iGEM 2023 — Team yale

{% 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 %}

### Week 1

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[23-06-01 Miniprep Pcargo and Transformation of Entry Vectors](https://static.igem.wiki/teams/4947/wiki/23-06-01-miniprep-pcargo-and-transformation-of-entry-vectors-benchling.pdf)
[23-06-02 PCR Amplification and Diagnostic Age of Pz Promoter](https://static.igem.wiki/teams/4947/wiki/23-06-02-pcr-amplification-and-diagnostic-age-of-pz-promoter-benchling.pdf)
[23-06-03 Pz Fragment Purification Glycerol Stocks of Entry Vectors and Pz Miniprep of Genes Entry Vectors and Gga Reactions](https://static.igem.wiki/teams/4947/wiki/23-06-03-pz-fragment-purification-glycerol-stocks-of-entry-vectors-and-pz-miniprep-of-genes-entry-vectors-and-gga-reactions-benchling.pdf)
[23-06-04 Transformation of Assembly Vectors with Drop Dialysis](https://static.igem.wiki/teams/4947/wiki/23-06-04-transformation-of-assembly-vectors-with-drop-dialysis-benchling.pdf)

### Week 2

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[23-06-05 Re Gga of Initial Assemblies](https://static.igem.wiki/teams/4947/wiki/23-06-05-re-gga-of-initial-assemblies-benchling.pdf)
[23-06-06 Transformation of Re Gga for Assembly Vectors Plating of Equol Strains Inoculation of Eql Donor Strain](https://static.igem.wiki/teams/4947/wiki/23-06-06-transformation-of-re-gga-for-assembly-vectors-plating-of-equol-strains-inoculation-of-eql-donor-strain-benchling.pdf)
[23-06-07 Plating of Eql Strains Results Glycerol Stocks of Stig117 Transformation of Stig117 with Eql Plasmids with Plating of Stig118 Stig123](https://static.igem.wiki/teams/4947/wiki/23-06-07-plating-of-eql-strains-results-glycerol-stocks-of-stig117-transformation-of-stig117-with-eql-plasmids-with-plating-of-stig118-stig123-benchling.pdf)
[23-06-08 Re Transformation of Donor Re Plating of Transformations from 23-06-07 Screening of Assembly Colonies Part 1](https://static.igem.wiki/teams/4947/wiki/23-06-08-re-transformation-of-donor-re-plating-of-transformations-from-23-06-07-screening-of-assembly-colonies-part-1-benchling.pdf)
[23-06-09 Screening of Assembly Colonies Part 2 Inoculation of Successful Assembly Strains](https://static.igem.wiki/teams/4947/wiki/23-06-09-screening-of-assembly-colonies-part-2-inoculation-of-successful-assembly-strains-benchling.pdf)
[23-06-11 Inoculations of Strains in Eql Assay 2 Inoculations of Some Assembly Colonies](https://static.igem.wiki/teams/4947/wiki/23-06-11-inoculations-of-strains-in-eql-assay-2-inoculations-of-some-assembly-colonies-benchling.pdf)

### Week 3

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[23-06-12 Conjugation Prep of Donor and Recipient Strains for Eql Integrase Assay Miniprep of Gga Assemblies for Sequencing Glycerol Stocks PCR Diagnostic of Gga Assemblies Plasmidsaurus Sequencing](https://static.igem.wiki/teams/4947/wiki/23-06-12-conjugation-prep-of-donor-and-recipient-strains-for-eql-integrase-assay-miniprep-of-gga-assemblies-for-sequencing-glycerol-stocks-pcr-diagnostic-of-gga-assemblies-plasmidsaurus-sequencing-benchling.pdf)
[23-06-13 Conjugation Inoculation Plating of Strains for Eql Integrase Assay WPS Analysis Age of PCR Screening PCR Diagnostic of Assembly Plasmids Control Gga A111](https://static.igem.wiki/teams/4947/wiki/23-06-13-conjugation-inoculation-plating-of-strains-for-eql-integrase-assay-wps-analysis-age-of-pcr-screening-pcr-diagnostic-of-assembly-plasmids-control-gga-a111-benchling.pdf)
[23-06-15 Diagnostic PCR Age of Gga Controls Glycerol Stocks of Conjugated Recipients](https://static.igem.wiki/teams/4947/wiki/23-06-15-diagnostic-pcr-age-of-gga-controls-glycerol-stocks-of-conjugated-recipients-benchling.pdf)
[23-06-16 PCR Amplification and Purification of A B C Backbone Gibson Assembly Module A Plasmid](https://static.igem.wiki/teams/4947/wiki/23-06-16-pcr-amplification-and-purification-of-a-b-c-backbone-gibson-assembly-module-a-plasmid-benchling.pdf)
[23-06-17 Transformation of Gibson Assembly Entry Vectors](https://static.igem.wiki/teams/4947/wiki/23-06-17-transformation-of-gibson-assembly-entry-vectors-benchling.pdf)

### Week 4

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[23-06-19 PCR Colony Screening of Ga Plasmid Transformations](https://static.igem.wiki/teams/4947/wiki/23-06-19-PCR-colony-screening-of-ga-plasmid-transformations-benchling.pdf)
[23-06-20 Repeat of PCR Colony Screening Amplification of Pz B and Pz C Gga of Modular Plasmids and Transformation of Gga S](https://static.igem.wiki/teams/4947/wiki/23-06-20-repeat-of-PCR-colony-screening-amplification-of-pz-b-and-pz-c-gga-of-modular-plasmids-and-transformation-of-gga-s-benchling.pdf)
[23-06-21 Miniprep of Original Entry Vectors Plating of Modular Ggas Age on Gga PCR Assemblies Sanger Sequencing of Gibsons](https://static.igem.wiki/teams/4947/wiki/23-06-21-miniprep-of-original-entry-vectors-plating-of-modular-ggas-age-on-gga-pcr-assemblies-sanger-sequencing-of-gibsons-benchling.pdf)
[23-06-22 Miniprep of Original Entry Vectors Gga Controls and PCR Screening Using Gga Assemblies](https://static.igem.wiki/teams/4947/wiki/23-06-22-miniprep-of-original-entry-vectors-gga-controls-and-PCR-screening-using-gga-assemblies-benchling.pdf)
[23-06-23 Miniprep of Original Entry Vectors Troubleshooting](https://static.igem.wiki/teams/4947/wiki/23-06-23-miniprep-of-original-entry-vectors-troubleshooting-benchling.pdf)
[23-06-24 Miniprep of Ga Entry Vectors for Sequencing](https://static.igem.wiki/teams/4947/wiki/23-06-24-miniprep-of-ga-entry-vectors-for-sequencing-benchling.pdf)

### Week 5

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[23-06-26 Gga for BSAI Site Assay PCR PCR of All Modular Gga S Re-Send Gibsons Pcargo and Equol Strains for Sanger Sequencing](https://static.igem.wiki/teams/4947/wiki/23-06-26-gga-for-bsai-site-assay-PCR-PCR-of-all-modular-gga-s-re-send-gibsons-pcargo-and-equol-strains-for-sanger-sequencing-benchling.pdf)
[23-06-28 Enzyme Digestion Assay Gga Junction Assay Gibson Amplicon Creation EQL Calibration Curve](https://static.igem.wiki/teams/4947/wiki/23-06-28-enzyme-digestion-assay-gga-junction-assay-gibson-amplicon-creation-eql-calibration-curve-benchling.pdf)
[23-06-29 Enzyme Digestion Assay Retry Gga Amplicon Purification Retry Gga Amplicon PCR EQL Calibration Curve Analysis](https://static.igem.wiki/teams/4947/wiki/23-06-29-enzyme-digestion-assay-retry-gga-amplicon-purification-retry-gga-amplicon-pcr-eql-calibration-curve-analysis-benchling.pdf)
[23-06-30 Gibson Assembly and Transformation of WGA Modules EQL Calibration Analysis](https://static.igem.wiki/teams/4947/wiki/23-06-30-gibson-assembly-and-transformation-of-wga-modules-EQL-calibration-analysis-benchling.pdf)
[23-07-01 PCR Colony Screening of GA Modules](https://static.igem.wiki/teams/4947/wiki/23-07-01-pcr-colony-screening-of-ga-modules-benchling.pdf)

### Week 6

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[23-07-03 Mini-prep and WPS of Inoculated Colonies Diagnostic PCR on GA Products Feeding Experiment](https://static.igem.wiki/teams/4947/wiki/23-07-03-mini-prep-and-wps-of-inoculated-colonies-diagnostic-pcr-on-ga-products-feeding-experiment-benchling.pdf)
[23-07-04 Re-GA of Modular Plasmids, Extra DpNI Digest of PCARGO, Transform Initial GA Modular Plasmids, Trial Run of Equol Feeding Experiment](https://static.igem.wiki/teams/4947/wiki/23-07-04-re-ga-of-modular-plasmids-extra-dpni-digest-of-pcargo-transform-initial-ga-modular-plasmids-trial-run-of-equol-feeding-experiment-benchling.pdf)
[23-07-05 Plating of Re-GA Modular Plasmids and Re-electroporation of OG-GA-S, Colony PCR Junction Screening of OG-GA-S, Equol Feeding Experiment, and Extraction Positive Control](https://static.igem.wiki/teams/4947/wiki/23-07-05-plating-of-re-ga-modular-plasmids-and-re-electroporation-of-og-ga-s-colony-pcr-junction-screening-of-og-ga-s-equol-feeding-experiment-and-extraction-positive-control-benchling.pdf)
[23-07-06 Colony PCR Screening of Re-GA Modules, Amplification of GGA Amplicon, Equol Feeding, and Extraction Experiments (Round 1)](https://static.igem.wiki/teams/4947/wiki/23-07-06-colony-pcr-screening-of-re-ga-modules-amplification-of-gga-amplicon-equol-feeding-and-extraction-experiments-round-1-benchling.pdf)
[23-07-07 PCR Purification Assay, Mini-prep of Potential Colonies, Test GGA Transformation, Equol Feeding Experiment](https://static.igem.wiki/teams/4947/wiki/23-07-07-pcr-purification-assay-mini-prep-of-potential-colonies-test-gga-transformation-equol-feeding-experiment-benchling.pdf)
[23-07-08 Colony Junction Screening, Equol Feeding Experiments](https://static.igem.wiki/teams/4947/wiki/23-07-08-colony-junction-screening-equol-feeding-experiments-benchling.pdf)
[23-07-09 Colony Junction Screening, Test GA](https://static.igem.wiki/teams/4947/wiki/23-07-09-colony-junction-screening-test-ga-benchling.pdf)

### Week 7

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[23-07-10 (A) GGA Amplification, GA Attempt 1, Colony Screening Retry](https://static.igem.wiki/teams/4947/wiki/23-07-10-a-gga-amplification-ga-attempt-1-colony-screening-retry-benchling.pdf)
[23-07-13 Mini-prep, Glycerol Stock, Successful GA and GGA3 for Sequencing, Transformation of Test GGA](https://static.igem.wiki/teams/4947/wiki/23-07-13-miniprep-glycerol-stock-successful-ga-and-gga3-for-sequencing-transformation-of-test-gga-benchling.pdf)
[23-07-14 WPS Analysis of Sequenced Modular Plasmids B1-M and Full GGA, Junction Screening, Conjugation for EQL Feeding](https://static.igem.wiki/teams/4947/wiki/23-07-14-wps-analysis-of-sequenced-modular-plasmids-b1-m-and-full-gga-junction-screening-conjugation-for-eql-feeding-benchling.pdf)
[23-07-15 Mini-prep of Remaining Modular Plasmids for Sequencing, B1-M Junction Screening](https://static.igem.wiki/teams/4947/wiki/23-07-15-miniprep-of-remaining-modular-plasmids-for-sequencing-b1-m-junction-screening-benchling.pdf)
[23-07-16 More B1-M Colony PCR Screening](https://static.igem.wiki/teams/4947/wiki/23-07-16-more-b1-m-colony-pcr-screening-benchling.pdf)

### Week 8

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[23-07-17 Sending More Plasmids for WPS Backup, Colony PCR Screen, Daidzein Concentration Optimization](https://static.igem.wiki/teams/4947/wiki/23-07-17-sending-more-plasmids-for-wps-backup-colony-pcr-screen-daidzein-concentration-optimization-benchling.pdf)
[23-07-18 WPS Analysis, Electroporation of STIG117 with Correct Module Plasmids, IsoLig and Lig, DZN Standard Curves, Conjugation of P.utida Inducer Experiment, Equol](https://static.igem.wiki/teams/4947/wiki/23-07-18-wps-analysis-electroporation-of-stig117-with-correct-module-plasmids-isolig-and-lig-and-dzn-standard-curves-conjugation-of-putida-inducer-experiment-equol-benchling.pdf)
[23-07-20 Conjugation of Modular Plasmid Strains into DH10LP, Mini-prep of GA Module Strains, Transformation of ASD with All B1-G3 Plasmids, Standard Curves for Intermediates, Extraction, LCMS for Validation of Initial Equol Experiment](https://static.igem.wiki/teams/4947/wiki/23-07-20-conjugation-of-modular-plasmid-strains-into-dh10lp-miniprep-of-ga-module-strains-transformation-of-asd-with-all-b1-g3-plasmids-standard-curves-for-intermediates-extraction-lcms-for-validation-of-initial-equol-experiment-benchling.pdf)
[23-07-21 Amplification of Test Run, Full Assembly from Modular Plasmids, Inoculations](https://static.igem.wiki/teams/4947/wiki/23-07-21-amplification-of-test-run-full-assembly-from-modular-plasmids-inoculations-benchling.pdf)
[23-07-22 Plating of Final Module Strains, Full Assembly Amplification Optimization, GA of Full Assembly](https://static.igem.wiki/teams/4947/wiki/23-07-22-plating-of-final-module-strains-full-assembly-amplification-optimization-ga-of-full-assembly-benchling.pdf)
[23-07-23 Test Feeding Experiment, Full Assembly PCR Colony Screen](https://static.igem.wiki/teams/4947/wiki/23-07-23-test-feeding-experiment-full-assembly-pcr-colony-screen-benchling.pdf)

### Week 9

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[23-07-24 Homolog Optimization, LCMS, Begin Final Daidzein Plasmid Construction, LC-MS of Equol Validation Experiment](https://static.igem.wiki/teams/4947/wiki/23-07-24-homolog-optimization-lcms-begin-final-daidzein-plasmid-construction-lc-ms-of-equol-validation-experiment-benchling.pdf)
[23-07-25 Final Daidzein Plasmid Construction, Transformation, and Plating](https://static.igem.wiki/teams/4947/wiki/23-07-25-final-daidzein-plasmid-construction-transformation-and-plating-benchling.pdf)
[23-07-26 Final Daidzein Plasmid Construction, Colony Screen and Inoculation, Inoculation of STIG117](https://static.igem.wiki/teams/4947/wiki/23-07-26-final-daidzein-plasmid-construction-colony-screen-and-inoculation-inoculation-of-stig117-benchling.pdf)
[23-07-27 Final Daidzein Plasmid Construction, Mini-prep and WPS, Electroporation of STIG117 with Final Daidzein Plasmid](https://static.igem.wiki/teams/4947/wiki/23-07-27-final-daidzein-plasmid-construction-miniprep-and-wps-electroporation-of-stig117-with-final-daidzein-plasmid-benchling.pdf)

### Week 10

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[23-07-28 Inoculation of Transformed STIG117 for Glycerol Stock](https://static.igem.wiki/teams/4947/wiki/23-07-28-inoculation-of-transformed-stig117-for-glycerol-stock-benchling.pdf)
[23-07-31 Colony PCR of Full Assembly, Extraction of Modular Feeding, LC-MS on Daidzein Concentration Curve](https://static.igem.wiki/teams/4947/wiki/23-07-31-colony-pcr-of-full-assembly-extraction-of-modular-feeding-lc-ms-on-daidzein-concentration-curve-benchling.pdf)
[23-08-01 LC-MS on Equol Strains and Modular Strains, Homolog Optimization](https://static.igem.wiki/teams/4947/wiki/23-08-01-lc-ms-on-equol-strains-and-modular-strains-homolog-optimization-benchling.pdf)
[23-08-02 Induction and Feeding for Equol in Different Strains Experiment](https://static.igem.wiki/teams/4947/wiki/23-08-02-induction-and-feeding-for-equol-in-different-strains-experiment-benchling.pdf)
[23-08-04 LC-MS of Nissle vs. DH10B Equol Strains and Thorough Inventory](https://static.igem.wiki/teams/4947/wiki/23-08-04-lc-ms-of-nissle-vs-dh10b-equol-strains-and-thorough-inventory-benchling.pdf)

{% endblock %}

## Iteration 1

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### Design

By reading through primary literature, we identified homologs of the seven genes responsible for the biosynthesis of daidzein from p-coumaric acid. The seven genes included 4CL, CHS, CHR, CHI, CPR, IFS/HIS, and HID (Fig. 1a, 1b). We hypothesized that using different combinations of the selected gene homologs could optimize de novo biosynthesis of daidzein, which has never been done before in the heterologous host E. coli. De novo daidzein production can help increase the efficiency of in vitro equol production by removing the need to externally feed daidzein during whole cell reactions for equol production.

We designed entry vectors for each of the gene homologs for use with Golden Gate Assembly (GGA). Each entry vector contained BsaI restriction sites followed by a 4 base pair (bp) overhang on both ends, a ribosome binding domain, a promoter, and the gene homolog. The overhangs were designed to allow for correct assembly via GGA, where the downstream overhang of a gene complements the upstream overhang of the next gene within the daidzein pathway.

We first attempted to assemble the entire nine-part assembly that consisted of one homolog for each of the seven genes, a pZ promoter to initiate transcription of the first gene, and the pCargo backbone to allow for future integration of the plasmid into the E. coli genome. All parts of the assembly were ligated using GGA via BsaI digestion.

### Build

We assembled all of our pathways via GGA. We assembled eleven plasmids that had different combinations of homologs within the entire nine-part assembly. Following the GGA, we electroporated our assembly plasmids into TransforMax EC100D pir+ electrocompetent E. coli.

### Test

We cultured the transformed cells on agar plates containing kanamycin and carbenicillin antibiotics (both resistance markers of which are present in the pCargo backbone) to select for successful transformations. Initially, the first round of cloning yielded no growth, so we repeated the cloning process with some modifications, such as plating on only carbenicillin agar plates, to account for human error. On the repeated attempt, growth was seen only on the cells plated on carbenicillin.

To further test for correct assemblies, we ran PCR screenings on bacterial colonies, as well as the GGA reaction product, for all plates that showed growth using primers that screen for junctions of the gene inserts. Agarose gel electrophoresis (AGE) was then used to image the results of the PCR (Fig. 2). However, none of the AGE results showed the expected amplicon band lengths, suggesting that the assemblies were not successful. For further information, we sent in a select few of the colonies for whole plasmid sequencing based on the AGE results to determine if the pCargo backbone had self-ligated.

### Learn

The whole plasmid sequencing results for the assemblies that were sent showed that the pCargo backbone had self-ligated without any inserts. This explained the bacterial growth on carbenicillin. With no successful assemblies after multiple GGA attempts, it became evident that constructing the large nine-part assemblies was not a viable strategy to assemble the daidzein pathway.

From these results, we switched our strategy to a complete modular approach, while still continuing with Golden Gate cloning. In order to decrease the size of our assemblies, we decided to assemble the modules without the genes outside of each module.

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/notebook/safety-form-lab-photos/figure-1a.png)

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/notebook/safety-form-lab-photos/figure-1b.png)

Fig. 1. a: Biosynthetic pathway of daidzein from the phytochemical precursor p-coumaric acid. There are seven genes of interest within the pathway. b) Table containing list of homologs selected for each of the seven genes involved in converting p-coumaric acid into daidzein.

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/notebook/safety-form-lab-photos/figure-2.jpeg)

Fig. 2. PCR results of GGA product of a nine-part assembly containing the following homologs: At4CL, GmCHS7, GmCHR5, GmCHIB2, CrCPR, Ge2-HIS, and GmHID. Column 1 represents the junction screening of the first three homologs (At4CL, GmCHS7, GmCHR5) and the pZ promoter, with expected amplicon length of 5.3 kb. Column 2 represents the junction screening of the remaining four homologs, with an expected amplicon length of 5.7 kb. Column 3 represents the junction screening of all seven homologs and the pZ promoter, with expected amplicon length of less than 6 kb due to the large amplicon length. Column 4 represents the screening for a section of the pCargo backbone, with an expected amplicon length of 0.6 kb.

## Iteration 2

---

### Design

Based on the results of our initial round of Golden Gate cloning, we adopted a modular assembly approach. We divided the seven genes into three separate modules and assembled the modules through GGA (Fig. 3). The modules were named with the following nomenclature: Module A assemblies have the first three genes (4CL, CHS, CHR), Module B assemblies have the fourth gene (CHI), and Module C assemblies have the last three genes (CPR, IFS/HIS, HID). All modular plasmids were constructed along with the pCargo backbone and pZ promoter (Fig. 4a).

Unlike the original design which tested the complete daidzein production pathway in one plasmid, this modular approach allows for the production of daidzein biosynthesis pathway intermediates. Module A assemblies convert p-coumaric acid to isoliquiritigenin. Module B assemblies convert isoliquiritigenin to liquiritigenin. Module C assemblies convert liquiritigenin to daidzein. We hoped that shifting the focus on daidzein intermediates would provide insight on the specific interactions between different homologs and increase the likelihood of successful assemblies via Golden Gate cloning.

### Build

In order to successfully assemble the modular plasmids through GGA, we needed to alter the 4 bp overhangs of each gene entry vector, as some genes now ligated to different parts of the assembly construct than in the original design (specifically, the first and last genes of every module now ligate to the pZ promoter and pCargo backbone, respectively). To save on time and costs, we decided to design primers that would correctly alter the overhangs, instead of ordering new entry vectors. We conducted PCR amplifications using the designed primers in order to get the altered amplicons of each homolog’s entry vector. Using the homolog amplicons and Twist backbone amplicons, we then ran Gibson Assembly in order to construct the new homolog entry vectors compatible with the modular assembly approach to create stable stocks of the modified genes. We ran GGA using all different combinations of homologs for each module, and we electroporated the plasmids into Transformax EC100D pir+ E. coli.

### Test

We cultured the transformed cells on agar plates containing kanamycin to select for successful transformations. We observed no bacterial growth on the plates. However, to gain more insight on possible ligation issues during assembly, we ran diagnostic PCRs on the GGA reaction products of the modular plasmids. For the PCR, we used primers that screen for all gene inserts within each plasmid. We then ran AGE on the PCR products. Additionally, we ran diagnostic digests in order to test the functionality of our BsaI restriction enzyme.

### Learn

The gel results from the PCR of the GGA products failed to result in the expected corresponding band lengths. Instead, the results showed consistent bands at around 1.8kb, while the expected length was 5 kb for module A, 1.5 kb for module B, and 6kb for module C (Fig. 4b). This band length suggests the self-ligation of the pCargo backbone without any of the gene inserts.

The results of our diagnostic tests indicated complete digestion of the pCargo plasmid by the BsaI restriction enzyme. However, homolog entry vectors did not fully digest from BsaI. Upon further analyses of our gene homolog entry vectors, we discovered that there was a dcm methylation site overlapping the BsaI restriction site on the entry vectors that we ordered. This methylation site explained the partial BsaI digestion of the homolog entry vectors, as well as the previous failed GGA attempts.

With this crucial information, we looked for an alternative cloning technique that would allow us to continue using the entry vectors we had, but without the use of restriction enzymes. Thus, we made the switch to Gibson Assembly (Iteration 3). As a backup, we also worked on GGA using amplicons of the entry vectors (Iteration 4).

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/notebook/safety-form-lab-photos/figure-3.png)

Fig. 3. Modular approach to constructing the daidzein pathway. All possible combinations of homologs for each module were assembled.

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/notebook/safety-form-lab-photos/figure-4a.png)

Fig. 4. a: Junction screenings schematic for the PCR of GGA assembly plasmids showing expected amplicon lengths for successful assemblies. b) Gel electrophoresis results following PCR. All assemblies showed consistent bands at around 1.8kb, consistent with the positive control which results from screening the pCargo backbone itself.

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/notebook/safety-form-lab-photos/figure-4b.jpeg)

## Iteration 3

---

### Design

Based on the unexpected drawback of the methylation site overlapping our BsaI restriction site, we switched our cloning technique from Golden Gate Assembly to Gibson Assembly, which eliminated the need for BsaI digestion and allowed us to continue using our gene entry vectors. We continued with the modular assembly approach to increase chances of successful clones.

However, prior to running Gibson Assemblies, we needed to create amplicons of each gene homolog that contain appropriate homologous regions that overlap with the upstream and downstream genes within the modular assembly constructs. In order to do so, we designed primers that contain an annealing region, as well as a ~20 bp overhang as the homologous region.

### Build

We conducted PCR amplifications to obtain the amplicons of each gene homolog with the appropriate homologous regions, as well as amplicons of the pCargo backbone and pZ promoter. We then DpnI digested and PCR purified the products. Using these amplicons, we ran Gibson Assemblies to construct every combination of homologs for each module. The Gibson Assembly plasmids were then electroporated into Transformax EC100D Pir+ E. coli.

### Test

We cultured the transformed cells on agar plates containing kanamycin and carbenicllin antibiotics to select for successful transformations. We observed growth on all of the plates and ran PCR screenings on several of the bacterial colonies for each modular plasmid. We screened for junctions of the gene inserts for each module, and colonies that yielded bands of the correct length from the junction screen AGE were sent for whole plasmid sequencing (Fig. 5).

### Learn

In order to obtain bacterial colonies with the correct sequences, several colonies had to be picked and tested. Additionally, we ran several colony PCR screenings with different primer pairs in order to obtain correct gel results. Colony PCR screening failures may have risen due to large amplicon bands of over 3kbp (affecting PCR success), but a simpler reason would be due to incorrectly assembled plasmids.

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/notebook/safety-form-lab-photos/figure-5.jpeg)

Fig. 5. Representative results of colony PCR screenings yielding expected results. Module A212 contains genes Gu4CL, GmCHS7, and GuCHR1 with an expected amplicon length of 2.8kb. Module B1 contains gene GmCHIB2 with an expected amplicon length of 2kb.

## Iteration 4

---

### Design

In tandem with our Gibson Assembly approach to construct the modular plasmids, we strategized a method to bypass the dcm methylation site overlapping the BsaI restriction site in order to allow us to use Golden Gate Assembly. While it is not a conventional approach, we decided to run GGA using the amplicons of our entry vectors, instead of using the homolog entry vectors as a whole. By using the amplicons, the methylation is not preserved in the PCR and should allow for proper assemblies. For this approach, we continued with the modular approach to construct the plasmids.

### Build

We designed primers that would amplify upstream of the BsaI restriction site for each homolog entry vector, but ensured that the dcm methylation site would not amplify as well. Additionally, we designed the primers to alter the 4 bp overhangs of the homolog entry vectors in order to facilitate the modular assembly of the daidzein pathway through GGA. Performing PCR amplifications enabled us to move forward with the GGA using the amplicons.

### Test

We cultured the transformed cells on agar plates containing kanamycin to select for successful transformations. We observed growth on all of the plates and ran PCR screenings on several of the bacterial colonies for each modular plasmid. We screened for junctions of the gene inserts for each module, and colonies that yielded correct bands from the AGE were sent for whole plasmid sequencing (Fig. 6).

### Learn

The amplicon-GGA approach yielded a handful of successful assemblies that were sequence verified.

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/notebook/safety-form-lab-photos/figure-6.jpeg)

Fig. 6. Representative results of colony PCR screenings of GGAs through amplicons of homolog entry vectors. Four colonies were picked and screened for each modular plasmid. Module A212 contains genes Gu4CL, GmCHS7, and GuCHR1 with an expected amplicon length of 2.7kb. Module B2 contains GuCHI1 with an expected amplicon length of 1.8kb. Module C111 contains genes CrCPR, GeIFS, and GmHID with an expected amplicon length of 3.2 kb.

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

## Cloning Protocols

---

[Colony PCR Protocol](https://static.igem.wiki/teams/4947/wiki/colony-pcr-protocol.pdf)
[Dpni Digestion of PCR Products](https://static.igem.wiki/teams/4947/wiki/dpni-digestion-of-pcr-products.pdf)
[Drop Dialysis](https://static.igem.wiki/teams/4947/wiki/drop-dialysis.pdf)
[Gel Run Protocol](https://static.igem.wiki/teams/4947/wiki/gel-run-protocol.pdf)
[Gel Validation and Extraction Protocol](https://static.igem.wiki/teams/4947/wiki/gel-validation-and-extraction-protocol.pdf)
[Gibson Assembly Protocol](https://static.igem.wiki/teams/4947/wiki/gibson-assembly-protocol.pdf)
[Golden Gate Assembly Protocol for Using Nebridge Golden Gate Assembly Kit Bsai Hf V2 E1601](https://static.igem.wiki/teams/4947/wiki/golden-gate-assembly-protocol-for-using-nebridge-golden-gate-assembly-kit-bsai-hf-v2-e1601.pdf)
[Heat Shock Transformation for E Coli 10 Beta Competent Cells](https://static.igem.wiki/teams/4947/wiki/heat-shock-transformation-for-e-coli-10-beta-competent-cells.pdf)
[PCR Amplification for Cloning Gel Verification Protocol](https://static.igem.wiki/teams/4947/wiki/pcr-amplification-for-cloning-gel-verification-protocol.pdf)
[PCR Purification](https://static.igem.wiki/teams/4947/wiki/pcr-purification.pdf)
[Qiagen Plasmid DNA Miniprep Kit Protocol](https://static.igem.wiki/teams/4947/wiki/qiagen-plasmid-dna-miniprep-kit-protocol.pdf)
[Restriction Enzyme Digestion Protocol Bamhi](https://static.igem.wiki/teams/4947/wiki/restriction-enzyme-digestion-protocol-bamhi.pdf)
[Electroporation of E. Coli](https://static.igem.wiki/teams/4947/wiki/electroporation-of-e-coli-1.pdf)
[Lucigen Pir+ Electroporation of E. coli](https://static.igem.wiki/teams/4947/wiki/lucigen-pir-electroporation-of-e-coli-1.pdf)

## Daidzein Pathway Feeding Protocol

---

[Daidzein Feeding and Extraction Protocol](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/daidzein-feeding-and-extraction-protocol.pdf)

## Equol Protocols

---

[Conjugation Protocol](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/conjugation-protocol.pdf)

 [L. casei Conjugation](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/l-casei-conjugation.pdf)

 [Landing pad clone screen](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/landing-pad-clone-screen.pdf)

 [Quantification of Equol using HPLC](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/quantification-of-equol-using-hplc.pdf)

## Housekeeping Protocols

---

[Antibiotics Protocol](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/antibiotics-protocol.pdf)

 [Bacterial Glycerol Freezer Stock Protocol](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/bacterial-glycerol-freezer-stock-protocol.pdf)

 [Inoculation + liquid medium Protocol](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/inoculation-liquid-medium-protocol.pdf)

 [Lysogeny Broth (LB) Media Protocol](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/lysogeny-broth-lb-media-protocol.pdf)

 [Oligo Suspension + 10µM stock Protocol](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/oligo-suspension-10-m-stock-protocol.pdf)

{% endblock %}

## Overview of Entire Equol Project

---

### Assembly of Equol Biosynthetic Pathway (From Last Year)

The four genes involved in the equol biosynthetic pathway were obtained from the NCBI database: daidzein reductase (DZNR), dihydrodaidzein racemase (DDRC), dihydrodaidzein reductase (DHDR), and tetrahydrodaidzein reductase (THDR).

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/screenshot-2023-10-12-at-1-43-53-am.png)

A complete plasmid containing all the necessary gene inserts for the expression of the equol biosynthetic pathway was designed using CAD-SGE on Benchling. These genetic elements were inserted into a DNA backbone referred to as pCargo containing ampicillin resistance and several regulatory elements (promoters such as tef and SP6 and terminators such as fd):

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/screenshot-2023-10-12-at-1-44-14-am.png)

The pathway constituted into the above pCargo backbone was derived from the equol biosynthetic pathway in Adlercreutzia equolifasciens, a bacterial species isolated from human feces known to natively express the equol pathway. Attempts to assemble analogous plasmids with the biosynthetic pathway derived from Slackia isoflavoniconvertens and Lactococcus garvieae were unsuccessful.
The process of assembling the pathway derived from A. equolifasciens involved ordering the four gene fragments from IDT and assembling them into entry vector plasmids via Gibson assembly. These entry vector plasmids were then sequence-verified using whole-plasmid sequencing (WPS) with nanopore technology for downstream applications. This process involved the creation of 12 entry vector plasmids, which were subsequently transformed via electroporation into a strain of E. coli with a ΔASD mutation (aspartate-semialdehyde dehydrogenase). The ΔASD mutation created an obligate requirement for diaminopimelic acid (DAP auxotrophy) amongst the transformed E. coli strains, allowing for post-transformation counterselection. DAP is necessary for the biosynthesis of lysine and the construction of peptidoglycan.
Following the assembly and sequence-verification of the entry vector plasmids, the individual gene fragments were amplified by polymerase chain reaction (PCR) in order to assemble these amplicons into the final equol biosynthetic pathway with the backbone pCargo. Initially, these gene assemblies were performed using transformation-associated recombination (TAR) in yeast. TAR assembly involves using homologous recombination of gene “hooks” and linearization by an endonuclease. Following TAR assembly, PCR junction screens, in which primer amplify the junctions between adjacent gene inserts, were used to confirm the fidelity of the assemblies. Those clones that contained all the screened junctions were selected for sequencing by WPS.

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/screenshot-2023-10-12-at-1-44-24-am.png)

Initially, none of the sequenced plasmids were entirely mutation-free, so further experiments were designed to troubleshoot these mutations. Several restriction digests were performed to obtain mutation-free fragments that could be joined to produce a mutation-free plasmid. Two plasmids were used to provide sequence-verified fragments of the gene inserts in the equol biosynthetic pathway, and one other plasmid was used to provide a sequence-verified pCargo backbone.

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/screenshot-2023-10-12-at-1-44-36-am.png)

A landing pad genetic element was then integrated via bacterial conjugation into a strain of E. coli Nissle 1917 and via electroporation into various strains of Lactobacillus. However, the attempts to integrate the landing pad into Lactobacillus were unsuccessful. The landing pad consists of an antibiotic selectable marker, a T7 RNAP circuit, a pT7-GFP/nanoluc reporter, and phiC31 attP sites, and it is integrated into the recipient genomes by transposition (Himar). The landing pad is maintained on the R6K suicide origin of replication and then conjugated via the R6K oriT. In bacteria, the phiC31 integrase integrates the gene inserts into the landing pad at cognate attP sites. Essentially, the landing pad facilitates the integration and expression of genetic elements across diverse bacterial species and across kingdoms (e.g. yeast). Following the integration of the landing pad into the E. coli Nissle strains, clones were screened for landing pad inducibility based on GFP expression. Those clones with greater GFP expression were hypothesized to subsequently have greater expression of the equol biosynthetic pathway.

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/screenshot-2023-10-12-at-1-44-44-am.png)

The genomic integration of the landing pad into E. coli Nissle was generally successful, with 7 clones selected for later integration of the equol pathway. The clones selected and their respective GFP expression are shown below:

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/screenshot-2023-10-12-at-1-44-52-am.png)

The final plasmids assembled, containing the equol biosynthetic pathway and pCargo backbone were named FK5, AE6, AE7-1, AE7-2, AE11, and AE14. The initial sequencing data for these assembled plasmids was as follows:

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/screenshot-2023-10-12-at-1-45-07-am.png)

Based on this data, AE7-1, AE7-2, FK5, and AE11 were further investigated the following year. This decision was made on the basis of minimizing non-synonymous mutations in the equol biosynthetic pathway gene inserts.

### Testing and Optimization of Equol Expression (This Year):

In order to ensure that any mutations in the KanR antibiotic selectable marker were nonfunctional amongst the assembled plasmids in terms of impacting kanamycin or carbenicillin resistance, strains containing the plasmids AE7-1, AE7-2, FK5, and AE11 were plated on kanamycin, carbenicillin, or LB. Additionally, EC100D pir cells containing the pCargo plasmid (Lucigen pir), with both kanR and carbR, were utilized as a positive control across all three conditions, and a Lucigen pir strain with no antibiotic resistance was used as a negative control for the Kan and Carb conditions. All the strains (except the negative control) grew on both Carb and Kan, and all the strains (including the negative control) grew on LB, confirming that any mutations in the kanR selectable marker were nonfunctional and could, therefore, be ignored for downstream experiments. These integrated plasmids were then transformed by electroporation into a DAP auxotrophic recipient strain of E. coli, BW19851.deltaASD. The resulting plasmids were then sequence-verified by WPS:

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/screenshot-2023-10-12-at-1-45-17-am.png)

Based on these WPS sequencing results, AE7-1 was selected for downstream applications as it was the only plasmid without any nonsynonymous mutations in the equol pathway.The mutations in cen4, a yeast regulatory element, were ignored for our purposes of bacterial expression. An assay was subsequently performed on all the above strains in order to determine whether the mutation in the phiC31 integrase was functional and could impede the ability of the plasmid to integrate into the genomic landing pad. This integrase functional assay involved the conjugation of each of the donor plasmids (AE7-1, AE7-2, AE11, FK5, and a positive control strain known to integrate robustly) into the recipient plasmids (E. coli DH10b strain with the landing pad and a negative control without the landing pad). This conjugation was successful, resulting in colonies for each of the experimental conditions and confirming that the mutation in the phiC31 integrase was nonfunctional. Therefore, the plasmids could be further assessed for their ability to express equol.

Equol expression by the recombinant strains was quantified using the analytical technique liquid-chromatography mass spectrometry (LS-MS). The specific machine used at the Yale West Campus Analytical Core was the Agilent LCMS 6120B with 1260 HPLC, which uses electrospray ionization (ESI) to nebulize the LC analyte into the chamber at the atmospheric pressure in the presence of a strong electrostatic field and heated drying gas. The electrostatic field causes the dissociation of the analyte molecules while the heated drying gas causes the solvent in the droplets to evaporate, in turn causing the charge concentration to decrease as the droplets shrink and the ions are desorbed into the gas phase. The ions then pass through a capillary screening orifice before entering the quadropole mass analyzer, which mathematically deconvolutes the data to determine the m/z (mass-to-charge) ratio. The quadropole mass analyzer consists of four parallel rods arranged in a square with the analyte ions directed through the center. Voltages applied by the rods generate electromagnetic fields that allow only ions of specific m/z ratios to pass through the filter at any given time. Scanning mode was used to assess a broader range of m/z ratios, and the LC-MS instrument used allowed for scanning in both positive and negative ion mode. A reverse phase Nova-Pak C18 column was used with a gradient consisting of solvent A (water/acetic acid, 98:2 v/v) and solvent B (water/acetonitrile/acetic acid, 78:20:2, v/v/v) at a flow rate of 1 mL/min from the beginning to 55 min, and 1.2 mL/min from this point to the end. The gradient profile was 0–55 min, 100–20 % A; 55–70 min, 20–10 % A; 70–80 min, 10–5 % A; 80–110 min, 100 % B. Detection was performed by scanning from 210 to 400 nm with an acquisition speed of 1 s. A volume of 5 uL was injected.

A calibration curve was produced for equol by preparing a range of equol samples of various concentrations in acetonitrile solvent via serial dilutions. This calibration curve would allow us to correlate peak intensity on the mass spectrogram to equol concentration in solution for downstream experiments. Equol was measured in positive scanning mode with a m/z ratio of 243.2 and an elution time of ~3.3 minutes.

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/screenshot-2023-10-12-at-1-45-27-am.png)

A similar calibration curve for equol’s isoflavonoid precursor, daidzein, was also established using the same procedure of serial dilutions in acetonitrile. Daidzein was detected in negative scanning mode with an m/z ratio of 255.2 and an elution time of ~3.1 minutes.

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/screenshot-2023-10-12-at-1-45-42-am.png)

Having established a calibration curve for equol, feeding experiments involving the E. coli DH10b strains with the equol biosynthetic pathway (plasmid AE7-1) integrated into the landing pad were performed. Nutrient-rich brain-heart infusion media (BHI) was used to incubate the E. coli DH10b strains overnight at 37 ºC at 220 rpm in culture tubes. These were then backdiluted the next morning in larger flasks and incubated again at 37 ºC until reaching an OD600 in the range of 0.6-1.0. At this point the bacteria were fed solubilized daidzein at various concentrations (in triplicate samples). In order to solubilize the lyophilized daidzein, dimethyl sulfoxide (DMSO) was used. Immediately following the feeding, the bacteria were induced using 1 mM theophylline monohydrate and 50 ng/mL aTc (anhydrotetracycline), which elicit the expression of the integrated equol pathway. The induced samples were incubated overnight at 37 ºC and then centrifuged in order to pellet the cells. The supernatant was sterile filtered and then an organic extraction was performed using ethyl acetate. The organic phase was allowed to evaporate under an air stream overnight, and the remaining gel-like substance remaining was resuspended in acetonitrile in HPLC vials for LC-MS analysis. The results of the feeding experiment in E. coli DH10b testing various daidzein concentrations:

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/screenshot-2023-10-12-at-1-45-57-am.png)

The samples were also assessed for residual daidzein concentration in order to understand the efficiency by which the cells converted daidzein into equol:

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/screenshot-2023-10-12-at-1-46-06-am.png)

Following the success of this first experiment of equol biosynthesis, we investigated the ability of other bacterial strains to express this pathway through integration into the landing pad. We attempted to naturally transform the equol-producing (AE7-1) plasmid into B. subtilis using starvation media. However, our attempts initially failed as we did not observe any colonies of B. subtilis after plating on media. We also attempted to integrate the pathway into E. coli Nissle, E. coli BL21-DE3, and Pseudomonas putida. The following gel demonstrates via junction screening the successful integration of the pathway into P. putida and DH10b (already known positive control), but it fails to show integration for E. coli Nissle, meaning we will have to re-attempt this bacterial conjugation:

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/screenshot-2023-10-12-at-1-46-16-am.png)

### Daidzein Assemblies:

As outlined in the engineering section of our Wiki, the assembly of the daidzein biosynthetic pathway utilized a modularized approach, in which enzymatic steps with relatively stable pathway intermediates were assembled in parallel. The fidelity of these assembly reactions, involving numerous gene inserts, was not perfect, so several technical replicates of each assembly reaction were performed and diagnosed using gel electrophoresis. The following represent our assemblies of different modules via Golden-Gate assembly:

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/screenshot-2023-10-12-at-1-46-26-am.png)

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/screenshot-2023-10-12-at-1-46-35-am.png)

The bands labeled with A (2.7 kb) contained the gene inserts encoding for the coumarate ligase, chalcone synthase, and chalcone reductase enzymes. The bands labeled B (1.8 kb) contained gene inserts encoding the chalcone isomerase enzyme. Finally, the bands labeled C (3.2 kb) contained gene inserts encoding the cytochrome p450 reductase, isoflavonoid synthase, and hydroxyisoflavone enzymes. sacB was a yeast promoter included as part of each of our assembly reactions to facilitate the expression system existing in the strains conjugated using the landing pad technology. The bands labeled sacB contained no gene inserts beyond this sacB promoter, serving as a negative control. The numbers following the letters A, B, and C represent the specific combinations of gene homologs (orthologs from different plant species) assembled for downstream testing of respective functionality in catalyzing daidzein production from p-coumaric acid.

In parallel with these Golden-Gate assemblies, Gibson assemblies were also attempted in order to achieve successful assemblies for those that failed via Golden-Gate:

![PNG Image](https://static.igem.wiki/teams/4947/wiki/notebook/safety-form-lab-photos/screenshot-2023-10-12-at-1-46-50-am.png)

The same organizational, labeling system was used for these Gibson assemblies as for the previously described Golden-Gate assemblies. Between the Gibson and Golden-Gate assemblies, successfully assembled plasmids were obtained for each possible combination of homologs for each module. We are now just beginning to conduct feeding experiments, analogous to those used to quantify equol production (described above), in order to quantify the production of each daidzein pathway intermediate and elucidate the optimal homolog combination for each module.

### Future Directions Summary:

* Conduct feeding experiments for individual modules and select optimal homolog combinations for each
* Construct full pathway by assembling the three modules into a single plasmid and test for daidzein production
* Combine this strain containing the full pathway with the strain expressing the equol pathway to synthesize equol directly from p-coumaric acid
* Begin construction of the p-coumaric acid pathway using plant homologs that convert aromatic amino acids into p-coumaric acid (expressed on a plasmid)
* Improve endogenous malonyl-CoA by overexpressing the pathway (expressed on a plasmid)
* Optimize malonyl-CoA and p-coumaric acid production (while balancing using genetic engineering techniques such as overexpression and gene knockouts) to get an endogenous equol-producing strain
* Optimize daidzein and equol production by optimizing: pH, temperature, media, substrate concentration, time, sugar release, and additives
* Saved for the end after constructing the other pathways since they affect the carbon flux (makes culture conditions optimizations less tractable)
* Test in different strains, including ones that survive in the human gut microenvironment, like E. coli Nissle as a potential live therapeutic
* Test batch feeding experiments and test extraction for production (as opposed to detection)
* Further optimize cell viability
* Optimize feeding experiments to cut cost, improve sustainability
* Characterize mass equol production