iGEM 2023 — Team edinburgh

{% 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 %}

# Notebook

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### Weeks 1 to 5 (1st May to 4th June):

**Conductivity:** We started reading on MtrCAB and Cytochrome A (CymA) gene from Shewanella Oneidensis MR-1. Read on tests to quantify conductivity, settled on a ferrocyanide test. We made BG11 growth medium for Synechocystis sp. PCC 6803. Selected genes NC\_004347.2 from NCBI. Designed primers. We began in-silico extraction (in silico primers) and assembly of the individual Mtr genes and the CymA gene in Benchling. We received the genome of Shewanella Oneidensis MR-1 (courtesy of Horsfall lab). We started extraction MTR genes; MtrA, MtrB, MtrC and CymA via PCR extraction, however this failed. Due to the failed PCR result for MTR, we switched from Q5 to Superfly polymerase, which also failed, confirmed via gel electrophoresis. Moreover, we received the cyanogate parts from the McCormick lab. The parts were given in E. coli. The E. coli was allowed to grow overnight followed by purification of the parts using Qiugen miniprep. We also ordered universal cyanogate parts for level 0 and 1 assembly.

---

### Week 6 (5th June to 11th June):

**Conductivity:** We continued PCR extraction for MTR, however with OneTaq polymerase (NEB). The gel run showed successful results (refer results).

**Carbon Sink:** We began to brainstorm how to integrate a carbon sink into our system. Decided to use PHA operon originally found in Synechocystis sp. PCC 6803. We began in-silico extraction and assembly of the PHA operon in CyanoGate vectors.

**Hardware:** Reading and development of 3-D design.

**Biosafety:** Brainstorming and reading.

---

### Week 7 (12th June to 18th June):

**Conductivity:** The PCR product was cleaned using Wizard SV GEL and PCR Cleanup (Promega). The Cyanogate protocol was employed for assembly of level 0 constructs and transformation of said constructs into cells. While some white colonies appeared, screening via colony PCR revealed that the plasmids were not the correct size, likely as a result of them closing prematurely.

**Carbon Sink:** We made multiple attempts to extract the PHA operon using Q5 polymerase from the PCC 6803 genome via PCR. The attempt failed.

**Hardware:** We set up CO2 limited growth for Synechococcus 11901 using KOH 1M solution to locally reduce CO2 levels.

**Biosafety:** Brainstorming and reading.

---

### Week 8 (19th June to 25th June):

**Conductivity:** Enzymes were switched to troubleshoot level 0 assembly, going from Bpi (ThermoFisher) BsaI (NEB). The bands were still the incorrect size. Troubleshooting continued by simplifying the protocol with only vector, PCR product, water, DNA ligase, BsaI and T4 DNA ligase. This yielded the expected band sizes. Furthermore, we began the extraction of MTR operons (MTR CAB, CA and AB) using Q5 polymerase. As Q5 did not work, so we switched back to OneTaq, which worked for MTR CA and AB, but not CAB. This was determined to be due to the elongation time being 2 minutes.

**Carbon Sink:** We continued the PHA operon extraction.

**Hardware:** Preparation of Salt bridge using NaCl, crude measurements with Potentiostat testing various cyanobacterial culture & electrolytes concentrations.

**Salinity resistance:** We started brainstorming ways of improving the conductivity of our solar cell after hardware tests.

**Biosafety:** In-silico design and assembly of the NucA-NuiA toxin-antitoxin system.

---

### Week 9 (26th June to 2nd July):

**Conductivity:** Level 0 vectors were inoculated with promoters and terminators, and then purified using a Monarch MiniPrep kit (NEB). Purified vectors were confirmed to be the correct size by digestion with BsaI and PvuI. The MTR CA and AB samples were cleaned using Wizard SV GEL and PCR Cleanup. These 2 samples were PCR-ed with longer primers to introduce overhangs for level 0 assembly. Incorrect band sizes appeared on gels. Simultaneously, extraction of MTR CAB continued, this time with an elongation time of 6 minutes (previously 2 minutes). Extraction was successful (see results). Cleanup of all 3 samples was attempted, yielding correct band sizes. Another attempt at PCR with overhang primers was made, switching back to Q5 polymerase. Herein, 3 reactions with the Q5 polymerase PCR reaction were set up at different annealing temperatures for troubleshooting. Results showed correct band sizes.

**Carbon Sink:** We continued to do the PCR extraction of PHA. This was successful.

**Artificial Pollen:** We started brainstorming for Artificial pollen integrating our human practices. We decided to try and induce increased production of specific amino acids (Histidine and lysine) for increased nutritional content of the cyanobacteria.

**Salinity resistance:** Due to our meeting with Professor Jamie Marland, we decided to improve conductivity by increasing salt (NaCl) concentration. Further brainstorming led to the idea of improving the salt tolerance of synechocystis through directed evolution.

**Biosafety:** We checked the effect of varying Zn2+ ion concentration in the 0-16 micromolar range in the medium on the growth of Synechocystis sp. PCC 6803 in order to determine the optimal inducer concentration that could be used in the solar panel for antitoxin induction without large negative effects on growth. Additionally, the level 0 parts ordered from Prof. McCormick’s lab arrived.

**Hardware:** We continued to make the growth curve for Synechocystis sp. PCC 6803.

---

### Week 10 (3rd July to 9th July):

**Conductivity:** MTR assembly continued onto level 1 with Cyanogate protocol with adaptations applied from the simplified level 0 assembly (See week 8). We performed a gel extraction of G-blocks for later use in the cyanogate protocol. Confirmation by gel electrophoresis showed expected band sizes. We assembled level 0 plasmids for MTR CA, AB, and CAB and then transformed them into competent TOP10 cells. Cells were screened on spectinomycin plates and left overnight. Screening successfully returned blue colonies for controls and white colonies for experimental. We performed colony PCR using the Green Go-Taq protocol, returning correctly sized bands for CA, AB, and CAB level 0 plasmids. The cultures that yielded correct bands were left in colony PCR overnight.

**Artificial Pollen:** We selected genes for his-lys expression, settling on hisG for histidine (with hik8 as a backup) and dapA and lysC for lysine. We investigated qualitative tests for histidine-lysine (his-lys) and chose mass-spec analysis in the University of Edinburgh’s Waddington Laboratories. We started the assembly of his-lys genes in silico, ordering primers and preparation of E. coli MG1655 cells for genome extraction.

**Directed Evolution:** Fully saturated NaCl solutions were prepared and preliminary growth curves with E. coli K12 were taken for our salinity subproject. The results were unexpected, as literature suggested that colonies wouldn’t tolerate salt concentrations higher than 1.5%, but plates saw growth in concentrations as high as 5%. Investigation using light microscopy showed that concentrations above 3% had lysed most colonies after they expanded.

**Carbon Sink:** We assembled and transformed the PHA operon into level 0 acceptor vector plasmids. The transformation returned a few white colonies which were checked via colony PCR screening, showing potential positive result. Following, diagnostic digestions were performed. These digestions showed the requirement for domestication. Therefore, we started designing the domestication primers.

**Hardware:** Discovered Synechococcus 11901 does not form a biofilm, switched to 6803 for the same. Set up was made for 11901. We tried to induce biofilm formation on different surfaces including Metal, Glass, Activated Charcoal. However, through microscopic analysis, we determined the cells did not adhere to any surface measured via eYFP observation.

---

### Week 11 (10th July to 16th July):

**Conductivity:** We miniprepped MTR CA, AB, CAB, and B, all coming back with correct band sizes in gel runs. Digestion of the plasmids also showed correct sizes of G-blocks (our lab lead couldn’t emphasize how perfect the results were). This confirmed that we’d need to domesticate MTR C in blocks CAB and CA.

**Artificial Pollen:** We extracted target his-lys genes (hisG, lysC, and dapA) from the genomic DNA of E. coli MG1655 via PCR extraction. Gel run showed expected band sizes (results).

**Biosafety:** Level 0 assembly was performed using BbsI restriction enzyme of G-blocks, including constitutive Synechocystis promoter PrbcL1C, zinc-inducible promoter PcopM, NucA nuclease toxin, and NuiA antitoxin, for the zinc-inducible toxin-antitoxin Kill Switch (KS) system. Competent E. coli TOP10 cells were transformed and plated on IPTG, Xgal, and Spectinomycin containing plates for screening.

**Carbon Sink:** Moreover, we continued to assemble and transform the PHA genes into level 0 acceptor plasmids. However, the absence of white colonies demonstrated failure. Nonetheless, some of the blue colonies were colony PCR-ed to double-check the colonies. The blue colonies did not have the gene of Interest.

**Directed Evolution:** We designed ggpS and ggpP primers for extraction from the PCC 6803 genome for salinity resistance.

---

### Week 12 (17th July to 23rd July):

**Conductivity:** We assembled MTR AB in level 1 plasmids using the cyanogate protocol and inoculated them into cells via transformation and colony PCR. Gel runs showed incorrect bands. We subsequently domesticated PCR for MTR the required blocks (CAB/CA). Domestication PCR was successful.

**Artificial Pollen:** The amplified and gel-extracted his-lys genes were purified, and so we began level 0 and 1 plasmid construction in silico. We transformed the level 0 plasmids at the end of the week into TOP10 E. coli cells. The his/lys cultures were plated and left to grow. 3 White colonies from lv0 his-lys transformations were selected for colony PCR. No bands appeared for the positive control or the other colonies. Therefore, we troubleshooted the colony PCR by lowering primer concentration and increasing annealing temperature, with hopes of mitigating primer dimer formation. Nevertheless, the colony PCR did not work.

**Biosafety:** Five transformed white colonies were selected for colony PCR screening of each Level 0 KS construct, and overnight cultures of colonies giving correct cPCR bands were set up. Plasmids were subsequently purified from these cultures using a QIAprep Spin Miniprep Kit (Qiagen) and digested using Bsal and Pvul enzymes to confirm correct insertion. Digestion and gels showed that the level 0 constructs contained correct insertions of the expected size. We assembled level 1 constructs containing eYFP under the control of the constitutive promoter PrnpB and zinc-inducible PcopM promoter for further testing and characterization of the zinc promoter by measuring eYFP fluorescence. We also made growth media with smaller variations in Zn2+ concentration based on the previous growth curve.

**Hardware:** Assembly of DC-DC current amplification circuit, worked on 3D print design.

**Carbon Sink:** The level 0 PHA assembly and transformation were redone, which successfully returned white colonies. At the same time, we performed Nile Red staining of 6803 and 11901 followed by light microscopy for our controls and learning the technique. Staining was successful for only 6803 (Refer results).

**Directed Evolution:** We extracted ggpS and ggpP by PCR. While extraction and purification from gel was successful, concentrations were too low for us to transform cells into level 0.

---

### Week 13 (24th July to 30th July):

**Conductivity:** Due to multiple bands of the domestication process producing smears (insert reference), we proceeded with gel extraction (instead of PCR cleanup). A domestication cycle was done for the PCR product CAB and CA in the thermocycler. The domestication was checked via diagnostic digestion showing failed domestications. Moreover, the continuing forward with Lv1 AB, cPCR for performed, however, this did not work.

**Artificial Pollen:** Further troubleshooting was performed for his-lys genes level 0 transformations colony PCR. We adjusted the assembly and thermocycling procedures by replacing ATP volumes with water, as it may have been inhibiting T4 DNA ligase. We changed from NEB T4 DNA ligase to NEB High Fidelity T4 DNA ligase. Of the 108 colonies transformed among the 3 genes, colony PCR followed by gel electrophoresis showed no bands.

**Biosafety:** The growth of Synechocystis in media with varying Zn2+ concentrations, 0 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, was checked, and from the growth curves, it was decided to use 7 μM Zn2+ for KS induction. We also screened level 1 eYFP constructs from the previous week by cPCR. All screened colonies gave bands of expected size, so overnight cultures were set up. Plasmids were subsequently purified from these cultures using a QIAprep Spin Miniprep Kit (Qiagen) and digested using BbsI and NotI enzymes to confirm correct insertion. Digestion and gels showed that the level 1 eYFP constructs contained correct insertions of the expected size. We immediately began the level T assembly of eYFP constructs using BbsI enzyme, transformed TOP10 cells with these assemblies (same protocol as before), and plated transformed cells on LB plates containing IPTG, Xgal, and Spectinomycin for screening.

**Directed Evolution:** We repeated our extraction of ggpS and ggpP, but ran into issues during the PCR stage as the thermocycler failed. We repeated the experiment after troubleshooting with the PCR machines and purified a high enough concentration to move forward. We performed level 0 transformation and plated TOP10 cultures with ggpS and ggpP cultures. Only ggpS returned white colonies; we performed colony PCR with those cultures and made stock of these cultures.

**Carbon Sink:** We ran colony PCR on the successfully transformed level 0 PHA colonies. Gel run showed incorrect band sizes. Decided to drop the sub-project.

**Hardware:** 6803 Growth curve and finalized the first iteration of the 3D print design.

---

### Week 14 (31st July to 6th August)

**Conductivity:** In response to the failed AB level 1 plasmids, we used miniprep for 5 samples, and checked all 5 using BbsI and NotI digestions. After choosing the AB digestion with the best band sizes, we did One-Taq PCR to check, yielding correct bands in subsequent gel runs. We performed another domestication of CAB and CA in two separate water baths instead of a thermocycler. Unfortunately, this also failed, verified via digestions. Additionally, we performed digestions of domesticated MtrC plasmids, but this failed, with gel plates showing excision of our gene somewhere in the process (most likely an anomaly). Lastly, we obtained ferricyanide (courtesy of Jamie Marland) followed by initial experiments.

**Artificial Pollen:** We continued with troubleshooting for his-lys genes level 0 colony PCR. We increased the insert-to-vector ratio twice, once to 6:1, another time to 10:1. There were still no bands.

**Biosafety:** Level 1 KS constructs were screened by cPCR and restriction digestion. Results showed correct insertions of the expected size. Level T KS constructs were assembled and transformed into TOP10 cells, which were plated on LB agar plates containing IPTG, Xgal, and Spectinomycin for screening.

**Directed Evolution:** We repeated ggpP level 0 assembly; however, it remained unsuccessful. Frozen ggpS samples were put into PCR and run on gels to confirm correct plasmid size. 5 of the 8 white colonies produced the correct bands.

---

### Week 15 (7th August to 13th August)

**INTERLAB:** Calibration step for Interlab Study using fluorescent standards and Plate Reader. The IGEM interlab parts failed to grow, and hence interlab was scrapped.

**Conductivity:** This week we began Ferrocyanide testing on wild type Synechocystis sp. PCC 6803. Moreover, We attempted to move from level 0 assembly of Mtr A, B, and CymA to Level 1. The successful level 1 plasmids were put into colonies and overnight cultures. We ran digestions to confirm, which returned smeared but correct bands. We also attempted to move from level 1 AB plasmids to level T, but this failed.

**Artificial Pollen:** We troubleshooted lysC colony PCR for Level 0 transformation. The cPCR screening was unsuccessful. Nevertheless, the positive colonies for hisG and dapA were cultured and subsequently minipreped.

**Biosafety:** We screened Level T eYFP and KS constructs by cPCR, but it failed due to the wrong primers ordered previously. We ordered new level T primers for cPCR.

**Directed Evolution:** We repeated with level 0 transformations of stpA two more times, once with 2x level 0 vector concentration, and then again with 5x level 0 vector concentration. Both transformations were unsuccessful.

---

### Week 16 (14th August to 20th August):

**Conductivity:** We continued the domestication of MtrCAB and MtrCA, and performed digestions for level 1 A, B, and CymA plasmids, but the results were inconclusive due to abnormal band sizes.

**Artificial Pollen:** Level 0 restriction digestions were performed to verify assembly for hisG and dapA. The band sizes followed the expected sizes from the digestion in silico, validating the cloning from Level 0 and the constructs could then be progressed to Level 1. We performed Level 1 assembly and transformation of hisG and dapA and then did colony PCR on white colonies. Following, the positive colonies were cultured and miniprepped.

**Biosafety:** The newly ordered level T cPCR primers were used to screen level T eYFP and KS constructs. For each construct, there were colonies that gave a correct size amplicon. Cultures from these colonies were set up, plasmids were purified, and screened by restriction digestion for correct insertion. After digestion, while the correct number of bands was observed, they were all slightly larger than expected. Due to time constraints, we were only able to note this down and continue with protocols. Larger cultures of level T eYFP and KS constructs were set up to get enough plasmid DNA for Synechocystis transformations. We performed the transformation protocol received from ABOA-Turku’s iGEM team with some modifications. Synechocystis transformations failed - there was no green bacterial film on the plates after a week, the plates started drying out and had to be disposed of.

**Directed Evolution:** We began growing native PCC 6803 cultures at salt concentrations (0.25%-13% by volume) for a standard curve of our salinity project. Additionally, we miniprepped terminator sequences (pC0.082) for level 1 ggpS assembly. Promoter (ptrc10) miniprep was unsuccessful, as our cultures had been incubated in the incorrect antibiotic during the overnight culture. We attempted level 1 reactions anyway, using a lower ptrc10 concentration. We incubated more ptrc10 colonies in overnight culture, this time with the correct antibiotics.

**Riboflavin:** Lit review & Primer design for Riboflavin transporter & ordered cassette - failed to synthesize.

---

### Week 17 (21st August to 27th August):

**Conductivity:** Level T reactions were performed to move level 1 A, B, and AB into level T. These were then transformed, and white colonies were picked for colony PCR; however, these indicated unsuccessful assembly and transformation. We continued to domesticate MtrCAB and MtrCA.

**Artificial Pollen:** We digested the Level 1 miniprepped plasmid DNA using BsaI, BbsI, BpiI, and NotI. Failed to get bands. Reperformed digestion of Level 1 miniprep.

**Biosafety:** Sent plasmid samples to the Edinburgh Genome Foundry (EGF) for Oxford Nanopore sequencing and analysis.

**Directed Evolution:** Our low ptrc10 concentration reaction showed poor growth, with a single colony showing some growth. PCR showed no level 1 constructs present. The new ptrc10 colonies were ready to be used, so we attempted again using a higher concentration of ptrc10. The level 1 reaction was still unsuccessful as indicated by colony PCR and gel runs.

---

### Week 18 (28th August to 3rd September):

**Biosafety:** Since Synechocystis transformations failed, we decided to try and characterize the NucA-NuiA kill switch in E. coli. For this purpose, we ordered a G-block encoding the whole Kill Switch cassette with the same NucA and NuiA sequences but with Synechocystis promoters substituted with corresponding E. coli promoters, zinc-inducible PzntA promoter for antitoxin and constitutive Anderson promoter (BBa\_J23114) for toxin. The G-block also contained the required overhangs to allow direct assembly into a LvT acceptor vector. Level T assembly of E. coli KS was performed using a standard protocol, TOP10 cells were transformed and plated onto LB agar plates containing IPTG, Xgal, and Spectinomycin with or without added 400 µM ZnSO4. Transformations worked - plating of non-resuspended cells transformed with a KS gave 10 white colonies on each plate, with and without zinc. However, when cells were resuspended and plated again, the plate without added zinc had no colonies at all, while the plate with zinc had 268 white colonies and 35 blue ones (as expected since antitoxin under a zinc-inducible promoter should not be expressed, and the toxin should kill the cells).

**Artificial Pollen:** Performed Level T reactions for hisG and dapA to have cell strains expressing both genes. Following, we did plasmid miniprep and digestion using Bci, Ava, bbs, NotC.

**Directed Evolution:** In response to our failed level 1 ggpS reactions, we miniprepped new level 0 constructs. We performed transformations for PCR using the new level 0 plasmids. This was successful, confirmed by PCR and gel runs. Digested and undigested samples showed correct band sizes. We ligated ggpP inserts from TWIST directly into pSEVA level T acceptor vectors and transformed them into TOP10 cells. This was successful, so we ran colony PCR and froze samples over the weekend.

---

### Week 19 (4th September to 10th September):

**Conductivity:** Decided to switch from Cyano.

**Artificial Pollen:** Ninhydrin Test, first attempt using LacZ Level 1 plasmid control, HisG, and dapA level 1, and amino acid 2% stock solution. A darker purple color corresponds to higher levels of amino acids. Used a water bath at 80°C. Comparison of level 1 plasmid control vector to dapA. Level 1 insert which had a much darker color, suggesting a qualitative increase in amino acid production. Quantitative comparison was obtained by OD570 of samples using a spectrophotometer with the ninhydrin and sodium acetate.

**Directed Evolution:** We attempted error-prone PCR (epPCR) of ggpS using level 1 constructs and newly designed primers. We made multiple attempts with rounds of troubleshooting, but all were unsuccessful.

---

### Week 20 (11th September to 17th September):

**Conductivity:** We successfully domesticated CAB and CA plasmids this week. Brainstorming on E. coli transformation and characterization.

**Artificial Pollen:** Repeated the ninhydrin test with initial OD600 of cultures and standardization of population densities. Also, the concentration of ninhydrin in the buffer solution was reduced by 10 fold to allow for more sensitivity in recording results. Used a hot plate for more accurate temperature reading and it was set to 95°C. Also did an additional set with a 3 fold dilution of cultures & prepped samples to be sent for M/Z spec.

**Directed Evolution:** The salinity experiments continued with troubleshooting for ggpS epPCR reaction with no success.

---

### Week 21 (18th September to 24th September):

**Conductivity:** Revival of Edinburgh's 2012 iGEM ccmA-H genes in E. coli.

**Artificial Pollen:** Grew overnight cultures of level 1 plasmid control, hisG, and dapA level 1 strains. Pelleted and froze samples in stationary phase with similar OD600s. Handed samples off to Waddington for mass spec analysis. Set up some petri dishes with cyanobacteria cultures to dry out, at room temperature and 37°C, and on metal.

**Biosafety:** We ran cPCR of level T E. coli KS constructs. Positive control failed because no template DNA was added, but three colonies gave the expected amplicon size, so we decided to proceed with one of these.

**Directed Evolution:** We attempted PCR of ggpS using q5 polymerase, and we performed level 0 and 1 assembly again using new primers with a slightly different annealing region and overhangs. Results showed that the primers worked with all templates but the level 1, indicating there was something wrong with the pICH47732 backbone.

---

### Week 22 (25th September to 1st October):

**Conductivity:** Co-transformation of ccm plasmid with lv1 A, Lv1 B, and Lv1 AB into E. coli. Growth was observed in LB medium with Kanamycin and ampicillin resistance, suggesting co-transformation.

**Biosafety:** We set up two overnight cultures, one from one of the colonies that gave the correct cPCR band size for level T E. coli KS construct, and the other one from a blue colony containing the vector (WT+vector). We used these initial cultures to make new cultures in triplicates with or without added 400 µM Zn2+ for both KS and WT+vector control. For KS characterization, we grew these cultures in a shaking incubator at 37°C, 160 rpm, and took OD600 measurements at 1-hour intervals to produce growth curves. For checking the viability of cells containing the KS in conditions with and without added zinc compared to WT+vector control, we performed a colony forming unit (CFU) counting assay. KS and WT+vector cultures (in triplicates) were grown in media with or without zinc were sampled every 2 hours (for 6 hours total), diluted, and plated onto LB + Spec + 400 µM Zn2+ and LB + Spec (without zinc) plates, respectively. The next day, colonies on each plate were counted, and CFU/mL values were calculated.

**Directed Evolution:** Error-prone PCR was performed using the new primers with overhangs compatible with the JUMP PJ23100 Promoter and L3SAP51 terminator to transfer from the cyanogate system to the JUMP system to circumvent potential issues with the cyanogate parts. This error-prone PCR was successful. The parts were then digested and gel-purified. However, the concentration of the purified parts was low.

---

### Week 23 (2nd to 9th October):

**Conductivity:** Miniprep of the overnight E. coli cultures with mtr & ccm co-transformations. Restriction digestion analysis of plasmids for confirmation of plasmids.

**Directed Evolution:** Error-prone PCR was performed again successfully, followed by digestion and PCR cleanup. This digested error-prone PCR library was then ligated in an assembly reaction into the JUMP pJUMP29-1A acceptor vector with PJ23100 Promoter, L3SAP51 terminator, and pET Ribosome Binding Site. This was then transformed into TOP10 cells, and colonies that fluoresced green under UV light were picked for colony PCR, which indicated successful transformation.

TOP10 cells that had been transformed with stpA in level T were then made chemically competent for heat shock transformation using the CaCl2 method. These competent cells and the JUMP assembly reaction product were handed to the Edinburgh Genome Foundry to perform a high throughput assay and sequencing for analysis of beneficial mutations.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Throughout our project we wrote went through iterations of approaches to the various issues we'd face in practice{% endblock %}
{% block page\_content %}

## Context

Throughout the project, the engineering design cycle was utilized in the design of the solar panel.

## Biophotovoltaic Cell Alternatives

**1. A single-compartment cell with culture fixed as a biofilm at the anode**

**2. A dual-compartment cell with free-flowing (planktonic) culture at the anode compartment.**

## Biofilm-based System

## Cycle 1: Biophotovoltaic Circuit utilizing Synechococcus sp. PCC 11901 in a biofilm

**Design:** First attempt at a one-chamber system, utilizing biofilm formation on the anode.

**Build:** Despite conducting extensive research on Synechocystis sp. PCC 6803, the model organism most frequently studied in cyanobacterial bioengineering and BPV designs, we initially had only Synechococcus 11901 (WT, eYFP strains) at our disposal.

Since this strain was recently discovered, there is limited data available regarding its characteristics, except for its rapid growth.

Our primary focus for this design cycle was to confirm and evaluate the formation of biofilms on the selected materials. Additionally, we aimed to construct an electrolytic cell, drawing inspiration from Bombelli et al, 2022 (DOI: 10.1039/D2EE00233G).

To create conductive materials with a high surface area suitable for biofilm colonization, we utilized stainless steel plates, stainless steel plates coated with activated charcoal and PVA as a conductive binding agent, as well as microscope glass slides with the same activated charcoal and PVA paste. (Inspired from Imologie M Simeon et al, 2022 DOI: 10.1016/j.ceja.2022.100246.)

Next, we concentrated the eYFP cultures through centrifugation at 3000 rpm for 5 minutes and applied them to the surfaces, allowing them to grow.

![](https://static.igem.wiki/teams/4849/wiki/tompic1.png)
**Figure 1**: photo of attempt at Synechococcus sp. PCC 11901 eYFP expressing spotted on various surfaces to study biofilm formation capabilities.

For testing whether the formed biofilms would yield measurable results, we designed a replication method based on the paper mentioned above.

![](https://static.igem.wiki/teams/4849/wiki/tompic2.png)
**Figure 2**: Dimensions of BPV-chamber developed by Bombelli et al, 2022 (DOI: 10.1039/D2EE00233G) , drawn to inspire first printed model seen to the left.

**Test:** After a 10-day incubation period, the plates were delicately rinsed to eliminate loosely adhered cells. Subsequently, the surfaces were exposed to UV light.

Furthermore, before rinsing, we collected samples from the surfaces using a sterile swab to examine any remaining cells under a microscope.

**Learn:** We have found no increased fluorescent for any of the surfaces.

While we observed viable dividing cells, there was no significant difference in cell count compared to samples taken from the media on which the surfaces were incubated.

![](https://static.igem.wiki/teams/4849/wiki/tompic3.png)
**Figure 3**: photo taken of Synechococcus sp. PCC 11901 eYFP expressing under a microscope.

We reached out to the McCormick lab to inquire about the absence of biofilm formation. They informed us that despite their sedimentation capability, they also hadn't observed biofilm formation in the 11901 strain.

## Cycle 2A: Biophotovoltaic Circuit utilizing Synechocystis sp. PCC 6803 in a biofilm

**Design:** In this cycle, our design approach remained consistent, utilizing a known biofilm-forming strain.

**Build:** We applied WT 6803 cultures to the surfaces and allowed them to grow, this time incorporating carbon cloth in addition to the previously mentioned surfaces. As we used the WT strain, measuring fluorescence was no longer an option. Instead, we relied on visual observation and microscopic inspection following a gentle rinse. In parallel, we used a glass slide as a control since the literature suggests that its smooth surface is not conducive to 6803 biofilm formation.

**Test:** Because we used the WT strain, we couldn't measure emitted fluorescence. Instead, we inspected samples taken from the surfaces after a gentle rinse following a 15-day incubation period.

**Learn:** Our observations revealed few cells and a lack of division, indicating poor growth. Once again, the cell count was not significantly higher compared to the control. We also attempted to measure any current output from the carbon cloth, but this yielded no data beyond noise.

Due to the extended growth time of 6803, particularly in biofilm formation, we decided to shift our focus toward planktonic cells.

If biofilm-based systems were to be employed in a future design cycle, the usage of curli to induce/improve biofilm formation would be a relevant approach in increasing Synechocystis sp. PCC 6803 biofilm strength and amount, while a Spy-Tag system could be implemented in allowing biofilm formation within a conductive hydrogel.

## Planktonic-based System

---

## Cycle 2B: Two-Chamber System with Planktonic-Based Synechocystis sp. PCC 6803 Anodic Chamber

**Design:** The transition to a biophotovoltaic panel comprising a media-filled chamber presented new challenges, such as elevated internal resistance and the necessity for a redox complex to transport charge from cells dispersed in the media to the electrode. In this engineering cycle, although we haven't yet developed a bioengineered cyanobacterial strain expressing the electron pump mtrCAB, our goal is to generate current readings from the metabolic activity of cyanobacteria. To achieve this, we've considered new panel designs, drawing inspiration from the research conducted by the McCormick lab in Edinburgh and publications by Paolo Bombelli at Cambridge University.

**Build:**

Experimentation with a two-chamber setup connected by a salt bridge: the anode contains various cyanobacteria cultures at different optical densities (ODs), while the cathode contains different solutions. The new design necessitated a revised prototype with a chamber housing the anode and a separate cathode that serves to reoxidize and complete the electric circuit. To connect the two chambers, we initially intended to use a proton-exchange membrane for ion movement, but due to cost considerations, we opted to continue using a salt bridge filled with 1.5% agar saturated with Potassium Chloride to facilitate ion passage.

![](https://static.igem.wiki/teams/4849/wiki/tompic4.png)
**Figure 4**: Labelled design of 3D model purposed to serve as a testing apparatus for planktonic-based Synechocystis sp. PCC 6803 BPV system.

## Test & Learn:

We acquired a sensitive multimeter capable of detecting currents in the microamp range. Additionally, we established a Synechocystis sp. PCC 6803 culture in a CO2-limited environment using 1M KOH as a carbon dioxide sink, aiming to enhance cyanobacterial conductivity. This was inspired by a publication describing the induced expression of nanowires under starvation conditions, which are filamentous, pilli-like shaped conductive wires known to increase electron flow (Thirumurthy Miyuki A. et al, 2020 DOI: 10.3389/fmicb.2020.01344).

## Test:

Conductivity, power output, and resistance measurement: Testing involved inserting a carbon-cloth electrode into two petri dishes, one serving as the anode and the other as the cathode. The anodic dish contained various Synechocystis sp. PCC 6803 growth cultures at different growth phases, as well as the CO2-limited culture and a concentrated culture. The cathode chamber consisted of 2M potassium chloride salt, and a salt bridge connected the two dishes.

## Learn:

Testing revealed that all samples exhibited a conductivity value of 1 microamp when compared to the control (fresh growth media in the anodic dish). The recorded voltage was initially 80 millivolts, but it was later discovered that this was due to a potential difference between the two chambers.

Despite the outcome being unfavorable, one significant piece of data emerged: the circuit displayed a notable high resistance, approximately 20 kilo-ohms.

Recognizing our limitations in the field of bioelectronics, we sought advice from Prof. Jamie Marleen (Link to human practices), who stressed the necessity of an electron mediator. This led to the introduction of the final sub-project within our iGEM narrative - the FAD transporter. It enables the extracellular export of the endogenously expressed redox complex flavin adenine dinucleotide, allowing it to serve as an electron mediator.

In order to address the high resistance arising from the inadequately conductive growth medium of the freshwater cyanobacterial Synechocystis 6803, our team embarked on a new project branch with a focus on enhancing salinity tolerance through the engineering of osmolite expression in our cells. This effort aims to reduce resistance and maximize the theoretical voltage achievable from the circuit.

It is also worth noting that the lack of current output readings beyond the noise-detection limit may be attributed to the necessity for the growth culture to be in the precise and optimal exponential growth phase.

## Cycle 3: Bioengineered Synechocystis sp. PCC 6803 in an Adapted Planktonic-Based Biophotovoltaic Cell System

As we approach the conclusion of the iGEM timeline, all our hardware design and bioengineering efforts will converge to produce the final biophotovoltaic product. In this cycle, we introduced several bioengineering aspects:

* Expression of the mtrCAB cassette to facilitate electron transport from the cytoplasm extracellularly.
* Expression of ggps and stpa to enhance salt tolerance in the growth media, increasing it from the native 0.5% up to 6% and thereby decreasing internal resistance.
* Expression of the FAD transporter pump to export naturally produced FAD (redox complex) to the extracellular medium and carry the charge from reduction at the proximate site of the mtrCAB pump to the oxidation at the electrode.
* Implementation of a NucA nuclease - NuiA kill switch as a safety measure to prevent the escape of GMOs from the system once implemented.

## Design:

The requirements for the design aspect remained largely the same, with the addition of incorporating the bioengineering project and the desire to merge both chambers into a compact, singular, and modular product.

In preparation for the event of failure in generating current readings, we also decided to employ the ferrocyanide reduction assay. This assay will serve as a means to measure the reduction potential resulting from electrons traveling outside of the cell.

## Build:

The merger of the anodic and cathodic chambers aims to make the solar cell mobile and compact. This integration also allows for a significant reduction in resistance introduced by the salt bridge. This was achieved through the introduction of a proton-permeable membrane, enhancing the surface area between the two chambers while keeping internal resistance negligible.

Modifications were made to the cathode to further decrease resistance and enhance the device's compactness. An open-air cathode was utilized, with exposure to the open air serving as an alternative for reoxidation and circuit completion.

![](https://static.igem.wiki/teams/4849/wiki/tompic5.png)
**Figure 5**: (Upper) exploded & labelled view of the final 3d model designed for a planktonic, bioengineered Synechocystis sp. PCC 6803-based system. (Lower) view of the anodic chamber destined for the bacterial culture and the inter-panel space destined to contain bacteria-neutralizing detergent in the event of breakage. Credit to Björn Xu for helping to create the exploded view model, as well as providing insight into the physical engineering of the final design prototype.

**Test & Learn:**

Unfortunately, as the project concluded, we were unable to test the hardware with the bioengineered components.

{% 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 %}

# Protocols

---

## Preparation of 0.8% Agarose Gel

1. Mix 0.8 g of agarose and 100 ml of 1x TAE;
2. Heat up for 2 min;
3. Leave to cool down;
4. Add 5 µl of Ethidium Bromide (10mg/ml);
5. Put on gel caster;
6. Let it cool down and gelify;
7. If not to use right away, store in fridge.

## Running DNA on Gel

1. Put gel on running apparatus;
2. Load 10 µl of ladders
3. Load samples ( x amount sample + 6x loading buffer + H2O)
4. Run 90 mV for 45 min.

## Loading buffer

* 333 µl of 60% glycerol
* 10µl of 0.5M EDTA
* 20-100µl 6xDye
* Complete till 1ml with H2O

## Competent cell preparation

1. 1 ml of an overnight E. coli culture was transferred to 100 ml of LB media and incubated at 37 °C and 200 rpm until OD = 0.4 - 0.6.
2. The culture was poured into 50 ml falcon tubes, cooled on ice for 30 minutes and centrifuged at 4 °C and 4000 rpm for 5 minutes.
3. The media was discarded, and the pellet was resuspended in 25 ml 0.1 M of ice cold MgCl2.
4. The cells were cooled on ice for 30 minutes and spun down at 4 °C 4000 rpm for 5 minutes.
5. The supernatant was discarded, the pellet was resuspended in 25 ml 0.1 M of ice cold CaCl2.
6. The cells were cooled on ice for 30 minutes and spun down at 4 °C at 4000 rpm for 5 minutes.
7. The supernatant was discarded, and the pellet was resuspended in 1.25 ml ice cold CaCl2/glycerol solution (1.7 mL 0.1 M CaCl2, 0.3 ml 100 % glycerol).
8. The suspension was divided into 100 µl aliquots that were flash frozen in liquid nitrogen and stored at -80 °C.

## Minipreps

1. An overnight culture was grown and centrifuged at 4000 rpm for 5 minutes.
2. The media was removed, and the pellet resuspended in 250 µl P1 buffer.
3. 250 µl of P2 buffer were added, and the mixture was mixed well.
4. 350 µl of N3 buffer were added, and the mixture was mixed well.
5. The tube was centrifuged at 13000 rpm for 10 minutes, 800 µl of the supernatant were added to the QIAprep spin column.
6. The column was centrifuged at 13000 rpm for 1 minute.
7. 0.75 ml PE buffer were added to the column.
8. The tube was centrifuged at 13000 rpm for 1 minute.
9. The column was transferred to a new Eppendorf tube, 50 µl of EB buffer were added to the column.
10. DNA was eluted after centrifuging 13000 rpm for 1 minute.

## PCR Cleanup (Qiagen)

1. 6X PB buffer was added to the PCR mixture and moved to the QIAquick spin column.
2. The column was centrifuged at 13000 rpm for 1 minute, the flow through was discarded.
3. 0.75 ml PE buffer were added to the column.
4. The column was centrifuged again at 13000 rpm for 1 minute, the flow through was discarded.
5. The column was centrifuged at 13000 rpm for 1 minute to remove any residual buffer.
6. The column was transferred to a new Eppendorf tube and 30 µl of EB buffer was added.
7. Purified DNA was eluted by centrifuging the column at 13000 rpm for 1 minute.

## PCR Cleanup (Monarch, 5 μg)

All centrifugation steps should be carried out at 16,000 x g (~13K RPM in a typical microcentrifuge).

1. Dilute sample with DNA Cleanup Binding Buffer (ensure that isopropanol has been added, as indicated on the bottle label)\* according to the table below. Mix well by pipetting up and down or flicking the tube. Do not vortex. A starting sample volume of 20–100 μl is recommended. For smaller samples, TE can be used to adjust the volume. For diluted samples larger than 800 μl, load a portion of the sample, proceed with Step 2, and then repeat as necessary.

   \*Beginning in April 2021, the DNA Cleanup Binding Buffer will be changed to a concentrated format which requires the addition of isopropanol by the user. Please refer to the instructions inside of the product that you receive.

   | SAMPLE TYPE | RATIO OF BINDING BUFFER: SAMPLE | EXAMPLE |
   | --- | --- | --- |
   | dsDNA > 2 kb (plasmids, gDNA) | 2:1 | 200 μl:100 μl |
   | dsDNA < 2 kb (some amplicons, fragments) | 5:1 | 500 μl:100 μl |
   | ssDNA > 200 nt\*\* | 7:1 | 700 μl:100 μl |

   \*\* Please note that recovery of ssDNA < 200 nts can be increased by using the Oligonucleotide Cleanup Protocol, but doing so will shift the cutoff size for DNA binding to 18 nt (versus 50 nt).
2. Insert column into collection tube and load sample onto column and close the cap. Spin for 1 minute, then discard flow-through. To save time, spin for 30 seconds, instead of 1 minute.
3. Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional.
4. Repeat wash (Step 3).
5. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute to ensure traces of salt and ethanol are not carried over to the next step.
6. Add ≥ 6 μl of DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

   Note: Typical elution volumes are 6–20 μl. Nuclease-free water (pH 7–8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated. For larger size DNA (≥ 10 kb), heating the elution buffer to 50°C prior to use can improve yield. Care should be used to ensure the elution buffer is delivered onto the matrix and not the wall of the column to maximize elution efficiency. To save time, spin for 30 seconds, instead of 1 minute.

## Transformations

1. 10 µl of plasmid DNA or reaction mixture were added to 100 µl of competent E. coli cells and left on ice for 30 minutes
2. The cells were heat shocked at 42 °C for 30 seconds, then returned to ice for 30 seconds.
3. 1 ml of SOC media was added.
4. The cells were incubated at 37 °C and 200 rpm for 45 minutes.
5. The tube was spun down at 13000 rpm for 1 minute, and the media was removed.
6. Bacterial pellet was resuspended in 100 µl of SOC, then plated.

## High Efficiency Transformation Protocol (Genomic DNA Production)

This protocol was performed to produce genomic DNA from which genes could be extracted.

Use this procedure to transform MG1655 chemically competent cells. We recommend verifying the transformation efficiency of the cells using the pUC19 control DNA supplied with the kit. Do not use these cells for electroporation.

1. Remove competent cells from the -80 °C freezer and thaw completely on wet ice (10-15 minutes).
2. Aliquot 1-5 µl (1 pg-100 ng) of DNA to the chilled microcentrifuge tubes on ice.
3. When the cells are thawed, add 50 μl of cells to each DNA tube on ice and mix gently by tapping 4-5 times. For the pUC19 control, add 1 µl of (10 pg/µl) DNA to a chilled microcentrifuge tube, prior to adding 50 µl of cells. Mix well by tapping. Do not pipette up and down or vortex to mix, as this can harm cells and decrease transformation efficiency.
4. Incubate the cells with DNA on ice for 30 minutes.
5. After the 30-minute ice incubation, heat shock the cells at 42 °C for 45 seconds.
6. Transfer the tubes to ice for 2 minutes.
7. Add 950 µl of Recovery Medium or any other medium of choice to each tube.
8. Incubate tubes at 37 °C for 1 hour at 210 rpm.
9. Spread 50 μl to 200 μl from each transformation on pre-warmed selection plates. We recommend plating two different volumes to ensure that at least one plate will have well-spaced colonies. For the pUC19 control, plate 50 μl on an LB plate containing 100 μg/ml ampicillin. Use a sterilized spreader or autoclaved ColiRoller™ plating beads to spread evenly.
10. Incubate the plates overnight at 37 °C.

## Restriction Digests

1. For a typical 20 µl reaction using Restriction enzyme, the reaction was set up as follows:

| Reagent | Volume or Mass Added |
| --- | --- |
| DNA | 1 µg |
| 10X reaction buffer | 2 µl |
| Restriction Enzyme | 1 µl |
| H2O | Up to 20 µl |

2. The mixture was incubated at 37 °C for 1 hour.
3. The completed reaction was run on an agarose gel to visualize the products.

## PCR (Extraction, Domestication and Troubleshooting)

### For Q5® High-Fidelity DNA Polymerase (M0491)

Annealing temperatures were calculated on the NEB Tm calculator ([NEBTm Calculator](http://tmcalculator.neb.com/#!/#!/))

**Reaction Setup:**

| Component | 25 µl Reaction | 50 µl Reaction | Final Concentration |
| --- | --- | --- | --- |
| 5X Q5 Reaction Buffer | 5 µl | 10 µl | 1X |
| 10 mM dNTPs | 0.5 µl | 1 µl | 200 µM |
| 10 µM Forward Primer | 1.25 µl | 2.5 µl | 0.5 µM |
| 10 µM Reverse Primer | 1.25 µl | 2.5 µl | 0.5 µM |
| Template DNA | variable | variable | < 1,000 ng |
| Q5 High-Fidelity DNA Polymerase | 0.25 µl | 0.5 µl | 0.02 U/µl |
| 5X Q5 High GC Enhancer (optional) | (5 µl) | (10 µl) | (1X) |
| Nuclease-Free Water | to 25 µl | to 50 µl |  |

**Thermocycling Conditions for a Routine PCR:**

| Step | Temp | Time |
| --- | --- | --- |
| Initial Denaturation | 98°C | 30 seconds |
| 25–35 Cycles | 98°C 50–72°C 72°C | 5–10 seconds 10–30 seconds 20–30 seconds/kb |
| Final Extension | 72°C | 2 minutes |
| Hold | 4–10°C |  |

### OneTaq® DNA Polymerase (M0480)

Annealing temperatures were calculated on the NEB Tm calculator ([NEBTm Calculator](http://tmcalculator.neb.com/#!/#!/))

**Reaction Setup:**

| Component | 25 μl reaction | 50 μl reaction | Final Concentration |
| --- | --- | --- | --- |
| 5X OneTaq Standard Reaction Buffer\* | 5 µl | 10 μl | 1X |
| 10 mM dNTPs (#N0447) | 0.5 µl | 1 μl | 200 µM |
| 10 µM Forward Primer | 0.5 µl | 1 μl | 0.2 µM |
| 10 µM Reverse Primer | 0.5 µl | 1 μl | 0.2 µM |
| OneTaq DNA Polymerase | 0.125 µl | 0.25 µl | 1.25 units/50 µl |
| Template DNA | variable | variable | < 1,000 ng |
| Nuclease-free water | to 25 µl | to 50 µl |  |

**Thermocycling Conditions for a Routine PCR:**

| Step | Temp | Time |
| --- | --- | --- |
| Initial Denaturation | 94°C | 30 seconds |
| 30 Cycles | 94°C 45-68°C 68°C | 15-30 seconds 15-60 seconds 1 minute per kb |
| Final Extension | 68°C | 5 minutes |
| Hold | 4-10°C |  |

### SuperFi DNA Polymerase (M0480)

Invitrogen info sheet found [here](https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0018859_Platinum_SuperFi_II_DNA_Pol_UG.pdf).

**Reaction Setup:**

| Component | Final concentration | 20-µL Reaction | 50-µL Reaction |
| --- | --- | --- | --- |
| 5X SuperFit II Buffer | 1X | 4 µL | 10 µL |
| Forward primer | 0.5 µM | x µL | x µL |
| Reverse primer | 0.5 µM | x µL | x µL |
| 10 mM dNTPs | 200 µM each | 0.4 µL | 1 µL |
| Template DNA | 0.1-10 ng plasmid DNA (5-100 ng genomic DNA) | x µL | x µL |
| PlatinumTM SuperFiTY 11 DNA Polymerase | 1X | 0.4 µL | 1 µL |
| Water, nuclease-free |  | to 20 µL | to 50 µL |

**Thermocycling Conditions for a Routine PCR:**

| Cycle step | Temperature | Time | Cycles |
| --- | --- | --- | --- |
| Initial denaturation | 98°C | 30 seconds | 1 |
| Denaturation Annealing Extension | 98°C 60°C 72°C | 5-10 seconds 10 seconds 15-30 seconds per 1 kb | 25-35 || Final extension | 72°C | 5 minutes | 1 |
| Hold | 4°C |  |  |

## Colony PCR

1. Put 50 μl sterile water into PCR tubes. One for each of your chosen colonies.
2. Put 1 ml of LB media into a sterile Eppendorf tube. One for each of your chosen colonies.
3. Using your pipette set to 40 μl, pick up the colony of interest.
4. Resuspend the colony in 50 μl of water in a PCR tube.
5. Once the colony is resuspended, take 40 μl and inoculate the 1 ml of LB media.
6. You will be left with 10 μl of resuspended colony in your PCR tube. This is your DNA for your PCR.
7. Once you have done all your samples, freeze them at -20 °C for 10 minutes.
8. During this 10 minutes, make your master mix.

### GoTaq® DNA Polymerase

Promega info sheet found [here](https://www.promega.co.uk/-/media/files/resources/protocols/product-information-sheets/g/gotaq-dna-polymerase-m300.pdf?la=en23).

**Reaction Setup:**

| Component | Final Concentration | Volume |
| --- | --- | --- |
| 5x Green GoTaq reaction buffer | 1X (1.5 mM MgCl2)2 | 5 µL |
| dNTP mix (10 mM) | 0.2 mM | 0.5 µL |
| Forwards primer (cPCRFor) | 1:00 PM | 2.5 µL |
| Reverse primer (cPCRRev) | 1 µL | 2.5 µL |
| GOTaq G2 DNA polymerase (Demonstrator has this) | 1.25 u | 0.125 µL |
| Template DNA |  | 5 µL\* |
| Water |  | 9.375 µL |
| **Total** | **25 µL** |  |

**Reaction Setup:**

| Step | Temperature | Time | Number of Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 95°C | 2 minutes | 1 cycle |
| Denaturation | 95°C | 0.5-1 minute |  |
| Annealing | 42-65°C\* | 0.5-1 minute | 25-35 cycles |
| Extension | 72°C | 1 minute per kb |  |
| Final Extension | 72°C | 5 minutes | 1 cycle |
| Soak | 40°C | Indefinite |  |

\* Annealing temperature should be optimized for each primer set based on the primer Tm.

## MAD Medium (1 L)

| Component | Quantity |
| --- | --- |
| NaCl | 18 g |
| MgSO4·7H2O | 5 g |
| A+ modified 100X | 10 mL |
| Tris (125 g/L, pH 8.2) | 8.3 mL |
| Modified D7 Metal 1000X | 1 mL |
| NaNO3 | 7 g |
| KH2PO4(1 M) | 0.83 mL |

Autoclave OR sterilise using 0.22 µm filter and cool down to room temperature before adding the following:

| Component | Quantity |
| --- | --- |
| FeCl3·6H2O (480 mM) | 0.5 mL |
| Vitamin B12 - Cobalamin | 1 mL |

### MAD Medium Stock Solutions

**Modified D7 Metal 1000X (1 L)**

| Component | Quantity |
| --- | --- |
| H3BO3 | 2.86 g |
| MnCl2·4H2O | 1.81 g |
| ZnSO4·7H2O | 222 mg |
| Na2MoO4·2H2O | 1.26 g |
| CuSO4·5H2O | 79 mg |
| CoCl2·6H2O | 40.3 mg |

**A+ Modified 100X (1 L)**

| Component | Quantity |
| --- | --- |
| KCl | 60 g |
| NaNO3 | 100 g |
| CaCl2·2H2O | 37 g |
| Na2EDTA | 3 g |
| KH2PO4 | 5 g |

## BG11 100X Medium (1L)

All components, with exception of buffers, must be autoclaved

**Main Components**

| Component | Quantity |
| --- | --- |
| NaNO3 | 149.6 g |
| MgSO4.7H2O | 7.49 g |
| CaCl2 | 3.60 g |
| Citric acid | 0.60 g |
| Na2EDTA | 1.12 mL of 0.25M solution, pH 8.0 |

**Trace Elements**

| Component | Quantity |
| --- | --- |
| H3BO3 | 2.860 g |
| MnCl2·4H2O | 1.810 g |
| ZnSO4·7H2O | 0.220 g |
| Na2MoO4·2H2O | 0.390 g |
| CuSO4·5H2O | 0.080 g |
| Co(NO3)2·6H2O | 0.050 g |

### Stock Solutions (g/100ml)

**Iron Stock**

| Component | Quantity |
| --- | --- |
| Ferric citrate | 0.6 |
| (ALTERNATIVELY) Ferric ammonium citrate | 1.11 |

**Phosphate Stock**

| Component | Quantity |
| --- | --- |
| K2HPO4 | 3.05 |

**Na2CO3 Stock**

| Component | Quantity |
| --- | --- |
| Na2CO3 | 2.0 |

**Glucose Stock**

| Component | Quantity |
| --- | --- |
| Glucose | 9.0 g (= 0.5 M) |

store in the fridge

### Buffers (g/100ml)

**TES buffer**

| Component | Quantity |
| --- | --- |
| TES | 22.9 |

Adjust with 1 M NaOH to pH 8.2

**HEPES buffer**

| Component | Quantity |
| --- | --- |
| HEPES | 23.8 |

Adjust with 1 M NaOH to pH 7.8

## Other (g/100ml)

| Component | Quantity |
| --- | --- |
| NaHCO3 | 8.4 |

## Cyanogate Assembly Protocol

**Reaction Setup:**

| Bpil Assembly Components (Level 0, T) | Bsal Assembly Components (Level 1) |
| --- | --- |
| 50-100 ng of acceptor vector | 50-100 ng of acceptor vector |
| For each vector/part to insert, use a 2:1 ratio of insert: acceptor vector. | For each vector/part to insert, use a 2:1 ratio of insert: acceptor vector. |
| 2 µL 10 mM ATP (Table of Materials) | 2 µL 10 mM ATP (Table of Materials) |
| 2 µL buffer G (buffer for Bpil/Bsal) | 2 µL buffer G (buffer for Bpil/Bsal) |
| 2 µL BSA (10x) (Table of Materials) | 2 µL BSA (lox) (Table of Materials) |
| 10 units Bpil (1 µL 10 U/µL Bpil, Table of Materials) | 10 units Bsal (1 µL 10 U/µL Bsal, Table of Materials) |
| Bring to 20 µL with dH2O. | Bring to 20 µL with dH2O. |
| 200 units T4 DNA ligase (1 µL 200 U/µL, Table of Materials) | 200 units T4 DNA ligase (1 µL 200 U/µL, Table of Materials) |

\*Experimentation showed that ATP is optional.

**Thermocycler Protocol:**

| Level 0, T |  | Level 1 |  |
| --- | --- | --- | --- |
| 37 °C for 10 min 16 °C for 10 min | (cycle x 5) | 37 °C for 10 min 16 °C for 10 min | (cycle x 5) |
| 37 °C for 20 min |  | 37 °C for 20 min |  |
| 65 °C for 10 min |  | 65 °C for 10 min |  |
| 16 °C (hold) |  | 16 °C (hold) |  |

## JUMP Assembly (Level 1)

1. Create a reaction mix in a PCR tube with the following components:

| Reaction Component | Amount (μl) |
| --- | --- |
| ggpS epPCR digest (20fmol/μl) | 1 |
| pJUMP29-1A acceptor vector (20fmol/μl) | 1 |
| PJ23100 Promoter in level 0 (20fmol/μl) | 1 |
| L3SAP51 terminator in level 0 (20fmol/μl) | 1 |
| pET Ribosome Binding Site in level 0 (20fmol/μl) | 1 |
| T4 DNA ligase - HF | 0.25 |
| BsaI-HF | 1 |
| T4 Ligase Buffer | 2 |
| ddH2O | Fill to 20 |

2. Set the Thermocycler to run in the following cycles:

* 1 cycle at 37°C for 15 minutes
* 25 cycles at 37°C for 3 minutes and 16°C for 3 minutes
* 1 cycle of 37°C for 15 minutes, 50°C for 5 minutes, and 80°C for 5 minutes
* 25 cycles of 37°C for 3 minutes and 16°C for 3 minutes
* 1 cycle of 37°C for 15 minutes, 50°C for 5 minutes, and 80°C for 5 minutes
* Finally, hold the reaction at 4°C

## Preparation of NaCl Lysogeny Broth and BL21 Growth Curve

1. Prepare Sterile 34% (w/v) Saline stock solution.
2. Add Saline solution in increasing amounts to LB in a falcon tube to create a medium of varying salt concentrations and then inoculate with 1ml of overnight culture BL21 using the table below:

| Concentration of NaCl in LB media (w/v) | Volume of 34% Saline Solution (mL) | Volume of LB media (mL) |
| --- | --- | --- |
| 0.5% | 0 | 29 |
| 1% | 0.441 | 28.559 |
| 1.5% | 0.882 | 28.118 |
| 2% | 1.323 | 27.677 |
| 2.5% | 1.764 | 27.236 |
| 3% | 2.205 | 26.795 |
| 3.5% | 2.646 | 26.354 |
| 4% | 3.087 | 25.913 |
| 4.5% | 3.528 | 25.472 |
| 5% | 3.969 | 25.031 |
| 5.5% | 4.380 | 24.590 |
| 6% | 4.821 | 24.149 |

3. Take an initial OD600 from each of the cultures.
4. Incubate the cultures at 37°C, 250rpm.
5. Take a 1ml sample every hour and record the OD600 of each culture. If the OD600 of a culture is significantly more than 1, dilute the sample and measure the OD600.

## Error Prone PCR

1. Create a reaction mix in a PCR tube with the following components:

| Reaction Component | Amount |
| --- | --- |
| 10x NEB Taq DNA polymerase Thermopol buffer | 5μl |
| Genomic Forward Primer | 1μl |
| Genomic Reverse Primer | 1μl |
| Taq polymerase | 0.5μl |
| ggpS Level 0 Template DNA | 50ng |
| MgCl2 | 2.95mM |
| MnCl2 | 0.1mM |
| ddH2O | Fill to 50μl |

2. Set the thermocycler to have an initial denaturation at 95°C for 1 minute, 25 cycles of 30 seconds at 95°C, 45 seconds at 53°C, and 90 seconds at 68°C, a final elongation at 68°C for 5 minutes, and hold at 4°C.
3. Purify the product with the Monarch DNA Gel Extraction kit (New England Biolabs) and store at -20°C.

## Special Protocols

### Membrane fractionation by cold osmotic shock

The membrane fractionation protocol allows to separate the contents of E. coli to the cytoplasmic,periplasmic and membrane components.
Doing this in Escherichia coli (E. coli) would allow for the identification of wanted membrane,periplasmic proteins (mtrB,mtrA respectively) in our engineered cells. :

**Materials**

* Overnight culture of E. coli
* Microcentrifuge
* Icebath
* 0.2 µm filter & filtration syringe
* Cell fractionation buffer (CFB) 1 (pH 8):
  + 0.2M Tris
  + 200 g/L sucrose
  + 0.1M EDTA
* Cell fractionation buffer (CFB)  2.1 (pH 8) :
  + 0.01M Tris
  + 0.005M MgSO4
* Cell fractionation buffer (CFB) 2.2 (pH 8) :
  + 0.01M Tris
  + 0.005M MgSO4
  + 1% Triton
  + 2% SDS
* Cell fractionation buffer (CFB) 2.3 (pH 8) :
  + 0.01M Tris
  + 0.005M MgSO4
  + 1% Triton
  + 0.2% SDS
* Cell fractionation buffer (CFB) 2.4 (pH 8) :
  + 0.01M Tris
  + 0.005M MgSO4
  + 1% Triton
  + 1% SDS

* Taken from [iGEM Bielefeld 2013.](https://2013.igem.org/Team%3ABielefeld-Germany/Labjournal/ProtocolsPrograms#Cold_osmotic_shock)

1. Centrifuge *E. coli* cell suspension for 5 min at 14,000 g (4 °C) to collect the cells
2. Discard the entire supernatant
3. Resuspend the cells in ice-cold CFB1. The resulting volume should be 1/4 of the former suspension volume
4. Incubate for 20 min on ice. Invert the suspension at regular intervals to counteract sedimentation
5. Centrifuge the cell suspension for 15 min at 14,000 g (4 °C)
6. Discard the entire supernatant
7. Resuspend the cells in ice-cold CFB2. The resulting volume should be 1/4 of the former suspension volume
8. Incubate for 10 up to 20 min on ice under regular invertion
9. Centrifuge the cell suspension for 15 min at 14,000 g (4 °C)
10. Save the supernatant, which contains the periplasmatic proteins and membrane proteins when using 2.2 and 2.3
11. If the periplasmatic protein fraction is turbid, re-centrifuge and filter it through a 0.2 µm filter

## Ferricyanide Assay Protocol

### Materials:

* Potassium hexa-cyanoferrate (III) (CAS Number: 13746-66-2)
* 20 mL cyanobacterial cultures
* 0.22 µm syringe filter
* Spectrophotometer
* Aluminium foil
* Shaking incubator
* 15 mL Synechocystis sp. pcc 6803 cultures at exponential growth phase (OD~1 at 630 nm), doublets for each experimental condition

### Protocol:

1. Cultures were removed from the incubator and placed under dark conditions for 4 hours prior to the start of the experiment.
2. Ferricyanide stock solution was made using water to 100 mM.
3. Cultures in light-absent conditions were covered in aluminium foil.
4. Ferricyanide was added to the culture, reaching a final concentration of either 1 or 5 mM.
5. 1 mL volumes were removed and passed through a 0.22 µm syringe filter. The filtrate was used to blank the spectrophotometer at 390 nm.
6. Cultures were placed in a shaking incubator.
7. At specified time intervals, cultures were removed from the incubator, and a 1 mL volume was removed and passed through a 0.22 µm syringe filter. Filtrate absorbance was measured at 390 nm and compared against the relevant blank (1 or 5 mM ferrocyanide).
8. Recorded data were used to plot a linear regression graph for analysis.

## SDS-PAGE Protocol

### Procedure:

#### 1. Setting up the SDS-PAGE Tank

* The tank for SDS-PAGE is set up with the inner chamber and half of the outer chamber filled with 1X running buffer.

#### 2. Sample Preparation

* Mix 8 μl of sample lysate with 10 μl of 2X Laemmli sample buffer and 2 μl of β-mercaptoethanol under a fume hood.

#### 3. Boiling the Sample

* Boil the sample at 98°C for 5 minutes.
* Spin down the sample.
* You can either freeze the sample at -20°C or proceed to load it onto the gel.

#### 4. Gel Loading

* Load the prepared sample onto a precast 4-15% Mini-PROTEAN TGX SDS-PAGE gel along with a protein standard.

#### 5. Electrophoresis

* Connect the power supply unit to the electrodes.
* Run the electrophoresis at 180V until the desired separation of protein bands has been achieved.

#### 6. Gel Staining

* Remove the gel from the tank.
* Carefully break the gel out of the plastic mold.
* Wash the gel with water to remove excess buffer and impurities.
* Pour off the water.
* Add enough Coomassie Blue to cover the entirety of the gel.
* Incubate the gel while shaking at an orbital shaker at approximately 35 rpm until protein bands become clearly visible.

## Heme Stain Protocol

### Materials

* TMB (N, N, N', N'-tetramethylbenzidine)
* MeOH
* 0.5 M Na Acetate, pH 5.0
* H2O2

### Method

1. Dissolve 30 mg TMB (N, N, N', N'-tetramethylbenzidine) in 15 ml MeOH. (Make sure it dissolves well before the next step.)
2. Add 35 ml of 0.5 M Na Acetate (pH 5.0).
3. Carefully place the gel in the stain.
4. Wait for 3-5 minutes.
5. Add 300 µl of H2O2 and mix.
6. Wait for 5 minutes.
7. Check for blue bands.

### Notes

* Load 50 µg of total protein per well.
* Keep the reaction in the dark at all times. TMB is light-sensitive.

## Optimal Transformation Protocol for Synechocystis sp. pcc6803

### Before Starting:

* Make sure you have enough BG11, 2x BG11, and 2x BactoAgar for the protocol.
* When making plates for plating cells after transformation, use a defined volume of BG11+Agar per plate:

+ Use 30 ml BG11-Agar media. This amount makes a thick gel which prevents gel breaking in the next step. As in the next step, we add antibiotics (here CAM and Spect in 500 μl BG11) underneath the agar.

* Set up a fresh liquid Synechocystis culture in advance for the protocol.

### Making the Preculture:

* Set up a preculture of Synechocystis in BG11 media (e.g., 25 ml BG11 in 250 ml flasks) by taking a loopful of wild-type Synechocystis pcc6803 culture grown on BG11 plates.
* Let the culture grow for 2-4 days (to reach OD750=1-5).
* Use a diluted sample (1:10; 100 μl culture + 900 μl BG11 or H2O) to measure OD750.

### Making the Main Culture:

* Make the main culture with a starting OD750 of 0.1 or 0.2 (etc.) by inoculating BG11 with the preculture.
* Let the culture reach mid-logarithmic growth phase (OD750=0.8). If high transformation efficiency is required, grow the cells (OD750=0.8) in BG11 with 2 mM EDTA for 2 days before the transformation.
* Transformation in late latent growth phase (OD750=0.3-0.7) or plateau growth phase (OD750= >1) will lower the transformation efficiency severely.

### Transformation:

* Two days after the addition of EDTA to the culture with OD750=0.8:

+ Pipet 5 ml of culture into a sterile 12 ml falcon tube.
+ Pellet the cells with 3000 g, 5 min, RT centrifugation.
+ Resuspend the cell pellet in 1 ml of fresh BG11.
+ Add DNA 15-20 μg/ml (usually 3-20 μg/ml) to each sample.
+ Incubate the cells for 5 h in light conditions on a shaker (150 rpm, 30°C, 50 μE, 1% CO2).
+ Centrifuge at 3000 g, 5 min, RT, and discard the supernatant.
+ Resuspend the pellet in the remaining liquid.
+ Plate them in BG11 (plate contains 30 ml BG11) with NO antibiotics.
+ Keep at 30°C and 50 μE until a slight green film arises (ca. 2-4 days). Usually two days.

* Selection: Addition of antibiotics

+ Take a sterile spatula and lift the agar at one side.
+ Fill 500 μl BG11 containing antibiotics (in our project CAM and Spect) underneath the agar.
+ The amount of antibiotics is calculated for the final BG11+Agar volume, here 30 ml.
+ We start with 3 μg/ml CAM (≃ 2.6 μl of 34 mg/ml CAM stock, 8.8 % of the final concentration; 34 μg/ml) and 6 μg/ml Spectinomycin (≃ 3.6 μl of 50 mg/ml CAM stock; 12 % of the final concentration; 50 μg/ml).

* Cover the plates with paper sheets for 1-2 days to reduce light intensity (usually two days!).
* Colonies should appear in 10-30 days (usually transformants appear in three weeks, but it may take longer time).

### Increasing Selection Pressure:

* Pick the colonies from the previous step (grown in BG11 plate + 3 μg/ml CAM + 6 μg/ml Spectinomycin) by loop and streak them to BG11 plates containing a higher amount of antibiotics:

+ 6 μg/ml CAM and 12 μg/ml Spectinomycin. Continue cultivation until colonies appear (1-3 weeks).

* Pick the colonies from the previous step (grown in BG11 plate + 6 μg/ml CAM + 12 μg/ml Spectinomycin) by loop and streak them to BG11 plates with the final antibiotic pressure: 34 μg/ml CAM + 50 μg/ml Spectinomycin.
* Finally, axenic cultures/transformants will be tested by culturing them on antibiotic-free LBA. Keep the plate at 37°C for a couple of days as some bacteria need longer time to grow!

### Notes:

* I did not transfer the colonies to 12 μg/ml CAM and 24 μg/ml Spectinomycin and directly streak them from 6 μg/ml CAM + 12 μg/ml Spectinomycin to the final concentrations.
* All the colonies survived in plates containing 34 μg/ml CAM + 50 μg/ml Spectinomycin.

### Ninhydrin Assay

The ninhydrin test is a widely used method to detect the presence of amino acids in a sample. It is often used to determine amino acid expression or the presence of free amino acids. Here's a protocol for performing the ninhydrin test on a live overnight culture of Escherichia coli (E. coli) to determine amino acid expression:

**Materials**

* Overnight culture of E. coli
* Ninhydrin solution (0.2% in acetone or ethanol)
* 2M Sodium acetate buffer (pH 6.0-6.5)
* Test tubes
* Water bath or heat block
* Pipettes
* Microcentrifuge (optional)
* Spectrophotometer (optional)

**Protocol**

1. Prepare the Ninhydrin Solution:
   * Prepare a fresh ninhydrin solution by dissolving 0.2 g of ninhydrin in 100 ml of acetone or ethanol. Mix well until the ninhydrin is completely dissolved.
2. Prepare Sodium Acetate Buffer:
   * Prepare a 2M sodium acetate buffer solution at the desired pH (usually around 6.0-6.5). Adjust the pH using acetic acid or sodium hydroxide as needed.
3. Harvest Cells (Optional):
   * If necessary, harvest the E. coli cells from the overnight culture by centrifugation. Wash the cells with a suitable buffer (e.g., phosphate-buffered saline) and resuspend them in a small volume of the sodium acetate buffer.
4. Reaction Setup:
   * Label test tubes for each sample or standard you will be testing.
   * To each test tube, add:
     + 1-2 ml of the sodium acetate buffer (pH 6.0-6.5)
     + 100-200 µl of the E. coli culture (or equivalent volume of harvested cells)
     + If using standards, prepare a set of standards containing known concentrations of amino acids in the same buffer.
5. Add Ninhydrin Solution:
   * Add 1-2 ml of the prepared ninhydrin solution to each test tube containing the samples and standards.
6. Heating:
   * Place the test tubes in a water bath or heat block preheated to around 80-100°C.
   * Incubate the tubes for about 10-20 minutes. The exact incubation time may vary based on the temperature and the specific amino acids being tested.
7. Color Development:
   * After incubation, remove the tubes from the water bath and allow them to cool to room temperature.
8. Measurement:
   * Measure the color development in each tube using a spectrophotometer at an appropriate wavelength (usually around 570 nm). Use the buffer with ninhydrin as a blank.
9. Data Analysis:
   * Compare the absorbance readings of the samples with the standard curve generated using known concentrations of amino acids. The intensity of color development indicates the presence and quantity of amino acids in the samples.
10. Interpretation:
    * Calculate the concentration of amino acids in the E. coli culture based on the standard curve and the absorbance readings.

## Protocol for Preparing E. coli Samples for Mass Spectrometry

### Materials and Supplies:

* OP50 media - ~1200 ml (1L solution - 5g Tryptone, 2.5g Yeast Extract)
* LB plate (1)
* Autoclaved Test Tubes (2-4)
* OD600nm Spectrometer
* Autoclaved 250ml Beckmann centrifuge bottles (6)
* Centrifuge with rotor that holds 250ml bottles
* 37°C incubator
* Roller or shaker that holds test tubes
* Shaker for 1L Erlenmeyer flasks
* Ice buckets w/ ice (2)
* French Press with refrigerated cell (cleaned before use)
* Autoclaved 1.5ml Eppies
* Sterile 15ml Falcon conical tubes
* Microcentrifuge
* Disposable 25ml pipets
* Bio-Rad RC DC Protein Assay Kit (Cat# 500-0122)
* 0.2 % RapiGest SF Powder in 50mM Ammonium Bicarbonate pH 7.8 (Waters Corporation, 5 Pack of 1 ml Vials, 186001860)
* 50mM Ammonium Bicarbonate buffer pH 7.8
* 500mM DTT
* 500mM IAA (Iodoacetamide - light-sensitive)
* 500mM HCl
* 250 ng/μl Trypsin, modified, sequencing grade in 0.01% Acetic Acid (Roche, 4 x 25 μg, Cat # 1418033)
* 100 mM CaCl2

### Sample Preparation

#### DAY 1 - Plate Growth

1. Streak a plate of TJ2 strain of OP50 E. coli located in the freezer in J256.
2. Use an LB Agar Plate found in J180C 4°C room.
3. Grow up colonies overnight at 37°C (K231).

#### DAY 2 - Culture Growth part 1

1. Inoculate 1-3 test tubes with 5 ml OP50 media with single colonies from the plate. Grow up several cultures in case some don't grow.
2. Grow overnight (8-12 hrs) at 37°C on a roller or shaker (K231 or K310 hallway).

#### DAY 3 - Culture Growth part 2

1. Remove 10 ml from 1L of OP50 media to use as a zero for spectrophotometer and add 10 ml overnight cultures to the liter of media for a 1:100 dilution.
2. Grow bacteria at 37°C on a shaker for ~5 hours or until OD600nm reading on the spectrophotometer (in J180) is 0.5.
3. Pour 200 ml of bacteria into 5 x 250 ml Beckmann centrifuge bottles and use the extra bottle as a balance with 200 ml of water.
4. Chill on ice for 5 minutes (I usually chill the centrifuge bottles ahead of time as well).
5. Spin down samples at 3000 rpm for 5 minutes at 4°C using a Beckman centrifuge in either J180 or J160.
6. Remove supernatant from pellets.
7. Add 2 ml of cold 50mM Ammonium Bicarbonate pH 7.8 to each pellet, making sure to fully resuspend the pellets in the buffer.
8. Combine all resuspended pellets into a sterile 15ml Falcon conical tube.
9. Freeze the sample at -20°C until ready to lyse the sample.

#### DAY 4 - Lysis

1. Lyse a 10 ml sample with a French Press (J432) using Klevit lab cell in K464 (see separate protocol for French Press lysis technique).
2. Spin the sample at 4000 rpm for 30 minutes at 4°C using Waterston lab centrifuge in K313 hall.
3. Remove supernatant from pelleted debris.
4. Spin the sample at 14K for 10 minutes to separate insoluble fraction from soluble.
5. Use Bio-Rad Protein Assay Kit to quantitate protein (see separate protocol). The spectrophotometer is in Fangman/Brewer lab J135.

### Sample Digestion

1. Make 0.2% RapiGest diluted in 50 mM Ammonium Bicarbonate pH 7.8 (1 mg RapiGest per 500 μl 50 mM Ammonium Bicarbonate pH 7.8).
2. Using a locking lid microcentrifuge tube, add 100 μl of 0.2% RapiGest per 100 μl protein mixture (1:1) - [final concentration of RapiGest should be 0.1% (w/v)]. If protein is in pellet form, add 25-50 μl of 0.1% RapiGest. Vortex the sample.
3. Boil the sample at 99°C for ~2 minutes, then let it cool for a couple of minutes.
4. Quick spin the sample and add DTT to a final concentration of 5 mM.
5. Incubate the sample at 60°C for 30 minutes.
6. Cool the sample to room temperature.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Here are the results from this summer's work.{% endblock %}
{% block page\_content %}

# General Results

---

## Growth curve and cell growth dynamics

---

The OD730nm values of both strains (Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 11901) were done at the same time for ten days. No lag phase is observed for either strain, which means the cultures that they were inoculated from were probably not at the right phase. By the OD730mn values that the original cultures had, they were probably at the stationary phase when it is recommended that they are at exponential growth phase for inoculation to generate a growth curve. The logarithmic growth phase is advisable since the cells actively divide and the growth rate is somewhat constant, which makes the increase in cell numbers more consistent and predictable, thus it is easier to observe growth dynamics and quantify growth patterns. Since the cell density from the original culture was too high to inoculate, the lag phase was 'skipped' and the culture started exponential phase directly (graph 1).

The cells were grown at 30ºC, under 300 µmol m/2s, the cells were grown at 180 rpm. (Note: Synechococcus sp. PCC 11901 strain doubling times and growth rate were not found in published literature since this strain is very new in cyanobacterial research. )

![](https://static.igem.wiki/teams/4849/wiki/growth-curve.png)

**Graph 1 Growth curve of Synechocystis sp. PCC 6803 in BG-11 medium**: The cells were monitored by optical density (OD) measurements at 730 nm. The y-axis represents the optical density readings which serve as an indicator of cell density. The x-axis shows the duration of the experiment in days.

| Strain | Growth Rate (OD730nm/Day) | Doubling Time (Days) |
| --- | --- | --- |
| *Synechocystis* sp. PCC 6803 | 0.243 ± 0.009 | 2.853 ± 0.112 |
| *Synechocystis* sp. PCC 19901 | 0.501 ± 0.019 | 1.381 ± 0.044 |

Table 11. Growth rate and doubling time of *Synechocystis* sp. PCC 6803 and *Synechococcus* sp. PCC 11901. Calculations done using equations: Doubling Time (DT) = (t2 - t1) / log2(Off / ODi). Growth Rate (GR) = ln(2) / Doubling Time

| Study | Growth Rate (OD730nm/Day) | Doubling Time (Days) |
| --- | --- | --- |
| Cui et al.33 | 0.186 ± 0.006 | not published |
| Eungrasamee et al.43 | 0.099 ± 0.010 | 7.4 ± 0.4 |
| Alphen et al.49 | 0.07 ± 0.005 | 8 hours |

# MTR Results

---

## Gene extraction

The MTR target genes: MtrA, MtrB, MtrC and CymA, were extracted and amplified from the genome of Shewanella Oneidensis MR-1 using OneTaq (NEB) polymerase. The PCR primers were designed to introduce CyanoGate overhangs in the extracted operons allowing their integration into Lv0 acceptor plasmid vectors. The MTR operons: MtrCAB, MtrCA and MtrAB, were extracted via PCR extraction (One-Taq polymerase) but with primers that did not introduce overhangs. A second PCR (Q5 polymerase) was then performed on the initial PCR product with overhang primers to produce MtrCAB, MtrCA and MtrAB g-blocks (gene with overhangs). All extracted genes and operons were analysed using agarose gel electrophoresis to determine the presence and size of the fragments. Appropriate negative controls were used for all gel electrophoresis runs (negative control was water).

The PCR extraction was successful, seen in figure 1 and 2, but only after troubleshooting (which involved using different enzymes, different annealing temperatures; and different extension times). The annealing temperature was determined used NEB Tm calculator. While One-Taq polymerase worked on the first try, Q5 and Platinum SuperFi II DNA Polymerase were preferred due to their very high fidelity. Finally, the bands from the gel were extracted, cleaned and taken forward into level 0 Cyanogate acceptor plasmid vector. All the extracted genes have been uploaded onto the IGEM registry.

**Working Conditions for Q5 polymrase:**

| Gene/Operon | Annealing Temperature | Extension Time |
| --- | --- | --- |
| CAB | 68°C and 64°C | 3 minutes |
| CA | 71°C, 68°C, and 64°C | 1.5 minutes |
| AB | 71°C, 68°C, and 64°C | 1.5 minutes |

\*64°C not preferable due to noise

**Working Conditions for One-Taq polymerase:**

| Gene/Operon | Annealing Temperature | Extension Time |
| --- | --- | --- |
| CAB | 50°C | 6 minutes |
| CA | 50°C | 3 minutes |
| AB | 50°C | 3 minutes |
| A | 50°C | 5 minutes |
| B | 50°C | 5 minutes |
| C | 50°C | 5 minutes |
| CymA | 55°C | 5 minutes |

![](https://static.igem.wiki/teams/4849/wiki/mtr1.png)

**Figure 1**: PCR with OneTaq of MtrA, MtrB, MtrC and CymA. A. 1 Kb DNA ladder diagram (New England Biolabs). The ladder is not to scale and it is provided for reference purposes only.

![](https://static.igem.wiki/teams/4849/wiki/mtr2.png)

**Figure 2**: PCR with Q5 polymerase to introduce overhangs in extracted operons; MtrCAB, MtrAB, MtrCA.

| MTR Genes/Operons | Link to Parts Repository |
| --- | --- |
| MtrA | [http://parts.igem.org/Part:BBa\_K4849016](http://parts.igem.org/Part%3ABBa_K4849016) |
| MtrB | [http://parts.igem.org/Part:BBa\_K4849014](http://parts.igem.org/Part%3ABBa_K4849014) |
| MtrC | [http://parts.igem.org/Part:BBa\_K4849025](http://parts.igem.org/Part%3ABBa_K4849025) |
| MtrCAB | [http://parts.igem.org/Part:BBa\_K4849027](http://parts.igem.org/Part%3ABBa_K4849027) |
| MtrCA | [http://parts.igem.org/Part:BBa\_K4849028](http://parts.igem.org/Part%3ABBa_K4849028) |
| MtrAB | [http://parts.igem.org/Part:BBa\_K4849030](http://parts.igem.org/Part%3ABBa_K4849030) |

| Cym Gene | Link to Parts Repository |
| --- | --- |
| CymA | [http://parts.igem.org/Part:BBa\_K4849026](http://parts.igem.org/Part%3ABBa_K4849026) |

## Assembly of Mtr genes and Operons into Cyanogate Vectors:

Assembly into Level 0 vectors was sucessful for all 4 genes (MtrCAB subunits and CymA) and 3 operons after multiple attempts with troubleshooting involved. All reactions were done in silico to infer the size of the fragments. Initially colony PCR allowed identification of colonies with correct inserts which were then confirmed by diagnositc digestions shown in figure 3, 4, 5. While digestion with BsaI cuts out the gene from the vector via two cuts, digestion with PvuI leads to linerisation allowing checking of the construct’s size. It is important to note that MtrC gene had an extra BsaI site which was the restriction sites used in Lv0 -> Lv1 cyanogate assembly. Likewise, the 242bp faint bands visible for MtrCAB, MtrCA and MtrC, demonstrate the presence of an extra BsaI site and thus the requirement for domestication (figure 5 and 6). Also, while the single cut bands for MtrCAB, MtrCA and MtrAB look bigger than their expected size, the gel was run longer and the sizes were confirmed.

![](https://static.igem.wiki/teams/4849/wiki/mtr3.png)

**Figure 3**: Diagnostic digestion on MtrA and MtrB. 1 Kb DNA ladder diagram (New England Biolabs). The ladder is not to scale and it is provided for reference purposes only.

![](https://static.igem.wiki/teams/4849/wiki/mtr4.png)

**Figure 4**: Diagnostic digestion on MtrC and CymA. 1 Kb DNA ladder diagram (New England Biolabs). The ladder is not to scale and it is provided for reference purposes only.

![](https://static.igem.wiki/teams/4849/wiki/mtr5-1.jpg)![](https://static.igem.wiki/teams/4849/wiki/mtr5-2.png)

**Figure 5**: Diagnostic digestion on MtrCAB, MtrAB and MtrCA. 1 Kb DNA ladder diagram (New England Biolabs). The ladder is not to scale, and it is provided for reference purposes only.

![](https://static.igem.wiki/teams/4849/wiki/mtr6.png)

**Figure 6**: Diagnostic digestion on MtrC, showing 242bp band suggesting requirement for domestication.

Assembly into Level 1 vectors was sucessful for MtrA, MtrB, MtrAB, and CymA only. We were unable to take MtrCAB, MtrCA and MtrC into Level 1 vectors due to numerous failed attemps it took to domesticate the extra BsaI site in MtrC gene, thereby consuming vauable time. Similar to level 0 assemblies, colony PCR allowed identification of colonies with correct inserts (Figure 7 and 8) which were then confirmed by diagnositc digestions. Nenvertheless, we have added all the different level 1 composite parts onto the IGEM registry. Importantly, we used the PtrC10 protomor (BBa\_K4849013) from the Cyanogate toolkit as it gave us the option to express the Mtr compex in both Synechocystis sp. PCC 6803 and E. coli.

![](https://static.igem.wiki/teams/4849/wiki/mtr7.png)

**Figure 7**: Positive colony PCR results showing Level 1 MtrA and Lv1 CymA. MtrA size with the promotor, and terminator is approximately 1500 bp. Colonies 4 and 5 were successful. CymA size with the promotor, and terminator is approximately 800 bp. Colonies 1 and 3 were successful.

![](https://static.igem.wiki/teams/4849/wiki/mtr8.png)

**Figure 8**: Positive colony PCR results showing Level 1 MtrB. MtrB size size with the promotor, and terminator is approximately 2100 bp. Colony 1 and 2 were successful.

Finally, as level 1 vectors could be used in E. coli and our teams decision to scrap any more attemps to chrecterise Mtr in Synechocystis sp. PCC 6803 (due to failed attemps with Biosafety and time taken to transform Synechocystis sp. PCC 6803), only MtrAB was taken into Level T cyanogate vector (figure 9). The sucessful assembly was confirmed via diagnostic digestions.

![](https://static.igem.wiki/teams/4849/wiki/mtr9.png)

**Figure 9**: Positive colony PCR results showing Level T Mtr AB. MtrAB size with the promotor, terminator and the end linker approximately 3300 bp. Colonies 5, and 6 were successful.

**Important Note**: All the assemblies were checked via whole plasmid sequencing (againt reference) conducted by the Edinburgh Genome Foundry. The genome sequencing report can be found at the bottom of the page.

| MTR Chassis (LvT) | Link to Parts Repository |
| --- | --- |
| Ptrc10 - MtrA - TrrnB - End-linker 1 | [http://parts.igem.org/Part:BBa\_K4849031](http://parts.igem.org/Part%3ABBa_K4849031) |
| Ptrc10 - MtrB - TrrnB - End-linker 1 | [http://parts.igem.org/Part:BBa\_K4849033](http://parts.igem.org/Part%3ABBa_K4849033) |
| Ptrc10 - MtrC - TrrnB - End-linker 1 | [http://parts.igem.org/Part:BBa\_K4849034](http://parts.igem.org/Part%3ABBa_K4849034) |
| Ptrc10 - MtrCAB - TrrnB - End-linker 1 | [http://parts.igem.org/Part:BBa\_K4849035](http://parts.igem.org/Part%3ABBa_K4849035) |
| Ptrc10 - MtrCA - TrrnB - End-linker 1 | [http://parts.igem.org/Part:BBa\_K4849036](http://parts.igem.org/Part%3ABBa_K4849036) |
| Ptrc10 - MtrAB - TrrnB - End-linker 1 | [http://parts.igem.org/Part:BBa\_K4849037](http://parts.igem.org/Part%3ABBa_K4849037) |

| Cym Gene | Link to Parts Repository |
| --- | --- |
| Ptrc10 - CymA - TrrnB - End-linker 1 | [http://parts.igem.org/Part:BBa\_K4849038](http://parts.igem.org/Part%3ABBa_K4849038) |

**[Sepcial LvT Synechocystis MTR constructs:](http://parts.igem.org/Part%3ABBa_K4849039)**

* i) pSEVA421 acceptor - Ptrc10 - MtrCAB - TrrnB - Ptrc10 - CymA - TrrnB - End-linker 2

**Other LvT Synechocystis MTR constructs:**

* i) pSEVA421 acceptor - Ptrc10 - MtrA - TrrnB - Ptrc10 - CymA - TrrnB - End-linker 2
* ii) pSEVA421 acceptor - Ptrc10 - MtrB - TrrnB - Ptrc10 - CymA - TrrnB - End-linker 2
* iii) pSEVA421 acceptor - Ptrc10 - MtrC - TrrnB - Ptrc10 - CymA - TrrnB - End-linker 2
* iv) pSEVA421 acceptor - Ptrc10 - MtrCA - TrrnB - Ptrc10 - CymA - TrrnB - End-linker 2
* v) pSEVA421 acceptor - Ptrc10 - MtrAB - TrrnB - Ptrc10 - CymA - TrrnB - End-linker 2

**Domestication: MtrCAB, MtrCA and MtrC**

Domestication involves using primers to make precise modifications to a DNA sequence, in this case, changing a single base pair so the DNA sequence does not have a recognition site for a restriction enzyme. Domestication was done by PCR followed by restriction digestion and ligation. Primers were designed allowing elimination of the restriction site by changing a single base pair. The domestication PCR for MtrCAB and MtrCA can be seen in the gel below (figure 10). The domestication PCR also introduced new cut sites on the linear DNA allowing digestion and then re-ligation into a plasmid.

Due to the band smears, gel extraction was chosen over PCR cleanup. Once the linear domesticated DNA was cleaned, it was digested and re-ligated in a thermocycler. However, diagnostic digestion gels of the domesticated plasmid showed that the domestication did not work. Moreover, the digestion and re-ligation step was performed in a water bath, but the domestication did not work checked via digestions. However, after several attempts, the domestication for MtrCAB and MtrCA was successful but due to time constraints and previous difficulty in taking our genes from Lv0 to Lv1 parts, we decided to drop MtrCAB, MtrCA and MtrC.

![](https://static.igem.wiki/teams/4849/wiki/mtr10.png)

**Figure 10**: Domestication PCR conducted at different temperatures (trouble-shooting).

| Primer Name | Sequence |
| --- | --- |
| MtrA Forward Primer | CAGTGAAGACataATGAAGAACTGCCTAAAAATG |
| MtrA Reverse Primer | CAGTgaagacATaagcTTAGCGCTGTAATAGCTTG |
| MtrB Forward Primer | CAGTGAAGACAtAATGAAATTTAAACTCAATTTGATCAC |
| MtrB Reverse Primer | CAGTgaagacATaagcTTAGAGTTTGTAACTCATGCTC |
| MtrC Forward Primer | CAGTgaagacATaATGATGAACGCACAAAAATC |
| MtrC Reverse Primer | CAGTgaagacATaagcTTACATTTTCACTTTAGTGTGATC |
| MtrC Domestication Forward Primer | CAGTgaagacATtggacTCGCAAATTTAGAAAT |
| MtrC Domestication Reverse Primer | CAGTgaagaCAGTCCAATCACTGGCATGTC |
| CymA Forward Primer | CAGTgaagacATaATGAACTGGCGTGCACTA |
| CymA Reverse Primer | CAGTgaagacATaagcTTATCCTTTTGGATAGGGGTG |
| CyanoGate Level 0 Primers - Forward | GTCTCATGAGCGGATACATATTTGAATG |
| CyanoGate Level 0 Primers - Reverse | TTGAGTGAGCTGATACCGCT |

**Ferricyanide assay: (Mtr Characterisation in *Synechocystis*)**

The potassium ferricyanide assay provides a biochemical alternative to electrical measurements, enabling the recording of the rate at which electrons are produced and secreted extracellularly by the metabolically active Synechocystis sp. PCC 6803.

Once dissolved in a solution, potassium ferricyanide dissociates, leaving the ferricyanide ion. This ion is freely present in the culture media but cannot enter the cells. This property makes ferricyanide suitable for the assay.

In this assay, potassium ferricyanide in a water solution (displaying a yellow hue) is reduced by a single electron to produce transparent potassium ferrocyanide. This change in color reduces the recorded absorbance at 420 nm. By plotting a linear regression line over time, the rate of electron escape from the cyanobacterial cells can be estimated. (AC Gonzalez-Aravena et al, 2018, DOI: 10.1039/c8ra00951a)

![](https://static.igem.wiki/teams/4849/wiki/mtrf.png)

**Formula 1**: reduction reaction of ferricyanide ion to ferrocyanide

To become familiar with the technique, we attempted the assay in the wildtype Synechocystis sp. PCC 6803 (figure 11). However, due to time constraints, we were unable to repeat the experiment in both the wild-type and bio-engineered strains to characterize the successful functionality of the different MTR composite systems, most importantly, MtrCAB, MtrCAB-CymA, CymA.

**Table 1**: Absorbance of ferricyanide at 420nm over time in minutes

| Time (minutes) | Absorbance (420nm) |
| --- | --- |
| 35 | -0.003 |
| 85 | -0.0235 |
| 140 | -0.033 |
| 200 | -0.04 |
| 260 | -0.0555 |
| 320 | -0.0685 |

![](https://static.igem.wiki/teams/4849/wiki/mtr11.png)
**Figure 11**: Linear regression plot of 1mM ferricyanide reduction by Synechocystis sp. PCC 6803 , measured as absorbance (420nm) over time(minutes). Data shown is the mean of experiment done in technical triplicates, OD(630nm) of Synechocystis sp. PCC 6803 cell culture was 1.44, blanking done at time =0.

**Co-Tranformation of Ccm and Mtr: (Mtr Characterisation in E. coli:)**

Attempting to charactize the mtrAB cassette in E. coli, we needed to introduce the cytochrome c maturation gene cascade (ccmA-H) which is lacking in E. coli (but present in Cyanobacteria) to the organism, as heme folding is crucial for the individual mtr subunits appropiate assembly and therefore transport to the membrane/periplasmic regions.

Using ccmA-H cassette in pSB1C3 vector (obtained from iGEM Edinburgh 2012, BBa\_K917006), we have underwent co-transformation of the ccmA-H genes with mtrA,B,AB in lvl 1, this was possible as each plasmid had conferred different antibiotic resistance selection (chloramphenicol for ccm, ampicillin for level 1 mtr).

We have then miniprepped the samples and did diagnostic digestions using NotI restriction enzyme which linearises the cyanogate vector, and EcoRI which linearizes the ccm vector. While we strongly believe that the co-transformations were succesful as multiple colonies had grown on the LB plates with both antibiotics added, the quality of the digestions was rather poor (figure 13). A potential reason for the survival of colonies on ampicillin colonies although lack of ccmA-H shown in digestion could be due to presence of the vector without the gene.

![](https://static.igem.wiki/teams/4849/wiki/mtr12.png)

**Figure 12**: Diagnostic digestion on MtrA, MtrB and MtrAB co-transformed with Ccm.

**Membrane Fractionation and Heme Staining (Future Experiments – in both Synechocystis and E. coli):**

Continuing the characterization efforts, membrane fractionation of the co-transformant E. coli would have been carried out to yield the cytoplasmic, membrane, and periplasmic fractions, each enriched with proteins typically localized there.

Running the fractionated samples on an SDS-PAGE would enable the separation of protein bands based on their size. We could then confirm the presence of mtrA and mtrB in the periplasmic and membrane fractions by staining the resulting gel with a heme-staining solution, which selectively stains proteins containing a heme group.

# *hisG-dapA* Results

---

## Finding the optimal ligation efficiency for hisG and dapA using CyanoGate

The most optimal insert:vector ratio has been described in this work, concluding that a higher ratio may be more suitable for the E. coli MG1655 genes hisG, dapA and lysC when applying the CyanoGate system for transformation - with a 10:1 insert:vector ratio showing highest success potential of bands appearing through colony PCR following a white-blue screening using Golden Gate assembly, although a 2:1 insert:vector ratio showed higher colony counts. Being native E. coli genes, this finding may be more relevant for further cloning experiments using genes from E. coli. Additionally, an increased number of cycles through thermal cycling following assembly could be more appropriate for these genes - suggesting an adjustment to the recommended 5 cycles by Vasudevan et al. (2019). This project may therefore demonstrate useful troubleshooting guidance for further cloning experiments using the newly developed CyanoGate system, or for cloning experiments specifically involving the genes hisG, dapA and lysC.

![](https://static.igem.wiki/teams/4849/wiki/hl-t4.png)
![](https://static.igem.wiki/teams/4849/wiki/hl-t5.png)

![](https://static.igem.wiki/teams/4849/wiki/hl-f17.png)

![](https://static.igem.wiki/teams/4849/wiki/hl-f18.png)

## Considerations for replicating the experiments

The proposed increase of the insert:vector ratio for level 0 and level 1 transformations should be considered when replicating the experiments using native E. coli MG1655 genes hisG and dapA following the modular platform CyanoGate. The presented modifications relating to thermal cycling should also be considered for further repeats of this experiment.

## Ninhydrin Test For Amino Acids

![](https://static.igem.wiki/teams/4849/wiki/hl-f2.png)

Ninhydrin test on dapA and hisG overexpressed strains. Shows 2% amino acid standard solution (2% AA), Wild type (WT), dapA level 1 overexpressed strain (dapA) and hisG overexpressed strain (hisG) both under Ptrc10 promotor. dapA shows a 1.48x increase compared to WT and hisG is 1.57x. Error bars are standard deviations. p=4.73E-5.

Ninhydrin test to quantitatively determine relative levels of amino acids between level 1 transformed strains and wild type (WT Control) E.coli (TOP10). A 2% amino acid standard was prepared to provide further comparison. The ninhydrin reaction involves ninhydrin (C9H604) reacting to amines or a-amino acids. The development of a deep blue colour indicates the presence of amino acids in the sample. This can be quantified with a spectrophotometer reading absorbance at 570nm, and provided the initial OD of the cell cultures for comparison is known, quantitative results can be drawn.

Our results, obtained using the ninhydrin protocol in the protocol section on an overnight culture of E.coli cells in stationary phase, suggest that the transformants are successful in overexpressing hisG and dapA and that this does correspond to increased amino acid expression. Compared to the wild type there were significant differences in the absorbance of the analytes from dapA and hisG. Stationary phase was used as this would mimic the cell culture makeup of the panel waste. After use the culture would have grown stagnant and so based on our growth curves, an overnight culture was sufficient to plateau the growth rate into a stationary phase.

![](https://static.igem.wiki/teams/4849/wiki/hl-f3.png)

![](https://static.igem.wiki/teams/4849/wiki/hl-f4alt.png)

![](https://static.igem.wiki/teams/4849/wiki/hl-f5.png)

BYJU'S (2022). Ninhydrin Test - Reaction, Principle, Procedure, Result Interpretation. [online] BYJUS. Available at: https://byjus.com/chemistry/ninhydrin-test/.

## Mass Spectrometry for Amino Acid Production

![](https://static.igem.wiki/teams/4849/wiki/mass-spec.jpeg)

Mass Spectrometry processed M/Z results displaying decreased production of L-Cystine between control strain (WT) and overexpressed strain (*dapA*) and increased production of L-Lysine between control strain and overexpressed strain.

To further verify that increase in amino acid concentration was due to histidine and lysine concentration respectively, we used mass spectrometry. Samples were sent off to the university's Waddington Laboratories for M/Z analysis. Overxpression of target amino acids was expected to reduce expression of other amino acids. Cystine production was tested to confirm this, and results came back positive, indicated by the reduced expression of Cystine. Results showed an increase in lysine production in the *dapA* overexpressed strain, and expected decrease in cystine production.

# Biosafety Results

---

## Completed Level T Constructs:

---

## LvT eYFP constructs:

---

pSEVA421 Acceptor

PrnpB

eYFP

TrrnB

End-linker 1

pSEVA421 Acceptor

PcopM195-BCD

eYFP

TrrnB

End-linker 1

## LvT Synechocystis KS constructs:

---

pSEVA421 Acceptor

PcopM195-BCD

NuiA

TrrnB

PrnpB

NucA

TrrnB

End-linker 2

pSEVA421 Acceptor

PcopM195-BCD

NuiA

TrrnB

PrbcL1C

NucA

TrrnB

End-linker 2

## LvT E. coli KS construct:

---

pSEVA421 Acceptor

PzntA

NuiA

TrrnB

BBa\_J23114

NucA

TECK120029600 Terminator

## Biosafety Analysis

---

Level T eYFP constructs were made in order to characterize PcopM195-BCD zinc-inducible Synechocystis promoter by measuring eYFP fluorescence. Level T Synechocystis KS constructs were supposed to be transformed into Synechocystis to characterize kill switch activity. Level T eYFP and KS constructs were screened by colony PCR. For each construct there were colonies that gave a correct size amplicon (Figures 1 and 2).

![](https://static.igem.wiki/teams/4849/wiki/fig1-biosafety.png)

**Figure 1.** cPCR of Level T eYFp constructs.

![](https://static.igem.wiki/teams/4849/wiki/fig2-biosafety.png)

**Figure 2.** cPCR of Level T Synechocystis Kill Switch constructs.

**Table 1: Expected amplicon sizes:**

| Construct | Expected Amplicon Size |
| --- | --- |
| LvT-PrnpB-eYFP (TY1) | 1494 bp |
| LvT-PcopM-eYFP (TY2) | 1544 bp |
| LvT-PrnpB-NucA (KS1) | 2401 bp |
| LvT-Prbc-NucA (KS2) | 2356 bp |

Cultures from colonies that gave the correct cPCR band were set up, plasmids were purified and screened by restriction digestion for correct insertion (Figure 3 and 4). After digestion, while the correct number of bands was observed, they were all slightly larger than expected. Due to time constraints, decided to continue with Synechocystis transformations. Importantly, we later also sent the Level T constructs to Edinburgh Genome Foundry for sequencing by Oxford Nanopore technology and analysis. The sequencing report showed that most reads of the Level T Kill Switch constructs were of the approx. expected size, which suggested that larger than expected size bands observed after restriction digestion were likely an experimental error.

![](https://static.igem.wiki/teams/4849/wiki/fig3-biosafety.png)

**Figure 3.** Restriction digestion of Level T eYFP constructs with BciVI and AvaI enzymes.

![](https://static.igem.wiki/teams/4849/wiki/fig4-biosafety.png)

**Figure 4.** Restriction digestion of Level T Synechocystis Kill Switch constructs with BciVI and AvaI enzymes.

**Table 2: Expected band sizes following digestion:**

| LvT Plasmid | AvaI Digest (AvaI) | BciVI Digest (BciVI) |
| --- | --- | --- |
| LvT-PrnpB-eYFP | 4857 bp | 2617 bp and 2240 bp |
| LvT-PcopM-eYFP | 4907 bp | 2667 bp and 2240 bp |
| LvT-PrnpB-NucA | 5764 bp | 3524 bp and 2240 bp |
| LvT-Prbc-NucA | 5719 bp | 3479 bp and 2240 bp |

![](https://static.igem.wiki/teams/4849/wiki/fig5-biosafety.png)

**Figure 5.** Colony PCR of Level T E. coli Kill Switch construct.

We performed the *Synechocystis* transformation protocol received from ABOA-Turku’s iGEM team with some modifications. First, the main Synechocystis sp. PCC 6803 culture used for transformations was a mixture of two cultures that were at OD630=0.8, and not at OD750=0.8 (mid logarithmic growth phase) as suggested in the original protocol. Second, the recommendation was to keep plates at 30°C and 50 µE until slight green film arose (ca. 2-4 days), however, we only had a shaking incubator with a light source at our disposal, so we kept the plates there. Synechocystis transformations failed - there was no green bacterial film on the plates after a week, the plates started drying out and had to be disposed of. Importantly, even if we were able to grow the film of cyanobacteria it would not have been possible for us to select for transformed cells because it requires multiple rounds of addition of antibiotic by increasing its concentration and waiting for colonies to grow, which is a slow process for Synechocystis and can take up to a couple of months, which in the case of iGEM timelines is unfeasible.

Because Synechocystis transformations failed we decided to try and characterise NucA-NuiA kill switch in E. coli. For this purpose, we ordered a G-block encoding the whole Kill Switch cassette with the same NucA and NuiA sequences, but with Synechocystis promoters substituted with corresponding E. coli promoters, zinc-inducible PzntA promoter for antitoxin and constitutive Anderson promoter (BBa\_J23114) for toxin. The G-block also contained the required overhangs to allow direct assembly into a LvT acceptor vector. We ran cPCR of level T E. coli KS constructs (Figure 5). Positive control failed because no template DNA was added, but three colonies, 2, 3, and 8, gave the expected amplicon size (1930 bp), so we decided to proceed to Kill Switch characterization with colony 2.

We set up two overnight cultures, one from colony 2 that gave the correct cPCR band size for level T E. coli KS construct, and the other one from a blue colony containing the empty vector (WT+vector). We used these initial cultures to make new cultures in triplicates with or without added 400 µM Zn2+ for both KS and WT+vector control. For KS characterisation, we grew these cultures in a shaking incubator at 37C, 160 rpm, and took OD600 measurements at 1 hour intervals to produce growth curves (Figure 6). From growth curves, cells containing Kill Switch plasmid or empty vector grew better without added zinc, indicating that the addition of 400 μM ZnSO4 slows down cell growth. Importantly, there was no apparent decrease in growth of cells containing the Kill Switch in media without added zinc compared to those grown with added zinc. This could be due to nuclease not being expressed, thus unable to kill the cells. Notably, NucA requires divalent metal ions like Mn2+ or Mg2+ as cofactors, the optimal concentration for these being around 5 mM, while NucA activity was shown to decrease with increasing concentration of monovalent salt. LB media contains a high concentration of NaCl but no divalent salts, meaning that NucA might not function due to lack of cofactors. Alternatively, zinc-inducible promoter might not function as expected, leading to constitutive expression of antitoxin, thus inhibiting nuclease. In future experiments, NucA and NuiA expression should be verified by Western Blotting before characterizing the Kill Switch. Also, LB media should be supplemented with appropriate concentrations of divalent salts to ensure the presence of cofactors for NucA.

![](https://static.igem.wiki/teams/4849/wiki/fig6-biosafety.png)

**Figure 6.** Growth curves of E. coli TOP10 cells with or without Kill Switch plasmid in media with or without added 400 μM ZnSO4.

![](https://static.igem.wiki/teams/4849/wiki/fig7-biosafety.png)

**Figure 7.** CFU/mL (log10) values for E. coli TOP10 cells with or without Kill Switch plasmid in media with or without added 400 μM ZnSO4. KS and WT+vector cultures (in triplicates) were grown in media with or without zinc were sampled every 2 hours (for 6 hours total), diluted, and plated onto LB + Spec + 400 µM Zn2+ and LB + Spec (without zinc) plates, respectively.

For checking the viability of cells containing the KS in conditions with and without added zinc compared to WT+vector control we performed colony forming unit (CFU) counting assay (Figure 7). In general, the experiment was unsuccessful since the numbers of colonies observed after plating 10 µL of culture diluted by the factor of 107 were too low to be able to accurately deduce the CFU number in original undiluted samples. Thus, in the future the experiment should be redone by plating lower dilution or higher volume of cells. Moreover, as noted previously, since NucA requires divalent metal ions like Mn2+ or Mg2+ as cofactors, ideally the plates should be supplemented with them. From our results, at 6 hours, no significant difference in CFU/mL numbers was observed between KS-containing cells grown with or without added zinc (2 sample t-test, p-value > 0.5).

A comprehensive list of the parts designed can be found on our [contributions page](https://2023.igem.wiki/edinburgh/contribution).

# Salinity Results

---

### Salt growth curve of BL21 (DE3) cells

To determine the level of growth and salt tolerance in Escherichia coli BL21 (DE3), a growth curve was made, incubating the E. coli in various concentrations of NaCl in LB media. 29ml LB media was inoculated with 1ml overnight culture of E.coli and the initial OD600 was measured. This was then followed by measurements taking 1ml of media every hour for 6 hours. 1% salt has been removed as the initial OD600 was significantly higher than those of the others, which may have affected the results. The results showed a significant decrease in growth after 6 hours of the E.coli BL21 after 3% NaCl (w/v) LB (Figure 1). The doubling time of the E.coli can be seen to steadily increase until 3.5% where the doubling time begins to increase significantly as the concentration of salt in the medium rises (Table 1). The E.coli grown in 0.5% salt (no additional salt), grew at the fastest rate for the first 5 hours with a doubling time of 1.05 hours in comparison to 1.5% salt with a doubling time of 1.09 hours. However, more testing would be necessary to determine if this difference is due to increased salt in the medium. The E. coli grew at 0.5% then saw a decline between hours 5 and 6, which was not mirrored in other cultures at different concentrations. However, a decline before recovery had been seen in other cultures, such as 3.5% in hour 5.

![](https://static.igem.wiki/teams/4849/wiki/sal-fig1.png)

**Figure 1**: 6 hour Growth Curve of BL21 (DE3) Cells In LB Media of Varying Salt Concentrations. 1% Salt LB was removed due to a likely error in protocol affecting results. LB media containing various salt concentrations were inoculated with BL21 (DE3) Overnight culture and the OD600 was measured every hour to measure the growth of the Escherichia coli.

Table 1 - Doubling Times of Salty E.coli Cultures at 6 hours. Doubling times were calculated using the formula Dt = hours/ log2( final OD600/ Initial OD600)

| Lysogeny Broth Salt Concentration (%) | Doubling Time at 6 Hours (Hours) |
| --- | --- |
| 0.5 | 1.29 |
| 1.0 | 1.38 |
| 1.5 | 1.23 |
| 2.0 | 1.26 |
| 2.5 | 1.32 |
| 3 | 1.40 |
| 3.5 | 1.65 |
| 4 | 2.05 |
| 4.5 | 2.35 |
| 5 | 3.85 |
| 5.5 | 2.27 |

### stpA level T

Due to being unsuccessful in extracting purified stpA gene from genomic DNA, a gene block was designed to be inserted into a cyanogate level T vector with correct cut sites and overhangs for an assembly reaction wherein the restriction enzyme BbsI would cut the gene block revealing sticky ends which could then be ligated into the level T vector. A level T reaction was conducted and the reaction product was transformed into TOP10 cells which were then spread onto XGAL-IPTG plates for blue/white selection. White colonies were then picked and colony PCR was conducted to confirm successful transformation. The PCR product was then run on 1% agarose gel and the gel was analysed using a UV imager (Figure 2). The results showed bands of the expected size across the white colonies indicating that the plasmid had been successfully constructed and transformed.

![](https://static.igem.wiki/teams/4849/wiki/sal-fig2.png)

**Figure 2**: Imaged Agarose gel electrophoresis. Colony PCR was performed generating bands at the expected positions between 1.5 and 2kb.

The ggpS gene was extracted from synechocystis sp. PCC 6803 by PCR from genomic DNA. The gene was then purified by gel extraction and ligated in an assembly reaction into the CyanoGate level 0 backbone. A further digestion and ligation was performed to assemble the gene along with the constitutive promoter ptrc10, which functions in both synechocystis as well as E.coli and the terminator pC0.082. This level 1 assembly was then transformed into E. coli TOP10 cells and confirmed by colony PCR and digestion. However, the final plasmid was found to be around 1kb larger than anticipated (Figure 3). Nevertheless, the correct band sizes indicating that ggpS has been successfully ligated into the level 1 vector and transformed were present. The decision was then made to proceed with error-prone PCR using ggpS in this backbone.

![](https://static.igem.wiki/teams/4849/wiki/sal-fig3.png)

**Figure 3**: Agarose Gel Electrophoresis image of undigested Level 1 construct and digested with NotI and BbsI. Plasmid miniprep was digested with NotI and bbsI and run on 1% agarose gel in gel electrophoresis and imaged. Repeats can be found on the right.

However, after repeated attempts, this error-prone PCR was unsuccessful as when run with dye on 1% Agarose gel and imaged, the bands indicated two PCR products, one at 4.5kb and one at 2.5kb consistently. In silico there was considered no reason to believe that primer mis-annealing should take place and so it was hypothesised that either the plCH47732 level 1 acceptor vector, the PTRC10 promoter, or pC0.082 terminator in level 0 was incorrect. A PCR reaction was performed using the level 0, level 1, and genomic PCR extract as template DNA and all templates except the level 1 were successful (Figure 4).

To circumvent the plCH47732 backbone, epPCR of ggpS was performed again using the level 0 assembly as a template and NEB Taq polymerase. The genomic Forward and Genomic Reverse primers were used. These primers when digested, would generate the overhangs necessary to assemble into a pJUMP29-1A acceptor vector with a constitutive PJ23100 promoter, pET ribosome binding site, and L3SAP51 terminator. The resulting library was digested with BbsI, creating 1.5kb double-stranded linear DNA fragments. These digested fragments were then used in a level 1 JUMP assembly reaction to generate level 1 constructs with a GFP selection marker. Fluorescent colonies were picked for colony PCR to check for successful assembly and transformation. However, the first attempt was unsuccessful.

After a subsequent attempt utilising longer extension times, the colony PCR indicated successful assembly and transformation of the epPCR library (Figure 5). The bands were at approximately 1.8kb as expected in colonies 1, 2, and 4. These results were not demonstrated in our final report. The error-prone PCR library was then transferred to the Edinburgh Genome Foundry, along with chemically competent cells for heat shock containing the stpA level T plasmid for transformation and subsequent performance of the assay.

![](https://static.igem.wiki/teams/4849/wiki/sal-fig4.png)

**Figure 4**: Agarose gel electrophoresis image: Q5 PCR of ggpS from ggpS, lvl0 and lvl1P1 templates, and Q5 of pICH47732 backbone from lvl1 template.

Sanger sequencing of the digested ggpS epPCR library showed that the consensus sequence from the sequencing matched the expected sequence from in-silico testing.

![](https://static.igem.wiki/teams/4849/wiki/sal-fig5.png)

**Figure 5**: UV Imaged Agarose Gel electrophoresis. 4 Colonies were picked for colony PCR using P1 and P2 primers. Results show colony 1,2 and 4 with the expected band size.

## Salinity Report

---

## Sequencing Report

---

{% endblock %}