no evidence to suggest that the yeast mannitol with higher concentrations of Ca would yield higher rhizobium ODs.

![Model of Bacterial ODs](https://static.igem.wiki/teams/4649/wiki/screenshot-2023-10-11-144505.png)
![Model of Bacterial ODs](https://static.igem.wiki/teams/4649/wiki/screenshot-2023-10-11-144516.png)

### Bacterial Transformation

The plasmid that we are using for cloning, pBBR1MCS-2, has a LacZ gene in it, so we can do blue-white screening in E. coli to see if our genes were inserted. Our blue-white screening showed that our plasmids were successfully assembled in E. coli. From there, we screened the plasmids by PCR, isolated the plasmids from E. coli, and then transformed them into Rhizobia. We then screened our Rhizobium by PCR, and our PCR and gel electrophoresis experiments showed that our transference to rhizobia was successful. We were able to confirm the length of inserted fragments. We expected a fragment length of around 3700 base pairs for pstSCAB and a fragment length of around 3200 for PqqC + gcd. Colonies that begin with the numbers 1 & 2 contain pstSCAB (~3700), and colonies that begin with the numbers 3 & 4 contain PqqC+gcd (~3200) From the image below, samples 1-1, 1-2, and 2-1 showed successful gene insertion of pstSCAB genes; samples 3-3, 3-4, 4-2, 4-3, 4-4, 4-5, and 4-6 showed successful gene insertion of PqqC+gcd genes.

![E. coli Transformation 8/5/23](https://static.igem.wiki/teams/4649/wiki/20230807-133107.jpg)
![E. coli Transformation 8/5/23](https://static.igem.wiki/teams/4649/wiki/copy-of-20230807-133114.jpg)
![R. tropici Transformation 8/5/23](https://static.igem.wiki/teams/4649/wiki/20230807-133121.jpg)
![R. tropici Transformation 8/5/23](https://static.igem.wiki/teams/4649/wiki/20230807-133128.jpg)
![Gel 8/12/23](https://static.igem.wiki/teams/4649/wiki/picture1.png)

### Phosphate Assays

To determine whether inserting the pstSCAB and PqqC+gcd genes increase phosphate uptake from the media, we looked that the cells’ ability to uptake phosphate (making halos) from plates with high levels of tricalcium phosphate. We tried various bacterial dilutions with varying bacterial spot amounts. We used R. tropici as a negative control, so no results were expected. We used P. fluorescens as a positive control as literature indicated it had phosphate uptake abilities (K.‐H. Park, C.‐Y. Lee, H.‐J. Son, Mechanism of insoluble phosphate solubilization by Pseudomonas fluorescens RAF15 isolated from ginseng rhizosphere and its plant growth‐promoting activities, Letters in Applied Microbiology, Volume 49, Issue 2, 1 August 2009, Pages 222–228, https://doi.org/10.1111/j.1472-765X.2009.02642.x). However, P. fluorescens is highly motile. We concluded that the agar plates were too "wet" to achieve good results, and the P. fluorescens appeared to move across the entirety of the plate, making it nearly impossible to detect phosphate uptake if any occurred. So, on October 1, we used the laminar flow hood to remove excess liquid. Then, we proceeded with 9 assay plates with the samples listed in the table below. After 7 days of incubation, the bacteria grew well and appeared concentrated enough to provide clear results. However, no halo was visible for any of the samples, proving our experiment did not work as planned. We believe this is due to the media we used (low calcium yeast mannitol) as the literature from which we derived the experiment used different media with tryptophan. For future experiments, we would like to try these plate assays again with the media used in the literature and see if we can produce similar results. (plate images in order from 1-9; 1L & 2L contain PqqC+gcd and 4L contains pstSCAB)

![Table of Plate Samples](https://static.igem.wiki/teams/4649/wiki/screenshot-2023-10-08-154555.png)
![Plate 1 10/7/23](https://static.igem.wiki/teams/4649/wiki/img-5681.jpg)
![Plate 2 10/7/23](https://static.igem.wiki/teams/4649/wiki/img-5682.jpg)
![Plate 3 10/7/23](https://static.igem.wiki/teams/4649/wiki/img-5686.jpg)
![Plate 4 10/7/23](https://static.igem.wiki/teams/4649/wiki/img-5685.jpg)
![Plate 5 10/7/23](https://static.igem.wiki/teams/4649/wiki/img-5684.jpg)
![Plate 6 10/7/23](https://static.igem.wiki/teams/4649/wiki/img-5683.jpg)
![Plate 7 10/7/23](https://static.igem.wiki/teams/4649/wiki/img-5687.jpg)
![Plate 8 10/7/23](https://static.igem.wiki/teams/4649/wiki/img-5688.jpg)
![Plate 9 10/7/23](https://static.igem.wiki/teams/4649/wiki/img-5689.jpg)

### Protein Gels

Our PCR test showed that our Rhizobium contained our genes, but we wanted to see if the proteins were actually produced from the inserted genes. Our first protein gel did not provide conclusive results. The ladder we used, (SMOBIO ExcelBand™ 3-color Regular Range Protein Marker, PM2500), allowed us to see that most of our proteins were under 25000 Da, which is not expected because the masses of our proteins are greater than 25000Da. In addition, there appeared to be a lot of debris in the gel-- either from cell lysis or proteins congregating. We ran another protein gel, ensuring cell debris is not inside the gel. This yielded more success as we saw promising results for gcd protein synthesis. We expected 87 kDa for GDH enzyme and saw a corresponding band for one of our PqqC+gcd samples- the arrow marks the band in the image below. It is worth noting that we were expecting to see the proteins running at their individual sizes, but because pstA, pstB, and pstC form the pstABC transporter and, therefore, form strong associations, we were also aware that we might see them still associating in the gel and a protein complex at a larger size. This would correspond to around 95 kDa.

![Expected Protein Masses](https://static.igem.wiki/teams/4649/wiki/screenshot-2023-10-08-164332.png)
![Protein Gel 2 10/10/23](https://static.igem.wiki/teams/4649/wiki/screenshot-2023-10-11-170801.png)

### Plant Experiments

Our ultimate goal is to measure the micro symbiosis between our Rhizobium and our various plant species (Common Bean, Soybean, Showy Pink Trefoil, American Senna.). We picked the common bean and soybean because these are plants that are know to participate in symbiosis with Rhizobium. We also wanted to know if we could implement the project using native plants as our micro symbionts. So we picked showy pink trefoil because they can grow in almost any soil, are indifferent to soil acidity, and germinate in 2-3 weeks. We also picked American Senna because it grows in both New York and Maryland, is a perennial whose normal life span exceeds 2 years, it has special value to bumble bees, it grows well in dry to moist soil, and it grows well in sunshine to partial shade.

We are also trying to measure if our Rhizobium is actually beneficial for the plant's growth or if it experiences phosphate poisoning when we cause it to uptake more phosphate. If we find negligible differences in growth over time, or if we see no differences, then we will know that either the Rhizobium never took root or that the Rhizobium doesn’t help the plant that much. We need to do this in order to see if our “built in killswitch” theory holds true that the plants would die from picking up too much phosphate and could then be reused as a source of phosphate themselves.
In the beginning, we planned a series of experiments to test the optimum conditions for our Rhizobium to survive. This included assessing what the best plant age for inoculating is. Unfortunately, our first Rhizobium check proved fruitless aside from a single set of nodules on a showy pink trefoil.

We are not sure why this is happening, but we looked over other protocols and saw that most experiments used a greenhouse, which we do not have. For the past week or two we have been studying a hypothesis that it may be the lower temperature that caused our lack of success since we are growing our plants at room temperature. As a result, we are now incubating our plants at 29° C. We will then contrast the growth rates with our previous rates to see if the warmer temperatures help nodules to form as expected.

![Protein Gel 1 10/7/23](https://static.igem.wiki/teams/4649/wiki/nodules.jpg)

## Future Plans

With more time, we would conduct replicates of all our experiments to get the most reliable results. Rather than using the plate assays for the phosphate assays, we instead are using a Phosphate Colorimetric Kit (Sigma MAK030) to measure phosphate levels with a color-based assay. Additional experiments we would conduct include inoculating plants with our modified rhizobia containing the genes, experiments varying the cfu count when inoculating plants with rhizobia to determine the optimal cfu count for rhizobia inoculation in different species, and experiments measuring inoculation in different pHs and temperatures to determine the optimal conditions for rhizobia nodulation. We would perform these future experiments in a growth room where the plants would have more space and we could more easily control temperature, humidity, and light schedule to yield more accurate results.

## Analysis of Results

Although many of our experiments did not go as planned, we learned from our mistakes and plan to use these lessons to successfully perform future experiments. We saw early successes in determining an ideal growth media for our bacteria and for our experimental purposes. When we began transformation, we encountered some issues with gene insertion. Upon review, we found an error in our gene constructs-- there was an issue with using restriction digest to make our recombinant DNA because we had an extra restriction site inside our gene. So, we modified our approach and used PCR to amplify the portion of the plasmid that did not include the multiple cloning site, and we used HiFi DNA assembly to integrate our genes. This led to successful creation of recombinant DNA and, therefore, allowed us to insert our genes into R. tropici and successfully clone our bacteria. Upon colony PCR, we validated that numerous colonies had successfully accepted our genes. However, when we performed phosphate plate assays and a protein gel to confirm the production and functioning of our genes, we encountered unexpected results. The plate assays took 7 days to measure due to the incubation period. This meant we were unable to foresee issues, such as the wet plates. Therefore, over the course of a month, we were unable to produce viable results. However, we experimented a lot with different bacterial solutions, bacterial spot amounts, and steps to remove extra liquid on the plates. So, we have knowledge that we can base our future experiments on with the hopes of producing viable results.

## Project Achievements

### Successful

* We found the doubling time of rhizobia in different medias
* We successfully ligated the pqq and gcd DNA into our plasmid and confirmed this with PCR and gel electrophoresis
* We successfully transformed our plasmid into E.coli
* We successfully transferred the plasmid from E.coli into rhizobium

### Unsuccessful

* We could not interpret our phosphate assays
* We have not yet gained results of colorimetric phosphate assays and the protein gels
* We were not able to observe consistent nodulation of rhizobia in our plant experiments

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