iGEM 2023 — Team stony-brook

{% extends "layout.html" %}
{% 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 %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4647/wiki/headers/notebook2.jpg)

## Notebook

![Description](https://static.igem.wiki/teams/4647/wiki/logo.png)

# Stony Brook University

## iGEM 2023

## Team Notebook

![](https://static.igem.wiki/teams/4647/wiki/collage-removebg-preview.png)

[Click here to view our team notebook](https://static.igem.wiki/teams/4647/wiki/team-notebook-1.pdf)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4647/wiki/beaker.png)

## Engineering

![](https://static.igem.wiki/teams/4647/wiki/cycle.png)

## Designing and Implementing Nitroscillator

Iterative design involves four major processes: **design, build, test, and learn.** We have made several changes to our design plans throughout the entirety of our project, with the help and feedback of our countless mentors and the work of our team.

## Design

First, we decided on which oscillator would be most robust for generating the oscillations we needed to design our bacterial system. The first oscillator we considered was designed by **Hasty et. al in 2010 [1]**. After feedback from our mentors, we decided against using this oscillator for two reasons:

1. The oscillator promotes **synchrony between individual cells**— which involves *diffusion of transcription factors across cells.* This may have been too many moving parts and variables, which could have contributed to *failure of the oscillations*.

2. The 2008 oscillator [2] is **easier to tune**— with different levels of arabinose and IPTG. We thought this would let us control the oscillations and gene expression with a **higher degree of control** than with the 2017 oscillator.

Initially, based on literature review [3], we assumed that since the only coding regions of nitrogenase were nifH, nifD, and nifK, that these were the only genes necessary for full expression of the enzyme. Thus, *our original plan was to simply put those three genes as a fused complex under the control of one hybrid promoter.* However, based on further literature search, **we found that there were multiple genes that comprise the full expression of nitrogenase [4],** and that the quantity and type of genes that each diazotroph possessed differed. From here, we decided on the best functioning and minimal cluster our engineered bacteria could possess to have a fully functional enzyme, drawing inspiration mainly from past iGEM teams and papers who considered doing the same.

After more literature searching, we settled on the **9 gene minimal MoFe nitrogenase cluster from the 2018 iGEM team from Nanjing-China.** We found that their work had several advantages: (a) it was previously successfully assembled, (b) it was characterized to have some function and (c) the sequence was relatively shorter than most minimal clusters.

![](https://static.igem.wiki/teams/4647/wiki/nifs.png)

*Created using Biorender.com*

From then: we developed a plan to incorporate the cluster to have it expressed under the dual plasmid system. **We developed three separate plans to do so:**

1. Placing nifH, nifD, and nifK under the control of the hybrid promoter and the rest of the operon in a separate plasmid.

2. Putting the entire operon on a separate plasmid with the hybrid promoter cloned alongside it

3. Integrating our operon and hybrid promoter into the genome

We strongly pursued the third plan, which would prevent the population of E. coli with successfully cloned plasmids from being lost due to fitness costs.

---

## Build

**We started by coming up with a plan to clone the operon.** Since IDT and TWIST both had a limitation on how many base pairs could be chemically synthesized at once, we had to think of ways to clone our genes altogether.

We considered this problem from the three plans that we planned to pursue. **Due to the number of and large base pair sizes of the genes and complete operon,** we had to eliminate strategies such as using restriction enzymes to stitch together the genes. We strongly considered other strategies that have been popular amongst other iGEM teams, such as *Gibson Assembly and HiFi Assembly.* Ultimately, based on feedback from our mentors, in particular AJ Sillato, we considered other ways to clone our operon. AJ gave us the ideas of pursuing cloning with **in-vivo assembly and sequence and ligation independent cloning [5][6],** which *were both cost-effective, less time-consuming, and less hands-on work,* which could *improve our results and introduce less confounding variables to the experiments.* Then, primers to pursue these methods were designed. For future teams, we characterized these primers as well as described how they were designed.

![](https://static.igem.wiki/teams/4647/wiki/jerome.jpg)

Another issue with our plans involving plasmids (plans 1 and 2), was the **conflict between origins of replications.** It is known that there are *three major categories of origins of replication,* and excluding some exceptions, origins of replications from the same category **cannot be co-transfected [7].** Due to our activator plasmid and repressor plasmids, (pJS167 and pJS169), *2 out of 3 origins of replications were already accounted for.* Thus, a third origin of replication was needed to clone the genes into. This **severely limited our choices for plasmid characteristics** we could choose from to control important factors such as copy number.

---

## Test

After designing the necessary primers for both in-vivo assembly (IVA) and sequence and ligation independent cloning (SLIC), **we tested our designs by running PCR and gel electrophoresis with our obtained constructions.** We found that our primers were very successful in amplifying the genes with overhangs, although with slightly less specificity than we would have preferred with some genes. For those, with feedback from our mentors, we decided to *run gel extractions or readjust the PCR conditions we had used,* **especially in regard to annealing temperature and time.**

We then set out to clone our operon in its entirety. Although we had all the components necessary to clone the operon in one experiment cycle, we decided against that plan based on feedback we received and literature-based evidence. Both cloning methods, IVA and SLIC, **have significantly reduced efficiency** when more fragments are attempted to be assembled all at once. Out of the two methods, SLIC had the higher efficiency when 10 or more fragments are assembled [8].

Thus, we attempted to clone our genes in chunks of three. **Nine nif genes were divided into three separate segments;** nifB-nifH-nifD, nifK-nifE-nifN, and nifX-hesA-nifV. We were able to successfully clone these three segments, and attempted to construct the entire operon by ligating these three segments together.

---

## Learn

**We learned and improved many parts of our system along the way.** Most significantly, based on the results we obtained from cloning our assembly together, we realized a few major points about the techniques we used.

1. **IVA -**

1. *Limitations in Fragment Assembly.* Although assembly of the three segments (nifB-nifH-nifD, nifK-nifE-nifN, and nifX-hesA-nifV), individually did not require much troubleshooting— i.e. most attempts worked either by the first round of assembly or second, the assembly of the total operon required much troubleshooting to see a possible product. We think that this is possibly due to two issues:

1. **One, the larger products is not as efficiently taken up by *E. coli* as the smaller constructs.** We tried electroporating the cells for delivery of the segments, and found that this generally worked better than chemical transformation.

2. **Two, larger products are harder to assemble by *E. coli.*** Using a strain designed for large fragment assembly, such as DH10B, increased efficiency. CFUs were extremely low for transformed strains with DH5-alpha, where majority products were either annealed vector backbones or if fragments were assembled, only one or two of the fragments were ligated together into the vector.

2. *Possible Construction of Plasmid Library.* We did not notice this issue for smaller scale assemblies, but we noticed an interesting phenomenon for the large operon assembly. Instead of one consistent plasmid product like we usually notice/expect, an unequal mixture of correct product and incorrect assemblies were found within one isolated colony. Our PCR screening step first brought this result to our attention, and our sequencing results cemented them (see our results page for further details). It is a drawback that a plasmid library could be constructed within one cell instead of one homogenous product— and perhaps should be combated by deploying in-vitro assembly methods instead.

2. **SLIC -**

1. *Need for troubleshooting “chew-back” digestion times.* While generally 30 minutes of incubation with T4 exonuclease is recommended for a 20 basepair homology overhang, we found that 30 minutes was not sufficient for our gene products. Although multiple attempts were done to ligate our genes into a vector, we found that most were either empty vectors that were results of the 2BcT vector re-ligating with itself. (See our results page for how our attempts with SLIC progressed.) A time-course should be run with different periods of incubation are compared for efficiency.

---

## References

1. Danino, T., Mondragón-Palomino, O., Tsimring, L., & Hasty, J. (2010). A synchronized quorum of genetic clocks. Nature, 463(7279), 326–330. https://doi.org/10.1038/nature08753

2. Stricker, J., Cookson, S., Bennett, M. R., Mather, W. H., Tsimring, L. S., & Hasty, J. (2008). A fast, robust and tunable synthetic gene oscillator. Nature, 456(7221), 516–519. https://doi.org/10.1038/nature07389

3. Scolnik, P. A., & Haselkorn, R. (1984). Activation of extra copies of genes coding for nitrogenase in Rhodopseudomonas capsulata. Nature, 307(5948), Article 5948. https://doi.org/10.1038/307289a0

4. Scolnik, P. A., & Haselkorn, R. (1984). Activation of extra copies of genes coding for nitrogenase in Rhodopseudomonas capsulata. Nature, 307(5948), Article 5948. https://doi.org/10.1038/307289a0

5. García-Nafría, J., Watson, J. F., & Greger, I. H. (2016). IVA cloning: A single-tube universal cloning system exploiting bacterial in vivo assembly. Scientific Reports, 6(1). https://doi.org/10.1038/srep27459

6. Li, M. Z., & Elledge, S. J. (2012). SLIC: A method for sequence- and ligation-independent cloning. Methods in Molecular Biology (Clifton, N.J.), 852, 51–59. https://doi.org/10.1007/978-1-61779-564-0\_5

7. Plasmids 101: Origin of Replication. (n.d.). Retrieved October 11, 2023, from https://blog.addgene.org/plasmid-101-origin-of-replication

8. Li, M. Z., & Elledge, S. J. (2007). Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nature Methods, 4(3), 251–256. https://doi.org/10.1038/nmeth1010

{% 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 %}

![](https://static.igem.wiki/teams/4647/wiki/drawings.png)

## Experiments

## Background Research

Our final project was chosen at the end of our Spring Readings Course, a weekly, one-hour meeting where potential
project ideas, ins-and-outs of iGEM, and synthetic biology was discussed. Much deliberation, presentations,
and feedback from our mentors and advisors was incorporated into the development and implementation
outline of our project.

After our project was briefly outlined, the first challenge we tackled was the realization that nitrogenase,
although only coded by three genes, nifH, nifD, and nifK (or some other analogs in other organisms),
required other nif-family related genes to fully mature and express. During this time, we also contemplated
the potential downsides of being able to successfully express this protein. One major point of concern was the
sensitivity of the fully assembled protein to gases naturally found under normal laboratory conditions, especially
oxygen.

![](https://static.igem.wiki/teams/4647/wiki/exp-1-removebg-preview.png)

***Figure 1:*** Student generated figure

We spent countless hours on researching the necessary steps and proteins/genetic elements needed for full maturation of nitrogenase, and ultimately decided on a minimal Mo-Fe nitrogenase operon stitched together by the 2018 Nanjing China iGEM Team. Their project developed a nine gene operon (nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA, and nifV) controlled by a wildtype nif promoter (Pnif) to successfully express mature nitrogenase.

Because the part was documented but not yet available for distribution to other iGEM teams, ideas on how to assemble the operon were discussed. Based on feedback from advisors, especially AJ Sillato, we decided on concurrently attempting In Vivo Assembly (IVA) [1] and Sequence and Ligation Independent Cloning (SLIC) methods [2].

---

## Primer Design

Primer design is key for both IVA and SLIC. For this reason, an extended period was devoted to meticulously designing primers necessary for both methods.

See the below figure for specifications on primer design followed.

![](https://static.igem.wiki/teams/4647/wiki/exp/exp-2-removebg-preview.png)

***Figure 2:*** Student generated figure. Created using Biorender.com

---

## Assembly of Intermediate nif Gene Clusters

After all genes were successfully able to be amplified with the specific homology regions (either IVA or SLIC), chunks of genes were assembled. Since nine genes make up the total operon, we attempted to split the genes into triples. Thus, nifB-nifH-nifD were assembled as one cluster, nifK-nifE-nifN as another cluster, and nifX-hesA-nifV as a final cluster. Both IVA and SLIC methods were used to approach these intermediate assemblies. Protocols directly from the original IVA or SLIC papers were closely followed. Any deviations from the protocol are detailed under the protocols page.

---

## Assembly of the Full Nitrogenase Operon

After the three intermediate segments were assembled, the complete assembly of the operon was attempted. Primers to attach homology regions to the segments and plasmid 2Bc-T were used to amplify the segments. Because more success was achieved with the in vivo assembly method, this method was pursued primarily. All cloning results were screened initially with colony PCR or single restriction enzyme digests. Final screening was verified through whole plasmid sequencing through a third-party sequencing service, Plasmidsaurus. See our results page for our sequencing results and analysis!

---

![](https://static.igem.wiki/teams/4647/wiki/exp/exp-3-removebg-preview.png)

## Integration into the E. coli Genome

The original paper, which details this process more in depth, uses a dual-plasmid system— pCas (addgene #62225) and pTargetF (addgene #62226— to introduce either gene deletions or insertions through a coupling method between lambda-red technology and CRISPR/Cas9. Lambda-red recombineering technology is a method of genome engineering based on homology-directed repair. It requires three genes (exo, beta, gam) and a healing fragment to successfully integrate the fragment into the genome. However, this method of cloning is highly inefficient. Coupled with CRISPR/Cas9, where the cell has no choice but to repair its double-strand break with a healing fragment, the efficiency increases to almost 100%.

***Figure 3:*** Student generated figure

Concurrently to the assembly of the full nitrogenase operon, a plan to integrate the full operon in place of the E. coli gene araC was developed. Firstly, we chose this target region because we wanted to accomplish the following: (a) to knock out araC, which would deter predictability of the oscillation cycles according to our dry lab by expressing in baseline levels and (b) to knock-in the operon in a well-characterized region of the genome with complete control over copy number. Based on papers we found on multigene-editing through lambda-red/CRISPR coupled technology we designed a plan (see below figure) to integrate our assembled system into the E. coli genome [3,4].

---

## Protein Expression Tests for Nitrogenase Activity

Due to limited time, a coomassie stained gel was initially used to determine protein expression within the nitrogenase operon. (See figure on how coomassie works). This qualitative method involved over-expressing our nitrogenase genes through addition of IPTG, inducing for a range of times, and running the protein lysates through a SDS-PAGE. Then, a dye known as coomassie blue was added to the gel for subsequent staining. The stain, which selectively binds to certain amino acids, making it useful for estimation of protein concentration in a sample. See the below figure for an overview on how coomassie staining works or check out our protocols page for further information!

![](https://static.igem.wiki/teams/4647/wiki/exp/exp-4-removebg-preview.png)

***Figure 4:*** Student generated figure. Created using Biorender.com

Coomassie staining is a widely used qualitative technique to estimate the level of proteins of interest present in the sample. In this case, E. coli housing the nif genes controlled under the T7 promoter will be overexpressed by inducing for various amounts of times and temperatures with IPTG. After a standard SDS-PAGE is run, the gel will be stained with coomassie blue, which binds specifically to arginine, tyrosine, lysine, and histidine with great affinity.

A more phenotypically approach was also pursued. Based on past iGEM team’s research, several nitrogen-deficient medias were evaluated. Our rationale for this assay was the following: if our engineered E. coli were able to survive in nitrogen-deficient media, then they must be expressing the enzyme, and the environment must be selecting for these phenotypes.

---

## Incorporating Nitrogenase into the Oscillator

First, we wanted to confirm our models matched up with the Stricker et. al paper by expressing the dual-plasmid system (activator and repressor plasmids) under a confocal microscope. We wanted to show, as a proof of concept, that there was oscillation observed under our laboratory conditions.

Second, plans to clone the hybrid promoter as developed by Stricker et. al into the assembled cluster was established. The plan involved replacing the T7 promoter found at the beginning of 2Bc-T with the hybrid promoter using in-vivo assembly methods. This cluster would also be attempted to integrate into the genome, as we believed that would provide the most flexibility for controllable expression while also protecting against selective pressures that would cause species with the nitrogenase enzyme system to lose its parts in the wild.

---

## Protocols

These are the protocols we used throughout the experiment:

* [50 uL Restriction Enzyme Digest](https://static.igem.wiki/teams/4647/wiki/50-ul-restriction-enzyme-digest-prt-rjskgdez.pdf)
* [DNA Gel Electrophoresis](https://static.igem.wiki/teams/4647/wiki/dna-gel-electrophoresis-prt-tktqyqyd.pdf)
* [IVA Protocol (Insertions, Deletions, Sub-cloning, Multisite Insertions/Modifications)](https://static.igem.wiki/teams/4647/wiki/iva-protocol-insertions-deletions-sub-cloning-multisite-insertions-modifications-prt-lfjkkinl.pdf)
* [Modified Invitrogen Miniprep Protocol](https://static.igem.wiki/teams/4647/wiki/apznzabtkahbe68jmb3qgkzvxjxmjonofyc1yamjye8d-fo6c4tddgefcha-ikkkfqo4yhiflmev54ilv3m8shsou09s8s5xfnul9numcgnu4st5hzkizxmfxmk5nyrrj20iqz5qrrwccgowhovokpp-zqluh4j6getutw46aqt1uz4cxmqwilagv-8ow-xzfyozbpkf3lr-lndkqrhcehpjbxkdzoencklcgm9fl79j-i6h.pdf)
* [Qiagen Miniprep Protocol](https://static.igem.wiki/teams/4647/wiki/qiagen-miniprep-protocol-prt-dv06qfbp.pdf)
* [SDS-PAGE Protocol](https://static.igem.wiki/teams/4647/wiki/sds-page-protocol.pdf)
* [SLIC Protocol (General)](https://static.igem.wiki/teams/4647/wiki/acfrogb2gsffudwzzrkz-tol1agdrw5wmrgjqeuphna4fbmgbhiu2xpgt4erxejxhhpofwpiw25y7foqtyfqcz3k5grtjyn34t-eoszxl-xobo-gkhrykmoklg1tvlr1jtwwi06uowf7f-pnjwjh.pdf)
* [Specification for Leica Confocal Microscopy](https://static.igem.wiki/teams/4647/wiki/acfrogaqqmgz9bnoqcu0emqkqi5u-c2op2w9zxuz-tai7a2z9-yukrus2-kia1qba5ihldgiiqasoexxlnbes38oppdsd9-l3xdngeik6s2oj5il0rdsi3ncl0flmbgm05xwqqfeykwjgxw-x03a.pdf)
* [Streaking Backteria from Stabs](https://static.igem.wiki/teams/4647/wiki/streaking-bacteria-from-stabs-prt-2tfk8w1n.pdf)
* [Transformation of Competent Cells](https://static.igem.wiki/teams/4647/wiki/transformation-of-competent-cells-prt-gnkxvpzb.pdf)

---

## References

1. García-Nafría, J., Watson, J. F., & Greger, I. H. (2016). IVA cloning: A single-tube universal cloning system exploiting bacterial in vivo assembly. Scientific Reports, 6(1). https://doi.org/10.1038/srep27459

2. Li, M. Z., & Elledge, S. J. (2012). SLIC: A method for sequence- and ligation-independent cloning. Methods in Molecular Biology (Clifton, N.J.), 852, 51–59. https://doi.org/10.1007/978-1-61779-564-0\_5

3. Pyne, M. E., Moo-Young, M., Chung, D. A., & Chou, C. P. (2015). Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli. Applied and environmental microbiology, 81(15), 5103–5114. https://doi.org/10.1128/AEM.01248-15

4. Jiang, Y., Chen, B., Duan, C., Sun, B., Yang, J., & Yang, S. (2015). Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Applied and environmental microbiology, 81(7), 2506–2514. https://doi.org/10.1128/AEM.04023-14

{% endblock %}

* [Sequencing](#s11)

* [PCR & Gel Electrophoresis](#s22)

* [Confocal Microscopy](#s33)

* [Nitrogenase Protein
  Expression](#s44)
* [Proof of
  Concept](#s55)

## Results

The results of our work regarding sequencing, PRC and gel electrophoresis, confocal microscopy, and nitrogenase protien expression are detailed on this page. Our overall goal was to introduce the nitrogenase gene cluster into our E. coli cells by assemnbling the cluster *in vivo* using IVA assembly. We assembled the cluster first in three subsections: BHD cluster (nifB, nifH, and nifD), the KEN cluster (nifK, nifE, and nifN) and the XAV cluster (nifX, hesA, and nifV). Afterwards, we assembled these three subsections into our final gene cluster, containing the nine core genes necessary for nitrogen fixation in *E. coli* [1]. Concurrently, we introduced an activator and repressor plasmid into our cells as detailed in *(Stricker et. al., 2008)* [2]. We did so with the goal of using this positive and negative feedback system to acheive oscillating expression of both nitrogenase and GFP. In the future, we aim to repeat and refine our experiments, and with success, we aim to use our system to oscillate the expression of nitrogenase in other bacteria that may be better suited for agricultural use. It is our hope that our data will be useful for future iGEM teams interested in Sticker's feedback circuit, or in working with the nitrogenase enzyme.

---

## Sequencing

For each of the plasmids that we sequenced, we cataloged the mutations we observed. In addition, we assessed whether or not the ligation was sucessful, or if we would have to attempt it again. When applicable, we documented when the plasmid that we sequenced was used in subsequent experiments.

**See the example below:**

![](https://static.igem.wiki/teams/4647/wiki/results/result.jpg)

***Figure 1:** Insertion of nif B, nif H, and nif D genes into the 2Bc-T plasmid vector using IVA (attempt #1)*

Mutation

* Within the nif gene cluster:
  + 1 missense mutation in nif K (C to A mutation)
  + 1 silent mutation in nif E (G to A mutation)
  + 3 silent mutations in nif N (2 G to A, one C to G mutation)
* Within the nif gene cluster:
  + 1 missense mutation in nif K (C to A mutation)
  + 1 silent mutation in nif E (G to A mutation)
  + 3 silent mutations in nif N (2 G to A, one C to G mutation)

This ligation **succeeded**; all 3 nif genes were inserted in the correct order.
This colony was used for subsequent assembly attempts.

**See the document attached below to explore the rest of our sequencing results:**

#document

---

## PCR & Gel Electrophoresis

Firstly, we wanted to make sure that our PCR primers were functional in attaching the homology overhangs needed for either SLIC or IVA.

**We found that all of our samples were able to be amplified with all of our IVA primers.** For sake of convenience and maximizing the most help for future iGEM teams, we *characterized the IVA primers into the parts pages (see our parts pages), and left the SLIC primers documented in a separate document.*

However, despite all the primers designed with the same guidelines, **we found that PCR troubleshooting was needed for each of the primer pairs.** We primarily changed *annealing temperature, annealing time, and number of cycles.*

**See the detailed work below:**

![](https://static.igem.wiki/teams/4647/wiki/results/screenshot-2023-10-12-105631.png)

---

## Confocal Microscopy

To ensure replication of the genetic oscillator system, we decided to show these results through confocal microscopy. Although much troubleshooting is needed to fully show the clear oscillations, we did obtain preliminary results that were very promising. In the coming weeks, before the Grand Jamboree, we will work on troubleshooting this exact result to show distinct changes in GFP signals.

For preliminary results, and as a proof of concept that the oscillator is replicable, we show the following video and images to show that the GFP signal is dimming in the time period it should.

We tried to image for longer, however, long periods of exposure of the fluorescence signal to the E. coli cells showed that the GFP signal attenuated very rapidly (within 30 minutes or so). We will be working on showing results that will be independent of this confounding variable, to show that the GFP signal both dims and starts to brighten up again, with reliable periods.

![](https://static.igem.wiki/teams/4647/wiki/results/screenshot-2023-10-12-110540.png)

***Figure 2:** Confocal microscopy results, time period: 3:32:58*

![](https://static.igem.wiki/teams/4647/wiki/results/screenshot-2023-10-12-110551.png)

***Figure 3:** Confocal microscopy results, time period: 3:39:28*

Despite severe attenuation of signal of GFP, this cell, which remained in solution near the same spot all throughout (verified by brightfield image, although not imaged here), showed an **increase in GFP signal.**

***We will improve upon these results after the wiki freeze, to show clearer oscillations and link full results with dry lab models and predictions.***

---

## Nitrogenase Protein Expression

![](https://static.igem.wiki/teams/4647/wiki/results/screenshot-2023-10-12-115419.png)

Finally, we ran a Coomassie stain with overexpressed protein concentrations. We expressed each of the three constructs: BHD, KEN, and XAV. This particular sample read from right to left: protein ladder, pre-induction BHD, 3 hours BHD, 9 hours BHD, pre-induction KEN, 3 hours KEN, 9 hours KEN, pre induction XAV, 3 hours XAV, and 9 hours XAV. We also found bands at possibly the right size, and protein concentrations increased with time. Unfortunately, we realized we had to troubleshoot to make sure that these bands are resolved much better. We plan to troubleshoot these results between the wiki freeze and the final Jamboree presentation.

---

## Proof of Concept

**Based on our compiled results; we realize the following proofs of concepts:**

**- We showed that oscillations occur in our system; with confocal microscopy and dry lab modeling.**

1. We had hoped for a very strong connection between dry lab’s model and wet lab’s replication and imaging of the oscillator. Regardless, we were able to show evidence of an oscillation given the level of arabinose and IPTG used.

**- We showed that there was some evidence of complete assembly of the minimal nif operon.**

1. First, we saw a **10kb product when we amplified the 2Bc-T vector with T7 forward and reverse primers.**

1. Although our sequencing results showed a majority product for another plasmid, with a smaller size (which we later characterized as 2Bc-T with XAV inserted inside), we found a very small number of plasmids that were around the size we wanted to see.

   ![](https://static.igem.wiki/teams/4647/wiki/results/screenshot-2023-10-12-112040.png)

2. We tried amplifying this construct with T7/T7 terminator primers. We saw the following gel results. We will attempt to gel extract this band, and use it for PCR to see if the whole operon can be obtained from this construct.

   ![](https://static.igem.wiki/teams/4647/wiki/results/screenshot-2023-10-12-112243.png)

1. We obtained colonies (albeit very low CFU) with JM109 DE3 transformed with the miniprep colony of the above gel result on nitrogen-free media.

1. Although further screening must be done: **there were two colonies on a 100 mm plate with cells induced with 1mM final concentration IPTG.** We hope this means our expression of nitrogenase was successfully complete, ***but further tests will be done after the wiki freeze.***

---

## References

1. Li, X., Liu, Q., Liu, X., Shi, H., & Chen, S. (2016). Using synthetic biology to increase nitrogenase activity. Microbial Cell Factories, 15(1). https://doi.org/10.1186/s12934-016-0442-6

2. Stricker, J., Cookson, S., Bennett, M. R., Mather, W. H., Tsimring, L. S., & Hasty, J. (2008). A fast, robust and tunable synthetic gene oscillator. Nature, 456(7221), 516–519. https://doi.org/10.1038/nature07389