iGEM 2022 — Team manchester

{% 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 %}
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# Notebook

This page contains the timeline of our progress as a team, inlcuding key dates throughout.

# January

*3rd:* Team members first meet one another

*10th:* Team members begin weekly meetings at 6pm each Monday

*14th:* Max joins the team!

# February

*7th:* Julia joins the team!

*9th:* The team has their first meeting with with the supervisors and advisors

# March

*12th:* Team members go to the New Scientist Event to participate and conduct outreach

*21st:* Teodora joins the team!

*23rd:* Team members present their initial batch of ideas to the supervisors and advisors

# April

*1st:* Team roles are assigned to all members

*9th:* Catherine joins the team!

# May

*11th:* Following months of generating project ideas, the team decides to embark on a project concerning microalgal co-cultivation. BloomAid is born!

*20th:* The application form for the iGEM impact grant opens, and the team begins work on filling this out

# June

*10th:* The team is allowed into the lab for the induction week!

*17th:*Ahmed leaves the team

*18th:* The impact grant deadline looms, and the team races to submit the form

*22nd:* The safety and security grant deadline looms, forcing the team to scramble to submit the form

*26th:* Jorge and Teodora are allowed into the lab to do their (albeit delayed) induction

# July

*7th:* The team meets with Professor Jon Pittman, to discuss the scientific details of the project. the feedback provided on the algological aspects of the project vastly changes much of our research, and is key in developing the project into BloomAid

*8th:*The team meets with an advisor from BMG labtech to discuss the project

*25th:*The team has a first presentation practice session with team Bath

*27th:*The team meets with Prof John Love to better understand the microalgae cultivation process in wastewater

*28th:*The team meets with Professor Philip Shapira, to gain some insight into the business feasibility of the project

# August

*1st:*The team recieves the news that they have won the Impact grant!

*2nd:*Members of the team recieve informal training on the use of the CLARIOstar Microplate reader, for use in the interlab study and experimental procedures

*5th:*The team attends Manchester Institute of Biotechnology monthly seminar

*13th:*Member of the team travel to London to attend a UK meetup of iGEM teams from various uiversities, in order to discuss and present project ideas

*16th:*The team recieves news that they won the safety and security grant!

*25th:*The team meets with Mr Olan Ryan from Q8 Aviation to discuss implementation of biofuel into sustainable aviation fuel

*26th:*The team completes their interlaboratory study experimental procedures

# September

*5th:*Member of team visits a microalgae cultivation plants as well as biofuel reactor in Almeria, Spain

*9th:*The team has a second presentation practice session with team Bath

*13th:*The team hosts a virtual symposium with iGEM teams from Bath, Hong Kong and Thessaly

*19th:*University begins for all members! this marks the end of the 9AM-5PM workdays which all members have become so accustomed to

*25th:*The team meets with Prof George Patrinos for better comprehension of bioethics and implement of our project into the real world

*24th:*The team begins to fill in the judging form, aiming to qualify for the modelling, entrepreneurship and sustainable development special prizes

# October

*1st:*The phosphate experiment final assay is performed

*3rd:*The team prepares to leave the lab by cleaning and tidying their lab space

*9th:*With the wiki deadline so close, the team finalizes changes to the wiki and recieves feedback from advisors and supervisors

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{% 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 %}
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# Engineering

# Introduction

Our project’s first aim is to increase microalgal growth, which will subsequently contribute towards the second part of our project: the accumulation of lipids, which can be used for biofuel production. Microalgal growth is supported by essential nutrients, including vibrioferrin (investigated by [TeamSUIS](https://2018.igem.org/Team%3ASUIS_Shanghai)), vitamin B12 or indole-3-acetic acid (IAA) (Guo et al., 2019). After comparing the availability of the chemicals and feasibility of replicating the experiment, we decided to use indole-3-acetic acid which is a plant hormone within the class of auxin.

The primary function of auxin in algae is the detoxification of reactive oxygen species (ROS) (Wang et al., 2021), as auxin indirectly modulates the homeostasis of ROS by inducing the expression of ROS detoxification enzymes, such as glutathione S-transferases (GST) and quinine reductases (Laskowski et al., 2002). Indole-3-acetic acid (IAA) is the most prevalent and best characterized naturally occurring plant hormone within the auxin class (www.ebi.ac.uk, ChEBI). By recruiting antioxidant enzymes and antioxidants, such as GST, which catalyzes the conjugation of the reduced form of glutathione (GSH) to xenobiotic substrates for the purpose of detoxification (Douglas, 2006), IAA can prevent the production of ROS. Since the generation of ROS inhibits the growth, development, and metabolism of the microalgae, IAA can be added to reduce the amount of cell damage caused by abiotic stress and enhance the growth of microalgae (Guo et al., 2019).

Moreover, several studies show that exogenous addition of IAA can both induce lipid accumulation and increase the production of fatty acids (Lin et al., 2022;Liu, Qiu and Song, 2016;Wang et al., 2021). As fatty acids are building blocks of the targeted biofuel, one of our goals is to engineer a bacterium that is able to produce IAA. Since E.coli does not naturally synthesize IAA, we decided to equip it with the genes ARO8, KDC, and AldH to allow E.coli to produce auxin (Guo et al., 2019).

According to the literature, varying IAA concentrations have been found in microalgae extracts and culture supernatants, leading to both stimulatory and inhibitory effects on the microalgae's growth and metabolism (Han et al., 2018). Low levels of auxin promote growth, an increase in biomass, and the biosynthesis of biomolecules (such as fatty acids), but higher levels of auxin can inhibit cell growth (300 µM for the Desmodesmus species, which are members of the Chlorophyceae class, to which our strain of microalgae belongs)(Lin et al., 2022). Therefore, controlling a suitable level of IAA production is also our objective in order to ensure the system runs effectively and efficiently by providing a decent level of auxin for microalgal growth, and the goal of this experiment is to demonstrate that our bacterium can produce auxin, IAA in particular, for algal development after transformation of the plasmid containing the three auxin genes (Aro8, Ald-H, and KDC).

# Design

In order to enable our bacteria to help microalgae grow, the primary purpose of our experiment was to test whether our bacteria can produce IAA using the implemented production pathway.

Based on the literature, we found an engineered pathway for IAA production from glucose (Guo et al., 2019)(Figure 1). To boost the biosynthesis of IAA and simplify the process, we used L-tryptophan as the precursor for IAA production, which is catalysed by three enzymes:ARO8, KDC, and AldH (Figure 1). The three enzymes perform different functions: Saccharomyces cerevisiae aminotransferase ARO8 for the conversion of L-tryptophan to indole-3-pyruvic acid, S. cerevisiae decarboxylase KDC for the decarboxylation of indole3-pyruvic acid to indole-3-acetaldehyde, and E. coli AldH for the oxidation of indole-3-acetaldehyde to the corresponding IAA (Guo et al., 2019). The coding sequences were retrieved from GenBank, optimised for E. coli codon usage and synthesised as gBlocks. For the plasmid design, the [RBS](https://parts.igem.org/Ribosome_Binding_Sites) for the three auxin genes are found in the iGEM page and they are the same for ARO8, KDC and AldH.

![](https://static.igem.wiki/teams/4123/wiki/images-large-jf-2019-02048d-0001.jpeg)

Figure 1: Metabolic pathway for the production of IAA from glucose (Figure was taken from Guo et al., 2019)

To enable the expression of the three pathway genes and subsequent phytohormone production, we designed a plasmid that will subsequently be built and transformed into the bacterium. We chose the E. coli DH5a strain and pBb8Ec-RFP (www.addgene.org, n.d.) as the vector backbone as they are well characterized. As microalgae take up auxin in the medium, an E. coli transporter may be required for exporting auxin into the medium. We found a member of the auxin efflux carrier (AEC) family that is expressed in E. coli but uncharacterized (Guo et al., 2019), as well as additional family members that are expressed in either Gram-positive bacteria or yeast with reference to the auxin secretion in the medium (‌Vandeputte et al., 2005). However, since there may already be pumps or channels where the auxin is externalized and there are not any properly characterized auxin efflux carriers expressed in any E.coli strains, we did not put an efflux carrier within our plasmid.

For the design of modeling, our plan was to both quantify auxin and determine the impact on the bacterial growth rate. We aimed to generate data showing the exact rate of auxin produced by our engineered bacterium. If the auxin expression is successfully tested, this could be done under a plate reader with different concentrations of cells and monitored at various timepoints consecutively. Another possible modeling was to check if the concentration of auxin reaches a certain amount, the accumulated auxin might impact bacteria growth rate. To do this, the engineered bacteria could be cultured with normal bacteria in different concentrations of auxin. The growth rate obtained by the experiment will be used to model the possible impact of auxin on bacteria growth.

# Build

In this phase, the main focus was to ligate the g-blocks and the vector to a complete plasmid.

![](https://static.igem.wiki/teams/4123/wiki/screen-shot-2022-10-12-at-00-15-45.png)

Figure 2: Plasmid map of [BBa\_K4123001](http://parts.igem.org/Part%3ABBa_K4123001)) carrying ARO8, KDC, and aldH under control of the arabinose-inducible promoter.

We used the Golden Gate DNA assembly method to insert ARO8, KDC, and aldH into the vector backbone pBbE8c-RFP. The vector backbone, lacking the rfp reporter gene, was amplified by PCR. We also added a FLAG tag to each insert as we might do western blot to check the expression of certain proteins.

To assemble the plasmid, we designed primers to remove the FLAG-tag by PCR and did the agarose gel extraction. According to the agarose gel (Figure 3), the size of the two fragments (normal Aro8 and vector backbone) were roughly the same as we expected (∼1.6kb and ∼2.6kb), so we performed an agarose gelDNA extraction and measured the DNA concentration using the Nanodrop.

![](https://static.igem.wiki/teams/4123/wiki/screen-shot-2022-10-09-at-14-58-29.png)

Figure 3: Agarose gel results under a transilluminator. (Lane 1: 1kb plus DNA Ladder. Lane 3: PCR mix of Aro8. Lane 5: PCR mix of vector)

The other two gBlocks, encoding KDC and aldH, were resuspended in nuclease-free water. The Golden Gate assembly reaction was performed by mixing 1:5 molar ratio of g-blocks and vectors and incubating them in the thermocycler for the cycle (37°C, 1 min → 16°C, 1 min) x 30 cycles→ 60°C, 5 min. In total, two Golden Gate reactions were set up. One reaction contains all three inserts required to build the IAA biosynthesis pathway. To the second reaction only two of the inserts, ARO8 and aldH, were added. This reaction should not yield a circular plasmid and was used as negative control. LB-agar plates were supplemented with L-arabinose to induce gene expression.

![](https://static.igem.wiki/teams/4123/wiki/screen-shot-2022-10-09-at-15-00-22.png)

![](https://static.igem.wiki/teams/4123/wiki/screen-shot-2022-10-09-at-15-01-19.png)

![](https://static.igem.wiki/teams/4123/wiki/screen-shot-2022-10-09-at-15-01-25.png)

Among the 8 plates, we expect colony growth on plate 1 as the mixture contains all the three inserts and were plated on the plate with the correct selective marker (chloramphenicol) and were induced by arabinose. Plate 2 was the negative control with only two of the inserts, ARO8 and aldH. Growth was not expected on this plate as it should not yield a circular plasmid. Plate 3 and plate 4 were cell competency tests, so we expected to see a lawn of bacteria on the plate as pBbE8c-RFP was chloramphenicol resistant. Plates 5 to 8 were positive controls and we expected a lawn of bacteria on the plates as they were plated on plates without chloramphenicol. Based on the results, we could conclude that the transformation worked as we expected and our bacteria were likely to have the plasmid we made at this stage.

In the next stage of the experiment, the main goal was to test whether our bacterium produces auxin. To check for correct assembly, we performed a diagnostic digest. The transformed cells were used to measure the level of auxin production using UPLC. Ideally, the UPLC result should show the production of auxin in the medium, indicating that the engineered bacteria was producing auxin as intended.

To do this, 6 transformants were tested for correct assembly. We extract the plasmids inside the bacteria using miniprep and we also use the original vector pBbE8c-RFP as a control. We chose two restriction digestion enzymes to cut the plasmid. One was Ndel which cut a single site on the plasmid (Figure 4). The other one was HindIII which cut twice on the plasmid, giving two DNA fragments (Figure 5).

![](https://static.igem.wiki/teams/4123/wiki/screen-shot-2022-10-05-at-15-01-52.png)

Figure 4: Unique restriction sites of the final auxin plasmid. Ndel was used to digest the plasmid into linear strand.

![](https://static.igem.wiki/teams/4123/wiki/screen-shot-2022-10-05-at-15-02-02.png)

Figure 5: Unique and double restriction sites of the final auxin plasmid. HindIII was used to digest the plasmid into 2 fragments.

We ran the restriction digestions in parallel and in the same agarose gel (Figure 6). The results indicate that the plasmids were assembled correctly with 3 fragments as they gave correct sizes. The 6 transformants also showed roughly the same results on the gel.

![](https://static.igem.wiki/teams/4123/wiki/screen-shot-2022-10-05-at-15-14-55.png)

Figure 6: Agarose gel results. (Lane 1: 1kb plus DNA ladder. Lane 2-7: 6 linearized samples with expected size of 8131bp. Lane 8 & 15: intact vector backbone (undigested). Lane 9-14: 6 samples cutting into 2 fragments with expected sizes of 2737bp and 5394bp.)

# Test

![](https://static.igem.wiki/teams/4123/wiki/judging-form-final-build/Phosphate-Uptake-Gif.gif)

After checking the correctness of plasmid assembly, we carried on checking if the bacteria with the plasmid are able to produce auxin (IAA). The 6 transformants with 3 inserts and the original undigested vector backbone were taken from the previous step. Referring to the synthesis pathway of IAA, we added 100μl of 10 g/L L-tryptophan as the limiting factor for our bacteria to produce the final product, IAA. 1% Arabinose stock was also added into the 6 samples to make up 0.1% arabinose for gene expression. Then we put them on the shaker for 24h, and checked if the medium contains IAA under UPLC.

To compare the wavelength of peaks between our samples and the control, we included pure IAA and L-Tryptophan to see if our samples contain these compounds. Based on the literature, IAA has a UV absorption spectrum characteristic of substituted indoles, with a strong maximum at 220 nm (E220 = 33 200) and a characteristic of three overlapping peaks with maxima at 274, 282, and 288 nm (E282 = 6060) (www.sciencedirect.com, n.d.), while tryptophan absorbs more light at 280nm, which is distinct from IAA (Biotek.com, 2020). Since we aim to detect IAA in the samples, all 6 samples (G1-G6, G stands for golden gate product) were focused on 280 nm which corresponds to IAA.

We ran the pure sample of both IAA (Figure 7) and L-Tryptophan (Figure 8) under the same settings, and the intact vector (Figure 9) was included as a control.

![](https://static.igem.wiki/teams/4123/wiki/screen-shot-2022-10-05-at-15-23-04.png)

Figure 7: UPLC-DAD chromatogram of IAA at 280 nm. The retention time of IAA is roughly between 0.55 to 0.6 min (0.563 in specific) .

![](https://static.igem.wiki/teams/4123/wiki/screen-shot-2022-10-05-at-15-23-18.png)

Figure 8: UPLC-DAD chromatogram of L-Tryptophan at 280 nm. The retention time of L-Tryptophan is roughly between 0.3 to 0.35 min (0.313 min in specific).

![](https://static.igem.wiki/teams/4123/wiki/screen-shot-2022-10-05-at-15-23-35.png)

Figure 9: UPLC-DAD chromatogram of the vector (pBbE8c-RFP) at 280 nm. The peaks were roughly between 0.25 to 0.3 min (0.258 in specific) and between 0.8 to 0.85 (0.820 in specific).

![](https://static.igem.wiki/teams/4123/wiki/screen-shot-2022-10-05-at-15-23-45.png)

![](https://static.igem.wiki/teams/4123/wiki/screen-shot-2022-10-05-at-15-23-59.png)

![](https://static.igem.wiki/teams/4123/wiki/screen-shot-2022-10-05-at-15-24-11.png)

![](https://static.igem.wiki/teams/4123/wiki/screen-shot-2022-10-05-at-15-24-22.png)

Figure 10: UPLC-DAD chromatogram of the 6 samples (G1-G6) at 280 nm.

According to the graphs (Figure 10), G1, 2, 5 of the 6 samples show differences with the vector control, while the other 3 samples showed a similar pattern with the vector control. Moreover, the peaks were not in a perfect overlap with IAA.

# Learn

![](https://static.igem.wiki/teams/4123/wiki/judging-form-final-build/Phosphate-Uptake-Gif.gif)

According to the results of UPLC, the results are inconclusive to show that our bacteria successfully produced IAA after the addition of L-tryptophan. In this case, we were considering additional tests to get more information from the experiment.

We could add more tryptophan for auxin synthesis. As we only added 0.1M L-tryptophan to the medium, this might not be sufficient for our bacteria to use as substrate for IAA production pathway. After comparing the excitation wavelength of the intermediates in the data analysis section, it is difficult to tell the exact compounds responsible for other peaks. Therefore, MS may be performed to see the details of the compounds corresponding to other peaks.

Another possible circumstance was that our plasmid did successfully assembled while several key sequences might have changed as the sequence was not checked. Although we checked the size of the plasmid before adding L-Tryptophan for IAA secretion, the results of the gel cannot guarantee the accuracy of sequences. The gel is not able to show us the specific difference if there were mutations in several sequences. To further check the sequences, we could do Sanger sequencing to the three samples (G1, G2, and G5) shown differences with the control in UPLC. If the sequences are what we desired, we could conclude that the plasmid was built successfully. As we still don’t know if the gene is expressing KDC, Aro8, and AldH, it might be necessary to check if the genes are functional. Therefore, presence of ARO8, KDC, and AldH may be validated by western blot analysis of FLAG-tagged proteins.

# **Reference list**

‌Biotek.com. (2020). Peptide and Amino Acid Quantification Using UV Fluorescence in Synergy HT Multi-Mode Microplate Reader | April 18, 2003. [online] Available at: https://www.biotek.com/resources/application-notes/peptide-and-amino-acid-quantification-using-uv-fluorescence-in-synergy-ht-multi-mode-microplate-reader/.

Douglas, K.T. (2006). Mechanism of Action of Glutathione-Dependent Enzymes. Advances in Enzymology - and Related Areas of Molecular Biology, pp.103–167. doi:10.1002/9780470123058.ch3.

Guo, D., Kong, S., Chu, X., Li, X. and Pan, H. (2019). De Novo Biosynthesis of Indole-3-acetic Acid in Engineered Escherichia coli. Journal of Agricultural and Food Chemistry, 67(29), pp.8186–8190. doi:10.1021/acs.jafc.9b02048.

Lin, Y., Dai, Y., Xu, W., Wu, X., Li, Y., Zhu, H. and Zhou, H. (2022). The Growth, Lipid Accumulation and Fatty Acid Profile Analysis by Abscisic Acid and Indol-3-Acetic Acid Induced in Chlorella sp. FACHB-8. International Journal of Molecular Sciences, [online] 23(7), p.4064. doi:10.3390/ijms23074064.

Liu, J., Qiu, W. and Song, Y. (2016). Stimulatory effect of auxins on the growth and lipid productivity of Chlorella pyrenoidosa and Scenedesmus quadricauda. Algal Research, 18, pp.273–280. doi:10.1016/j.algal.2016.06.027.

‌

Vandeputte O, Oden S, Mol A, Vereecke D, Goethals K, El Jaziri M, Prinsen E. Biosynthesis of auxin by the gram-positive phytopathogen Rhodococcus fascians is controlled by compounds specific to infected plant tissues. Appl Environ Microbiol. 2005 Mar;71(3):1169-77. doi: 10.1128/AEM.71.3.1169-1177.2005. PMID: 15746315; PMCID: PMC1065166.

‌

Wang, C., Qi, M., Guo, J., Zhou, C., Yan, X., Ruan, R. and Cheng, P. (2021). The Active Phytohormone in Microalgae: The Characteristics, Efficient Detection, and Their Adversity Resistance Applications. Molecules, 27(1), p.46. doi:10.3390/molecules27010046.

www.addgene.org. (n.d.). Addgene: pBbE8k-RFP. [online] Available at: https://www.addgene.org/35270/

www.ebi.ac.uk. (n.d.). indole-3-acetic acid (CHEBI:16411). [online] Available at: https://www.ebi.ac.uk/chebi/searchId.do?chebiId=CHEBI:16411#:~:text=Indole%2D3%2Dacetic%20acid%20(IAA%2C%203%2DIAA

‌

www.sciencedirect.com. (n.d.). Indole-3-Acetic Acid - an overview | ScienceDirect Topics. [online] Available at: https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/indole-3-acetic-acid#:~:text=IAA%20has%20a%20UV%20absorption

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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# Experiments

This page contains the notebooks that were kept for each wet-lab experiment, in addition to the induction week and interlab study protocols. For more details on these experiments, including detailed explanations as to their rationale, refer to the wet-lab section below.

[Introduction Week Protocols](https://static.igem.wiki/teams/4123/wiki/igem-induction-week-1-compressed.pdf)

[iGEM Manchester 2022 Lab Book](https://static.igem.wiki/teams/4123/wiki/lab-book-nikhil-min.pdf)

[Interlab Study Protocols](https://static.igem.wiki/teams/4123/wiki/igem-interlab-study-experiment-3-challenge-cell-measurement-min.pdf)

# Wet Lab Experiment Links

[![](https://static.igem.wiki/teams/4123/wiki/1024px-indol-3-ylacetic-acid.png)

Auxin Experiment

![](https://static.igem.wiki/teams/4123/wiki/judging-form-final-build/asset-14.png)](#Auxin-Experiment)

[![](https://static.igem.wiki/teams/4123/wiki/undraw-lightbulb-moment-re-ulyo.png)

Light switch experiment

![](https://static.igem.wiki/teams/4123/wiki/judging-form-final-build/asset-14.png)](#Light-Switch-Experiment)

[![](https://static.igem.wiki/teams/4123/wiki/rfp.png)

RFP-silencing experiment

![](https://static.igem.wiki/teams/4123/wiki/judging-form-final-build/asset-14.png)](#RFP-Experiment)

[![](https://static.igem.wiki/teams/4123/wiki/crisprcas9.png)

PhoU Experiment

![](https://static.igem.wiki/teams/4123/wiki/judging-form-final-build/asset-14.png)](#PhoU-Experiment)

# Standard Protocols

Throughout the experiments, our team used a set of standard protcols. This enabled us to produce batches of reagents in advance and troubleshoot our epxeriments to a greater degree.

# Click the link for the standard protocols

[Standardised procedures](https://static.igem.wiki/teams/4123/wiki/copy-of-standard-protocols-compressed.pdf)

# Quantification of Auxin biosynthesis in E.Coli

The auxin expression experiment aims to prove our bacteria are able to produce auxin for algae growth after taking up the plasmid with the 3 auxin genes (Aro8, Ald-H, and KDC). The first part of the experiment includes inserting all g-blocks (Aro8, Ald-H, and KDC) into the vector backbone (pBbE8c-RFP) through golden gate assembly followed by transformation. The second part of the experiment aims to show the auxin expression through measuring auxin concentration by UPLC.

# Click the link for the Auxin procedures

[Auxin Procedures](https://static.igem.wiki/teams/4123/wiki/auxin-protocols-1-compressed.pdf)

# Light SwitchCre-LoxP recombination Experiment

The Light Switch experiment aims to prove our bacteria are able to change their genetic expression from one set of genes to another, via induction using a specific wavelength of light. The first part of the experiment involved cloning the LoxP gblock into a vector backbone, before transforming into bacteria. The second part of the experiments then involved testing the fluorescence of the MKATE2 before inducing a genetic change by transfecting with the Cre Plasmid. Transformed and induced cells were then tested for the fluorescence expression of MKO2, which comprised the final section of the experiments.

# Click the link for the Light Switch procedures

[Light Switch procedures](https://static.igem.wiki/teams/4123/wiki/cre-lox-experimental-procedure-min.pdf)

# PhoU knockdown for phosphate accumulation

The phosphate-accumulation experiments aims to show that our bacteria are capable of taking up phosphate from a growth medium and sequestering it in their cells. We seek to starve the microalgae of phosphate to trigger lipid accumulation. The firs part of the experimental procedure is concerned with the clonning or our pdCas9 sysytem and the transformation of our competent E.Coli. The second section explains the use of our vanadate molybdate colorimetric assay to detect phosphate sequestration.

# Click the link for the Phosphate uptake procedures

[PhoU knockout procedures](https://static.igem.wiki/teams/4123/wiki/phosphate-experimental-procedure-min.pdf)

# RFP silencing via introduction of PdCas9 plasmid and light induction

The RFP silencing experiment aims to validate our genetic switch mechanism through the activation of LoxP, leading to the knockout of Red Fluorescent Protein encoded in the genome of the bacteria. This will provide a simple visual cue as to whether the genetic rearrangement leads to a change in expression. The first part of the experiment involved introducing the RFP pdCas9 plasmid into a strain of RFP genome encoded *E.Coli*. This was followed by induction the plasmid via Cre and IPTG. the second section of experiments involved identifying the loss of fluorescence of the RFP caused by the CRISPR knockout.

# Click the link for the RFP silencing procedures

[RFP silencing procedures](https://static.igem.wiki/teams/4123/wiki/rfp-silencing-experiment.pdf)

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{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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# Results

Below are the summarized results of each of our experiments: auxin biosynthesis, phosphate uptake, light switch, and RFP silencing. For a more detailed information on the experiments, including their experimental procedures and lab books, please refer to our experiments page:

[Experiments](https://2022.igem.wiki/manchester/experiments)

# Auxin Biosynthesis

![](https://static.igem.wiki/teams/4123/wiki/judging-form-final-build/Phosphate-Uptake-Gif.gif)

In the biosynthesis of auxin (IAA), the goal of this experiment is to demonstrate that our bacterium can produce auxin, IAA in particular, for algal development after the transformation of the plasmid containing the three auxin genes (*Aro8, Ald-H*, and *KDC*). After transformation, we checked the correct assembly of the plasmid (BBa\_K4123001) by performing a diagnostic digest, and the results indicate that the plasmids were assembled correctly with 3 fragments as they gave the correct sizes. After checking the correctness of the plasmid assembly, we carried on checking if the bacteria with the plasmid are able to produce auxin (IAA). By using L-tryptophan as a limiting factor, we compared the wavelength of peaks between our 6 Golden Gate samples (G1-G6) and the control using Ultra Performance Liquid Chromatography (UPLC). Since we aim to detect IAA, the samples were focused on 280 nm which corresponds to IAA. We also ran the pure sample of both IAA (Figure 1) and L-Tryptophan (Figure 2) under the same settings, and the intact vector (Figure 3) was included as a control. Only 3 of the 6 samples showed differences with the vector control, while the other 3 samples showed a similar pattern with the vector control. Moreover, the peaks were not in perfect overlap with IAA (Figure 4). Therefore, the results are inconclusive to show that our bacteria successfully produced IAA after the addition of L-tryptophan. In this case, we were considering additional tests to get more information from the experiment.

![](https://static.igem.wiki/teams/4123/wiki/screen-shot-2022-10-05-at-15-23-04.png)

*Figure 1: UPLC-DAD chromatogram of IAA at 280 nm. The retention time of IAA is roughly between 0.55 to 0.6 min (0.563 in specific).*

![](https://static.igem.wiki/teams/4123/wiki/screen-shot-2022-10-05-at-15-23-18.png)

*Figure 2: UPLC-DAD chromatogram of L-Tryptophan at 280 nm. The retention time of L-Tryptophan is roughly between 0.3 to 0.35 min (0.313 min in specific).*

![](https://static.igem.wiki/teams/4123/wiki/screen-shot-2022-10-05-at-15-23-35.png)

*Figure 3: UPLC-DAD chromatogram of the vector (pBbE8c-RFP) at 280 nm. The peaks were roughly between 0.25 to 0.3 min (0.258 in specific) and between 0.8 to 0.85 (0.820 in specific).*

![](https://static.igem.wiki/teams/4123/wiki/screen-shot-2022-10-05-at-15-23-45.png)

![](https://static.igem.wiki/teams/4123/wiki/screen-shot-2022-10-05-at-15-23-59.png)

![](https://static.igem.wiki/teams/4123/wiki/screen-shot-2022-10-05-at-15-24-11.png)

![](https://static.igem.wiki/teams/4123/wiki/screen-shot-2022-10-05-at-15-24-22.png)

*Figure 4: UPLC-DAD chromatogram of the 6 samples (G1-G6) at 280 nm.*

For more details, please go to the [engineering page](https://2022.igem.wiki/manchester/engineering) and the [lab book](https://2022.igem.wiki/manchester/experiments).

# Phosphate Uptake

![](https://static.igem.wiki/teams/4123/wiki/judging-form-final-build/Phosphate-Uptake-Gif.gif)

The two transformed bacterial colonies (Transformant A and Transformant B) were grown overnight alongside two controls: a colony composed by the pdCas9-sgRNA-RFP-containing bacteria and a colony containing commercially-competent bacteria. These were grown overnight in M9 minimal medium alongside the inducers arabinose and anhydrotetracycline (which were expected to activate the expression of the dCas9 and the PhoU-gRNA genes). The next morning, after calibrating our spectrometer using the vanadate-molybdate method and a set of phosphate dilutions, the growth medium from these bacterial cultures was extracted and compared. This was done through a set of serial dilutions to increase the sensitivity of the vanadate-molybdate reagent. The reading at 470 nm led to the conclusion that the bacterial cultures had roughly the same phosphate-sequestration capacity, as the absorbance levels presented by the spectrometer were highly similar (table 1). Therefore, we conclude that the experimental results did not provide evidence of the successful engineering of bacteria with a higher capacity to absorb and accumulate phosphate.

![](https://static.igem.wiki/teams/4123/wiki/screen-shot-2022-10-11-at-22-41-10.png)

*Table 1: Table presenting the absorbance values of each one of the serial dilutions of the four growth mediums. It can be observed how the phosphate concentration levels are highly similar between the four mediums, leading to the conclusion that our engineered bacteria did not have a higher capacity to sequester phosphate from the medium.*

For more details, please go to our [lab book](https://2022.igem.wiki/manchester/experiments).

# Light Switch

![](https://static.igem.wiki/teams/4123/wiki/judging-form-final-build/Phosphate-Uptake-Gif.gif)

The goal of this experiment was to determine whether our Opto-Cre-Vvd recombinase is able to correctly rearrange the genes within our FLEx cassete. To start, we first amplified our LoxP gBlock containing our FLEx cassette using PCR. Following the PCR of the LoxP gBlock, a clear band at the desired size of 1.7 kb was obtained.

![](https://static.igem.wiki/teams/4123/wiki/pcr.jpg)

*Figure 5: Gel from our PCR amplification of the gBlock. Lanes 6 and 8 show a band of 1.7kb, indicating the presence of the LoxP gBlock amplification*

We then conducted a gel extract of this PCR product, followed by a Gibson assembly with the vector backbone. The Gibson assembly product was then transformed into a fresh culture of competent cells, with both DH5a and Stellar competent cells being used. A number of colonies were formed, which we then used to grow overnight cultures to amplify the Gibson product. Images of the colonies formed are shown in the figure below.

![](https://static.igem.wiki/teams/4123/wiki/cell-image.jpg)

*Figure 6: Colonies formed when the competent cells were transformed with the Gibson product. We observed a lawn of colonies for the Stellar competent cells, and a lower amount of colonies for the DH5a competent cells.*

We then attempted to detect the presence of the Gibson product within these bacterial cultures using a fluorescence reading. We attempted to detect the presence of the mKate2 fluorescent protein via this fluorescence. However, only fluorescence levels comparable to blank were detected from all 10 samples, meaning our transformation was not successful. Unfortunately, determination of the erroneous part of the experiment was difficult due to the lack of control for the transformation stage. Therefore, a troubleshooting step was conducted via restriction enzyme digestion to figure out if our Gibson assembly had been successful. To do this, we decided to conduct a restriction digest. Upon restriction enzyme digestion with BbsI and XbaI, plasmid with loxP insert would be cut in two places to form two bands of equal size at around 1.7 kb. On the other hand, recircularized vectors with no insert would be cut to form two distinct bands, at 2.7 kb and 1 kb. Below is the result of the restriction enzyme digestion of the Gibson product transformants.

![](https://static.igem.wiki/teams/4123/wiki/resultslightswitch.png)

*Figure 7: gel electrophoresis results of restriction digestion of our transformants.*

As shown in figure 5, all 10 transformants failed to show a clear band at 1.7 kb, indicating that our transformation was unsuccessful and that our loxP gBlock was not cloned into the vector properly. Thus, as our Gibson cloning was unsuccessful, the bacterial colonies that we grew did not contain the fluorescent proteins of interest. This also explains blank fluorescence levels from plate reading. Unfortunately, we were unable to go back to the first stage of our experiment to try and generate a functional Gibson product due to time constraints, and we were forced to leave the experiment here. Given more time, we would have re-attempted the PCR of the loxP gBlock, followed by the Gibson cloning of this product. This would have hopefully yielded a plasmid that we could then transfect into bacteria, and complete the experiment according to the rest of the wetlab protocol.

For more details, please go to our [lab book](https://2022.igem.wiki/manchester/experiments).

# RFP Silencing

![](https://static.igem.wiki/teams/4123/wiki/judging-form-final-build/Phosphate-Uptake-Gif.gif)

Gibson assembly was carried out to inset the gBlock. The transformation was carried out and the results of this step are shown in figure 1. A and B are the experimental assembly pCas9. When 50 μL were plated, this did not produce any colonies. B the pellet was plated, and white colonies were observed, which correlates with what is expected as the insert insertion will disrupt the RFP gene (RFP gene preces make red colonies). C and D are the transformants with the assembly that do not contain the insert. No colonies were observed as suggested. E and F are the transformants with pdCas9-sgRNA-RFP. Red colonies are observed in both cases as expected, because the RFP gene will give a red-pink colour to the colonies. G and H are transformants of the Linearized-PCR pdCas9-sgRNA-RFP. No colonies were observed, as expected.

![](https://static.igem.wiki/teams/4123/wiki/screenshot-2022-10-11-at-22-58-57.png)

*Figure 8: Transformation results after Gibson assembly. A and B are transformants of the assembly pdCas9, 50 μL and pellet were plated respectively. White colonies are observed only in B. C and D are transformants of the assembly pdCas9 without insert, 50 μL and pellet were plated respectively. No colonies were observed. E and F are transformants of the pdCas9-sgRNA-RFP, 50 μL and pellet were plated respectively. Red colonies are observed in both cases. G and H are transformants of the linearized-PCR pdCas9-sgRNA-RFP, 50 μL and pellet were plated respectively. No colonies were observed.*

Note: This experiment was interrupted due to time limitation, However, the assembly was completed. Please direct to the [lab book](https://2022.igem.wiki/manchester/experiments) for information about the complete protocol.

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