iGEM 2024 — Team east-coast-biocrew

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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**March**

3/9: Began settling on main projects and planning a general overview of schedule for the coming months.

**May**

5/4: Narrowed down project idea.

5/11: Began researching chemicals involved in chocolate taste/smell.

5/18: Continued researching chocolate taste/smell chemicals and began researching possible pathways

5/28: Continued discussion on gene pathways. Finalized furaneol and phenylethylamine DNA sequences, looked more into concrete pathways for 2,3,5-trimethylpyrazine and came to the conclusion that 3-ethyl-2,5-dimethylpyrazine would be a more viable option.

**June**

6/4: Finalized genes and assembled list of gene sequences. Decided to use *Lactococcus lactis* over *E. coli* for actual expression of genes due to its relevance to the food industry.

6/8: Ordered gene constructs.

6/25: Designed and ordered primers for amplifying the pNZ8148 vector.

6/29: Ran PCR to amplify the vector in preparation for HiFi assembly for each gene insert. Gel showed 2 bands of unknown length and no visible ladder. Ladder was not vortexed before being inserted into gel, likely causing the lack of visible bands in the ladder. Annealing temperature was set to 63 C, but 3’ ends of primers have a melting temperature of around 60 C, so we planned to repeat at lower annealing temperature.

**July**

7/6: Re-ran PCR on all constructs at lower annealing temperature (55 C) and achieved successful vector linearization for Xmt-Mxmt-Dxmt, PtPDC, and PgPDC.

7/13: Ran HiFi assembly on all genes, including those which did not show a positive band from 7/6 gel electrophoresis as the lack of bands may have been due to a gel loading error. Found nanodrop concentrations and performed Dpn1 digestion in order to remove template DNA from PCR products.

7/14: Repeated PCR and achieved successful amplification for all constructs. The ladder showed up faint using 5 uL, so we plan to increase load to 10 uL for future gels.

7/20: Performed Dpn1 digestion and Hifi assembly on all PCR products.

7/27: Transformed Hifi assembly products into DH5a.

**August**

8/3: After unsuccessful transformations of Hifi assembly products, began a new round of PCR, Dpn1 digestion, and column purification on all gene constructs. Transformed pNZ8148 into *DH5a E. coli* to verify that the lack of growth is not because of issues with chloramphenicol plates (positive controls worked, but were grown on ampicillin plates). Also plated the rest of the transformed DH5a *E. coli* with higher amounts of bacteria.

8/10: Created new chloramphenicol plates at a chloramphenicol concentration of 10 ng/uL instead of the default 35 ng/uL. Transformed 7/20 Hifi assembly products into MC1061 *E. coli* and performed Hifi assembly on 8/3 PCR products. Began PCR reaction for EGFP construct. Gel showed a negative band.

8/16: Re-plated leftover transformed MC1061 cells using 200 uL instead of 100 uL of cell/SOC mixture. Transformation of 8/10 Hifi assembly products into MC1061 and DH5a, ensuring a heat shock time of 45 seconds for MC1061 cells instead of 30 seconds.

8/17: Growth on DH5a plates from 8/16 transformations. Colony PCR on 10 colonies from the PgPDC plate produced negative bands.

8/21: Ran colony PCR on 8/16 transformation growth and found 5/7 bands positive for Xmt-Mxmt-Dxmt and 1/7 bands positive for TDH-KBL (All others were negative). Transformed 8/10 Hifi assembly products into MC1061. Performed Dpn1 digestion and column purification on EGFP PCR products. After column purification, concentration of PCR product ended up around 8.5 ng/uL (not high enough for Hifi assembly), so sample was lyophilized for future resuspension in lower volume to ensure higher concentration. Also plated more leftover MC1061 transformed cells.

8/23: Found growth on plates from 8/21 and ran Colony PCR on colonies from original DH5a plates (for gene constructs SlPDC, PtPDC, and PgPDC, performed colony PCR on 20 colonies. For gene construct FBA-TPI, performed colony PCR on 5 colonies from DH5a and 15 colonies from MC1061 plates).

8/24: Ran colony PCR on gene constructs Xmt-Mxmt-Dxmt and TDH-LBL from 8/23 MC1061 cell growth. Made cell master plate for FBA-TPI MC1061 plate. Made liquid cultures for 4 FBA-TPI positive colonies from MC1061 plate in preparation for column purification. Performed a new round of PCR on all PDC gene constructs (as no successful amplification in *E. coli* had resulted for any of them). Transformed leftover Hifi assembly products of PDC genes, trying both 1 uL and 5 uL of DNA for transformations.

8/27: Performed Dpn1 treatment and column purification on all PDC PCR products. Resulting concentrations were too small, so PCR products were lyophilized for future resuspension in lower volume to ensure higher concentration for Hifi assembly. Performed plasmid prep for FBA-TPI, but resulting concentration was way too low (~5 ng/uL), likely because the *E. coli* was cultured in 1 mL media instead of 5 mL and we let E coli grow for 3 days until starting plasmid prep. Performed Hifi assembly on EGFP and transformed into MC1061 *E. coli* using 2 different DNA amounts (2 uL and 3 uL).

8/31: Plates from 8/27 transformation of assembled EGFP did not grow. Resuspended lyophilized PDC genes to ensure DNA higher concentration than previously.

**September**

9/1: Performed plasmid prep on Xmt-Mxmt-Dxmt, TDH-KBL, and FBA-TPI from positive MC1061 colonies and recorded Nanodrop concentrations.

9/7: Made electrocompetent *L. lactis* cells. Made and filter sterilized wash/electroporation solution from 0.5 M sucrose solution and 10% glycerol. Because of strange pink color of the presumed *L. lactis* cells, it was concluded that the cells were likely contamination. Performed Hifi assembly on EGFP construct. Transformed all genes into MC1061 *E. coli*. Performed colony PCR on genes to verify primer binding (since some purified plasmid samples sent for sequencing were unable to bind to primer).

9/12: Re-transformed all gene constructs after finding limited growth on plates from 9/7 round of transformations. Ran PCR on purified plasmid samples in order to re-confirm their successful assembly. Gel showed positive bands for inserts, suggesting successful assembly.

9/14: Found no growth from 9/12 transformations. Made new 100ug/ml ampicillin plates.

9/18: Recorded OD600 of *L. Lactis* growth in 2 different mediums: LB and M17. Made competent *L. Lactis* cells. Made 8 liquid cultures for Xmt-Mxmt-Dxmt, TDH-KBL, and FBA-TPI from original plates in preparation for plasmid prep.

9/21: Performed plasmid prep on FBA-TPI. Performed electroporation of Xmt-Mxmt-Dxmt, TDH-KBL, and FBA-TPI into *L. lactis*.

9/27: Found no growth from 9/21 electroporations. Received fixed plasmid (without RepC mutation) and performed PCR on fixed plasmid.

9/28: Performed Dpn1 digest on 9/27 PCR products.

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Design, Build, Test, Learn, Repeat{% endblock %}
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# Design

## Selection of Chassis and Plasmid

Our goal for this project was to genetically modify an organism to produce chocolate compounds in order to combat the climate and labor issues related with chocolate production. When we originally researched the pathways needed to create these compounds, we were debating between choosing *E. coli* or *Saccharomyces cerevisiae*, as many of the papers we read had successfully created these compounds in those species.

However, we ended up choosing *Lactococcus lactis cremoris* (CCUG 21954, ATCC 9625, NCTC 7019, LMG 7932, IFO 3427) as the host organism when we found that one of our compounds could be produced in *L. lactis*, specifically 4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) ([Hayashida et al. 2001](https://www.sciencedirect.com/science/article/pii/S1389172301801213)). Furthermore, we chose *L. lactis* because it has a long-standing reputation for being food-safe, producing many of the dairy products we enjoy such as cheese, butter, and buttermilk ([Madera et al. 2003](https://www.sciencedirect.com/science/article/abs/pii/S0168160503000424?via%3Dihub)). Additionally, the pathways for the production of other chocolate compounds documented in *E. coli* had common precursors presumed to also be present in *L. lactis* ([Xu et al. 2020](https://pubs.acs.org/doi/10.1021/acs.jafc.0c01706)).

We chose our plasmid, pNZ8148, because nisin, which induces our promoter, is also commonly used in the food industry ([Zhang et al. 2024](https://pubs.acs.org/doi/10.1021/acschembio.3c00577)). Nisin is a broad-spectrum bacteriocin produced by *L. lactis* used extensively as a food preservative ([O’Reilly et al. 2023](https://www.nature.com/articles/s41598-023-34586-x)). The combination of pNZ8148 and the use of *L. lactis*, on top of the purification of our compound, would ensure that our product would pose a lower risk to human consumption.

![Diagram of pNZ8148 Vector](https://static.igem.wiki/teams/5272/engineering-success-pics/pnz8148-vector-diagram.png)

Fig. 1: a diagram of our chosen pNZ8148 plasmid, containing the PnisA promoter, chloramphenicol resistance, a repC and a repA region.

## Design of Pathways

We created four pathways for the production of four chocolate compounds: 2-Phenylethylamine, theobromine, 4-Hydroxy-2,5-dimethyl-3 (2H)-furanone (HDMF) (also known as the trademark name Furaneol), and 3-ethyl-2,5-dimethylpyrazine. Each coding region is preceded by a ribosome binding site (either BBa\_B0032 or BBa\_J34801), which is further preceded by the inducible nisin promoter (BBa\_K4307009). Downstream of the coding region is the terminator found inside of the original pNZ8148 plasmid (Accession CP068658.2, Region 399302 - 399354).

![Synthesis pathway for 2-phenylethylamine](https://static.igem.wiki/teams/5272/engineering-success-pics/2pe-synthesis-diagram.png)

Fig. 2: Pathway for the production of 2-phenylethylamine. Phenylalanine decarboxylase (PDC) is an enzyme which catalyzes the production of 2-phenylethylamine and carbon dioxide from the amino acid phenylalanine. The PDC coding region is placed in between the nisin promoter, which is inducible with the biochemical nisin, and a terminator ([Xu et al. 2020](https://pubs.acs.org/doi/10.1021/acs.jafc.0c01706)).

Even though there’s only one PDC pathway, we had three different amino acid sequences which all coded for PDC from three different organisms: PDC from *Psychroflexus gondwanensis*, *Psychroflexus torquis*, and *Solanum lycopersicum*.

![Synthesis pathway for theobromine](https://static.igem.wiki/teams/5272/engineering-success-pics/tbr-synthesis-diagram.png)

Fig. 3: Pathway for the production of Theobromine. The enzyme 7-methylxanthosine synthase (XMT) catalyzes the reaction of xanthosine into 7-methylxanthosine. The coenzyme S-adenosyl-L-methionine (SAM) is also turned into S-adenosyl-L-homocysteine (SAH) in the function of the XMT enzyme. Once creating 7-methylxanthosine, XMT can once turn 7-methylxanthosine into 7-methylxantine using hydrolysis to split 7-methylxanthosine and water into 7-methylxantine and a ribose sugar. Upstream of the XMT coding region are the nisin promoter and a RBS. Before the next coding region, a 15 base pair spacer and another RBS are attached. The next coding region is the enzyme theobromine synthase (MXMT) which can turn 7-methylxantine into theobromine using the same SAM coenzyme. Downstream of the MXMT coding region is the terminator ([Leibrock et al. 2022](https://link.springer.com/article/10.1007/s10068-022-01082-3)). \*note: while only XMT and MXMT enzymes are required to synthesize theobromine, we accidentally included an extra DXMT enzyme which would have turned our theobromine into caffeine. We are working on fixing this issue in the future.

![Synthesis pathway for HDMF](https://static.igem.wiki/teams/5272/engineering-success-pics/hdmf-synthesis-diagram.png)

Fig. 4: Pathway for the production of 4-Hydroxy-2,5-dimethyl-3 (2H)-furanone (HDMF). Fructose-bisphosphate aldolase (FBA) splits fructose-1,5-bisphosphate into the triose phosphates dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Dihydroxyacetone is the preferred product, as it can be turned into furaneol by a not-fully understood metabolic pathway ([Pérez et al. 1999](https://pubs.acs.org/doi/10.1021/jf980404z)). The triose-phosphate isomerase (TPI) enzyme further increases the yield of furaneol by creating more dihydroxyacetone phosphate from glyceraldehyde 3-phosphate. The nucleotide sequence that codes for these enzymes is structured very similarly to previous pathways. The nisin promoter precedes, with an RBS, the first coding region (FBA), a 15-base pair spacer, a second RBS, the second coding region (TPI), and a terminator.

![Synthesis pathway for pyrazine](https://static.igem.wiki/teams/5272/engineering-success-pics/edmp-synthesis-diagram.png)

Fig. 5: L-threonine 3-dehydrogenase (TDH) converts the amino acid threonine to 2-amino-3-ketobutyrate (AKB) and AKB is metabolized to glycine by the acetyltransferase activity of 2-amino-3-ketobutyrate coA ligase (KBL) in usual conditions. However, in poor nutrition, at low CoA concentrations, and when the enzyme is overloaded, unstable AKB is gradually decarboxylated to aminoacetone (red) and the lyase activity of KBL decomposes threonine to acetaldehyde and Gly. 3-ethyl-2,5-dimethylpyrazine is synthesized chemoenzymatically via the condensation reaction of two molecules of aminoacetone and one molecule of acetaldehyde. Despite 3-ethyl-2,5-dimethylpyrazine not being the major product of the reaction, yields have been reported as high as 20.2% under normal conditions (not explicitly creating conditions with poor nutrition or low CoA concentrations) ([Motoyama et al. 2021](https://www.nature.com/articles/s42004-021-00545-8)). The structure of the nucleotide is as follows: the nisin promoter precedes, with an RBS, the first coding region (TDH), a 15 base pair spacer, a second RBS, the second coding region (KBL), and a terminator.

In addition to these pathways, the nisin signal transduction pathway found in our nisin promoter is as follows:

![NICE system](https://static.igem.wiki/teams/5272/engineering-success-pics/nice-system.png)

Fig. 6: The nisin-controlled gene expression (NICE) system in L. lactis. Nisin first binds to NisK, a membrane-bound receptor protein. NisK autophosphorylates and transfers a phosphate group to NisR, which activates the protein. Once activated, NisR can interact with the pNisA promoter, turning on expression of the gene. The gene is transcribed into mRNA, where it can then be translated into our chocolate-producing enzymes via free-floating ribosomes ([Bakari et al. 2014](https://www.researchgate.net/publication/265905327_Lactococcus_lactis_Recent_Developments_in_Functional_Expression_of_Membrane_Proteins)).

# Build

We successfully assembled our vectors inside of pNZ8148. We did this through HiFi assembly. We first designed primers specific to each pathway which would amplify pNZ8148 with a 5’ overhang homologous to the 5’ end of the coding region. For example, for the TDH-KBL pathway (Fig. 5) a reverse primer would have homology with the 5’ end of the TDH coding region and a forward primer would have homology with the 5’ end of the KBL coding region (see Fig. 7).

![Primer diagram](https://static.igem.wiki/teams/5272/engineering-success-pics/primer-diagram.png)

Fig. 7: A diagram showing the positioning of primers. A reverse primer amplifies pNZ8148 and includes a 20-base pair 5’ homology with the start of the first coding region. A forward primer would do the same at the end of the KBL gene in the TDH-KBL pathway.

This 20 base pair overhang is crucial for HiFi assembly. 3 enzymes constitute HiFi assembly: 5’ exonuclease, DNA polymerase, and DNA ligase. 5’ exonuclease chews back more of the 5’ end strand to create a longer overhang so that the two fragments anneal together, DNA polymerase fills in the gaps, and DNA ligase seals any nicks and covalently links the DNA fragments together.

In order to prepare the PCR products for HiFi assembly for higher efficiencies, two additional steps were taken: DpnI to digest methylated DNA, and column purification to further purify our DNA.

Once the plasmid was constructed, we first cloned the plasmid inside of *E. coli*. Then, we could extract the plasmid and transform it into our final host organism, *L. lactis*. Only then could we induce the plasmid by adding nisin to the media and start producing our chocolate compounds.

# Test

Unfortunately, we had many issues with cloning, both in *E. coli* and *L. lactis*.

We first tried cloning inside of DH5a *E. coli* cells. While each gene construct ended up growing up on an LB + chloramphenicol plate (Fig. 8), when we performed colony PCR, hardly any colonies were positive for our gene insert (Fig. 9).

![Plated transformed colonies](https://static.igem.wiki/teams/5272/engineering-success-pics/plated-transforms.png)

Fig. 8: Colonies of our transformed genes in dh5a cells. We see visible colonies in genes 1, 2, 3, 4, 6, and 7 in diagrams a), b), c), d), e), and f), respectively. Gene 5 is omitted due to a labeling error early on. All of our genes have colonies.

![Result of colony PCR on transformed cultures](https://static.igem.wiki/teams/5272/engineering-success-pics/colony-pcr-1.png)

Fig. 9: Colony PCR results. 7 colonies were picked from each of our 6 gene constructs. Gel a) has gene #1 on the left of the middle ladder, with gene #2 on the right of the middle ladder. Gel b) has gene #3 on the left and gene #4 on the right. The left gel in diagram c) is gene #6, and the right gel is gene #7. The genes correspond to: 1) SlPDC, 2) XMT-MXMT-DXMT, 3) TDH-KBL, 4) PtPDC, 6) FBA-TPI, 7) PgPDC.

We had successful insertion and cloning of gene #2 (five out of seven with a positive result), gene #3 (one out of seven), and #4 (one out of seven). However, we also learned that our plasmid required cloning inside of recA+ strains of *E. coli*, and DH5a competent cells lack the recA protein. The lack of recA results in plasmid instability and the potential deletion of the chloramphenicol resistance gene ([NICE Expression Handbook](https://www.mobitec.com/media/mobitec/old_content/NICE_Expression_System-Handbook.pdf) and [Volff et al. 1997](https://pubmed.ncbi.nlm.nih.gov/9079935/)). Therefore, we moved all future cloning attempts to be strictly inside of MC1061 cells.

![Plated clones of transformed cells](https://static.igem.wiki/teams/5272/engineering-success-pics/plated-clones.png)

Fig. 10: Cloning results in MC1061 cells. Only three genes grew: XMT-MXMT-DXMT (a), TDH-KBL (b), and FBA-TPI (c).

![Result of colony PCR on clones](https://static.igem.wiki/teams/5272/engineering-success-pics/colony-pcr-2.png)

Fig. 11: Colony PCR results for our MC1061 and DH5a cloning. The top 2 gels in (a) as well as the first 6 non-ladder lanes on the bottom left gel were taken from 20 DH5a PtPDC colonies. No colonies show positive results. The last lane in the bottom left gel in (a), the bottom right gel in (a), and the first 7 lanes in the top gel in (b) show positive results for 15/15 colonies MC1061 colonies with the FBA-TPI gene. The rest of the lanes in (b) indicate negative colonies for DH5a genes FBA-TPI and PgPDC. The left gel in (c) shows the gene XMT-MXMT-DXMT in MC1061. We hypothesize that the gel either punctured or the DNA stain wasn’t mixed properly which resulted in the empty middle. The right gel in (c) is the TDH-KBL gene in MC1061.

The colony PCRs show significant improvement of MC1061 compared to DH5a. While DH5a resulted in 0/45 colonies, MC1061 had positive results in every single lane (excluding the mishap on the left gel in Fig. 11 (c)). All of our positive colony PCR results in this latest round came from MC1061, indicating plasmid stability inside of MC1061.

However, cloning inside of MC1061 was far from perfect. Despite attempting transformation six different times, only one attempt resulted in colonies that grew. This highlights our underlying issues with the replication of our plasmid inside of *E. coli*. After growing liquid cultures from the one successful attempt and purifying that plasmid, we attempted transformation into *L. lactis*. Unfortunately, nothing grew.

# Learn

In addition to realizing the lack of plasmid stability in the DH5a recA- strain, there was an underlying issue with the overall replication and viability of our plasmid in both *E. coli* and *L. lactis*. After contacting NovoPro labs, the supplier of our plasmid, we determined that there was a mutation within the repC region. NovoPro labs conducted three generations of full-length sequencing on the pNZ8148 to find the mutation. This could be one of the major reasons why we’ve been having a lot of trouble cloning inside of both *E. coli* and *L. lactis*. They have since sent us a plasmid with the fixed repC region, and we plan to repeat many of our experiments with the fixed plasmid.

# Design Round 2

Going back to square one, we started on using re-building our construct with the fixed plasmid. We took this time to address many of our previous issues as well. Firstly, we designed new primers that would add BioBrick assembly prefixes and suffixes in order to make them BioBrick compatible. We also designed primers to address removing the DXMT enzyme (see the note in Fig. 3). Finally, we added a pathway for the expression of EGFP in order to characterize both the strength of our promoter (we would see EGFP expression at varying concentrations of nisin) and to check for any unwanted promoter expression when it is not being induced, as it is possible that our promoter is not fully repressed in *E. coli* ([NICE Expression Handbook](https://www.mobitec.com/media/mobitec/old_content/NICE_Expression_System-Handbook.pdf)).

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

### **Smell Testing:**

**Pyrazine**

* Add 200 mL of deionized water into a beaker.
* Add 1 milligram of the Pyrazine powder into the beaker.
* Mix well.
* In order to smell the compound, place it upright and cup your hand. Waft the vapor with the cupped hand and smell.

**Theobromine**

* Add 200 mL of deionized water into a beaker.
* Add the stir bar and turn the knob to an appropriate speed.
* Add 2 grams of Theobromine powder into a beaker.
* Mix well.
* In order to smell the compound, place it upright and cup your hand. Waft the vapor with the cupped hand and smell.

**Phenylethylamine**

* Add 200 mL of deionized water into a beaker.
* Add the stir bar and turn the knob to an appropriate speed.
* Add 200 mg of Phenylethylamine powder to the beaker.
* Mix well.
* In order to smell the compound, place it upright and cup your hand. Waft the vapor with the cupped hand and smell.

**Furaneol**

* Add 200 mL of deionized water into a beaker.
* Add the stir bar and turn the knob to an appropriate speed.
* Add 200 mg of Furaneol powder to the beaker.
* Mix well.
* In order to smell the compound, place it upright and cup your hand. Waft the vapor with the cupped hand and smell.

**To Create a Full Solution:**

Combine all compounds in the same ratio, except keep the amount of water the same.

### **PCR for vector amplification:**

PCR Reaction for a 25 uL reaction:

* In a PCR tube, add 12.5 uL of Phusion Hot Start Flex 2x Master Mix or Q5 High-Fidelity DNA Polymerase
* Add in 1.25 uL each of the forward and reverse primer at 10uM
* Add 20ng of template DNA, so in our case, 4uL at 5ng/uL of PNZ8148
* Adding enough water to get to 25uL (6uL of water)
* Run in the thermocycler with an initial denature at 98C for 30 seconds, then repeat the following cycle 35 times (denature at 98C for 10 seconds, anneal at 56C for 30 seconds, extend at 72C for 90 seconds). At the end of the cycles, hold at 72C for 5 minutes and then drop down to 4C forever.

### **Gel Electrophoresis**

Preparing the gel:

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

Preparing the Sample and running gel:

* On a piece of parafilm, spot 2 ul of DNA loading dye with 10 uL of PCR product and mix for each sample
* Transfer each mixed sample into wells
* Ensure that appropriate DNA ladder is inserted into well
* Run gel at 100V for ~20-30 minutes and view under UV light

### **Dpn1 Digestion**

**Overview**

* The objective of **DpnI digestion** is to selectively cleave methylated DNA, specifically recognizing and destroying DNA that was in bacteria. This is commonly used in molecular biology, particularly cloning, for the following purpose:
  + **Elimination of Template DNA**: After a PCR or mutagenesis reaction, DpnI digestion removes the parental (template) DNA, which is typically methylated if it was propagated in a bacterial strain like *E. coli* that methylates specific residues.
* After Dpn1-mediated digestion, it is believed that template DNA (methylated) will be cleaved, leaving only the intended PCR product.

**Materials:**

* 8uL PCR product (DNA)
* 1uL 10x rCutSmart Buffer
* 1 uL Dpn1

**Procedure:**

* Combine the PCR product, buffer, and Dpn1 enzyme into a 1.5 mL Eppendorf tube.
* Incubate at 37 C for 30 minutes
* Heat at 80 C for 20 minutes

### **Column Purification**

StrataPrep PCR Purification Kit
**Materials:**

* DNA-binding solution
* Wash buffer
* Microspin cups
* Receptacle tubes (2 ml)
* Microcentrifuge tubes
* Microcentrifuge

**Procedure:**

* Add a volume of DNA-binding solution equal to the volume of the aqueous portion of the PCR product to the microcentrifuge tube containing the PCR product and mix the two components.
* Using a pipet, transfer the PCR product–DNA-binding-solution mixture to a microspin cup that is seated in a 2-ml receptacle tube. Snap the cap of the 2-ml receptacle tube onto the top of the microspin cup.
* Spin the tube in a microcentrifuge at maximum speed for 30 seconds.
* Open the cap of the 2-ml receptacle tube, remove and retain the microspin cup, and discard the DNA-binding solution.
* Open the cap of the 2-ml receptacle tube and add 750 μl of 1× wash buffer to the microspin cup. Snap the cap of the receptacle tube onto the top of the microspin cup.
* Spin the tube in a microcentrifuge at maximum speed for 30 seconds.
* Open the cap of the 2-ml receptacle tube, remove and retain the microspin cup, and discard the wash buffer.
* Place the microspin cup back in the 2-ml receptacle tube and snap the cap of the receptacle tube onto the microspin cup.
* Spin the tube in a microcentrifuge at maximum speed for 30 seconds. On removal from the centrifuge, make sure that all of the wash buffer is removed from the microspin cup.
* Transfer the microspin cup to a fresh 1.5-ml microcentrifuge tube and discard the 2-ml receptacle tube.
* Add 50 μl of elution buffer directly onto the top of the fiber matrix at the bottom of the microspin cup. Avoid touching the fiber matrix with the pipet tip.
* Incubate the tube at room temperature for 5 minutes.
* Snap the cap of the 1.5-ml microcentrifuge tube onto the microspin cup and spin the tube in a microcentrifuge at maximum speed for 30 seconds.
* Open the lid of the microcentrifuge tube and discard the microspin cup. The purified PCR product is in the bottom of the 1.5-ml microcentrifuge tube. Snap the lid of the microcentrifuge tube closed to store the purified PCR product.

### **HiFi assembly**

* Setup the following reaction on Ice:

![Reccomended Amounts of Fragments used for Assembly](https://static.igem.wiki/teams/5272/protocols/hifi1.png)
***Note:** Optimized cloning efficiency is 50–100 ng of vector with 2-fold excess of each insert. Use 5-fold molar excess of any insert(s) less than 200 bp. Total volume of unpurified PCR fragments in the assembly reaction should not exceed 20%. To achieve optimal assembly efficiency, design 15-20 bp overlap regions between each fragment.*

* Incubate samples in a thermocycler at 50°C for 15 minutes (when 2 or 3 fragments are being assembled) or 60 minutes (when 4–6 fragments are being assembled). Following incubation, store samples on ice or at –20°C for subsequent transformation.

  **Note:** *Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).*

### **Heat Shock Transformation in *E. coli***

**Overview:**

The purpose of this protocol is to transform E. Coli to take up our plasmid DNA.

**Materials:**

* Frozen competent cells stored at -80C (25ul)
* Plasmid DNA
* Ice bucket
* 42C water bath
* SOC media
* Selection Plates
* Incubators
* Microfuge tubes

**Procedure:**

* Place a tube of 25 μL of cells on ice until thawed.
* Add 2 microliters of plasmid. Flick to tube 4-5 times to mix without vortexing.
* Place the mixture on ice for 30 minutes without mixing.
* Heat shock the mixture at 42C for exactly 30 seconds for DH5a cells, and 45 seconds for MC1061 cells.
* Place back on ice for 5 minutes. **Do not mix.**
* Add 475 μL of room temperature SOC to the mixture.
* Shake at 37 C for 120 minutes at 250rpm
* Warm selection plates to 37 C.
* Spread 100 μL of each dilution onto a selection plate and incubate overnight or for multiple days at 37 C.

### **Colony PCR**

**Materials:**

* 10uM primer VF2
* 10uM primer VR
* 2X Q5 HotStart Master Mix
* Distilled water
* Agar plate with bacterial colonies
* PCR tubes
* Toothpicks
* Collecting Bacterial Cultures + Reaction Setup:

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

* Take a toothpick and mark out 10 colonies that you want to analyze.
* Lightly touch the toothpick onto the colonies, dip into 100uL of water.
* Use 1uL of that water/cell concentration for template DNA for the rest of the PCR reaction.
* PCR reaction:
  + Used 2x PCRBIO HS Taq Mix Red
  + 12.5uL of 2x Taq
  + 1uL of Forward primer
  + 1uL of Reverse primer
  + 1uL of template DNA
  + 9.5uL of deionized water
* Run samples in thermocycler: 35 cycles at 56°C annealing temperature, 2 minutes for extension time

### **SM17 Medium Preparation**

**Overview:**

This protocol was used to prepare SM17 media, a version of M17 media that was supplemented with Sucrose for optimal growth of *L. Lactis,* acting as a carbon source for the bacteria. This protocol makes 1L of media.

**Materials:**

* M17 Broth Powders
* Sucrose
* Water
* Plates
* Bottles
* Agar
* Autoclave Tape

**Procedure:**

* Suspend 42.25 grams of M17 broth powder in 1000 ml of purified/distilled water.
* 171g of Sucrose was added to bring the solution to 0.5M.
  + Optional: If preparing plates, 15g of Agar could be added to make 1L worth of plates. If making plates, 20 ml of media should be used for each, totalling 50 plates per liter of media.
* The solution was separated into bottles of equal volume.
* Autoclave tape was added to all the bottles.
* The media was autoclaved at 15 lb of pressure (121 celsius) for 15 minutes.

### **Plasmid Prep**

**Overview:** The objective of miniprep was to extract plasmid DNA from E. coli.

**Materials/Reagents:**

* All those are included in the kit.

**Procedure:**

* Pellet 1ml of cells.
* Resuspend the pelleted cells in 250 µL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
  + **Note.** Ensure RNase A has been added to the Resuspension Solution
* Add 250 µL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.
  + **Note.** Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.
* Add 350 µL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times.
  + **Note.** It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate should become cloudy.
* Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
* Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate.
  + **Note. Close the bag with GeneJET Spin Columns tightly after each use!**
* Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
  + **Note.** Do not add bleach to the flow-through.
* **(For EndA+ strains which have high level of nuclease activity only - optional)**: Wash the GeneJET spin column by adding 500 µL of Wash Solution I (#R1611, diluted with isopropanol) and centrifuge for 30-60 sec. Discard the flow-through.
  + **Note.** This step is essential to remove trace nuclease activity.
* Add 500 µL of the Wash Solution (diluted with ethanol prior to first use as described on p.3) to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
* Repeat the wash procedure (step 8) using 500 µL of the Wash Solution.
* Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
* Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 µL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.
  + **Note.** An additional elution step (optional) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%. For elution of plasmids or cosmids >20 kb, prewarm the Elution Buffer to 70°C before applying it to silica membrane.
* Discard the column and store the purified plasmid DNA at -20°C.

### **Lactococcus Lactis electroporation and competent cell prep**

Using Rice IGEM 2018 Protocol

**Making electrocompetent cells:**

* Grow cells overnight at 30 °C to an O.D620 of 0.7.
* Wash twice with ice-cold washing solution.
* Resuspend cells in 1/100 volume of electroporation solution.
* Keep on ice.

**Electroporation of cells:**

* Add 0.25 µg plasmid DNA (in water) to 100 µl of electrocompetent cells.
* Homogenize by gently mixing with pipette several times.
* Transfer mixture into cuvette.
* Wipe moisture from the cuvette and insert the cuvette into the device.
* Electroporation:

| Mode | Prokaryotes |
| --- | --- |
| Voltage (V) | 2000 volts |
| Time Constant (T) | 5 minutes |

* Add 1 ml ice-cold complex medium, incubate 2 hours at 30 °C.
* Plate diluted cells on selective chloramphenicol plates.
* Incubate 2 days at 30 °C.

**Expected results:**
Transformation efficiency up to 1.4 x 106 transformants/µg of DNA.

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{% block lead %}Our achievements and future plans{% endblock %}
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# Cloning results in DH5a

We started all our initial transformations in DH5a strains of *E. coli*. Cloning in DH5a was never consistent, except for the positive controls. This suggested that something was wrong with our plasmid inside of DH5a.

Of the few transformations that were successful, such as (Fig. 1), we still had issues. When performing Colony PCR on these colonies, hardly any had actually taken up the plasmid (Fig. 2). While 5 out of 7 colonies tested for XMT-MXMT-DXMT plate and 1 out of 7 colonies tested for TDH-KBL plate, all the other genes were negative. Additionally, we had a hard time growing these colonies, potentially suggesting that they had somehow lost the plasmid entirely.

![Plated transformed colonies](https://static.igem.wiki/teams/5272/engineering-success-pics/plated-transforms.png)

Fig. 1: Colonies of our transformed genes in dh5a cells. We see visible colonies in genes 1, 2, 3, 4, 6, and 7 in diagrams a), b), c), d), e), and f), respectively. Gene 5 is omitted due to a labeling error early on. All of our genes have colonies.

![Result of colony PCR on transformed cultures](https://static.igem.wiki/teams/5272/engineering-success-pics/colony-pcr-1.png)

Fig. 2: Colony PCR results. 7 colonies were picked from each of our 6 gene constructs. Gel a: SIPDC left of middle ladder, Xmt-Mxmt-Dxmt right of middle ladder. Gel b: Tdh-Kbl left of middle ladder, PtPDC right of middle ladder. Gel c: FBA-TPI left gel, PgPDC right gel.

We eventually found out that the reason for the low plasmid stability over generations was due to our selection of a RecA- strain of *E. coli* such as DH5a ([Duarte et al. 2021](https://www.mdpi.com/1422-0067/22/3/1379)). The lack of the RecA protein can especially make the chloramphenicol resistance gene susceptible to deletion ([Volff et al. 1997](https://pubmed.ncbi.nlm.nih.gov/9079935/)). Therefore, we switched to a RecA+ strain of *E. coli* such as MC1061.

# Cloning results in MC1061

We found much more successful cloning in MC1061 compared to DH5a. Following a 3-day incubation at 30 C, MC1061 *E. coli* colonies with XMT-Mxmt-Dxmt, TDH-KBL, and FBA-TPI genes grew (Fig 3).

![Plated clones of transformed cells](https://static.igem.wiki/teams/5272/engineering-success-pics/plated-clones.png)

Fig 3: Cloning results in MC1061 cells. Only three genes grew: XMT-MXMT-DXMT (a), TDH-KBL (b), and FBA-TPI (c).

When performing colony PCR on these colonies, 15 out of 15 of all tested colonies of FBA-TPI showed successful insertion, 7 out of 7 of all tested colonies of TDH-KBL showed successful insertion, and 2 out of 7 of all tested colonies of XMT-Mxmt-Dxmt showed successful insertion (5 negative bands likely attributed to gel loading error). Transformations in MC1061 *E. coli* showed a much higher rate of positive colonies than transformations in DH5a *E. coli* (Fig. 4).

![Result of colony PCR on clones](https://static.igem.wiki/teams/5272/engineering-success-pics/colony-pcr-2.png)

Fig. 4: Colony PCR results for our MC1061 and DH5a cloning. The top 2 gels in (a) as well as the first 6 non-ladder lanes on the bottom left gel were taken from 20 DH5a PtPDC colonies. No colonies show positive results. The last lane in the bottom left gel in (a), the bottom right gel in (a), and the first 7 lanes in the top gel in (b) show positive results for 15/15 colonies MC1061 colonies with the FBA-TPI gene. The rest of the lanes in (b) indicate negative colonies for DH5a genes FBA-TPI and PgPDC. The left gel in (c) shows the gene XMT-MXMT-DXMT in MC1061. We hypothesize that the gel either punctured or the DNA stain wasn’t mixed properly which resulted in the empty middle. The right gel in (c) is the TDH-KBL gene in MC1061.

This higher insertion success rate in MC1061 cells than DH5a cells is likely due to the presence of a presence of the recA protein in MC1061 cells. The recA gene aids in the recombination of homologous DNA, but is engineered to be dysfunctional in DH5a cells in order to prevent unwanted recombination of the plasmid. However, the PNZ8148 plasmid requires the function of the recA gene in *E. coli* for proper amplification, so MC1061 *E. coli* being recA+ is likely the cause for the higher rate of positive colonies in MC1061 than DH5a ([Azizpour et al. 2017](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5650732/)).

Additionally, colonies grew much better for MC1061 cells when incubated at 30 degrees celsius compared to 37 degrees celsius. Initial cloning of assembled PNZ8148 in both DH5a and MC1061 cells with incubation at 37 degrees celsius only produced growth on the DH5a plates. We know transformation worked due to the fact that we were able to verify some colonies with colony PCR. Therefore, it must have been a lack of cloning efficiency that caused low growth in MC1061 cells. When repeated with MC1061 cells at 30 degree incubation, colonies grew (Fig. 3).

# Cloning conclusions from transformations in both MC1061 and DH5a

In all transformation attempts in MC1061 and DH5a, no colony PCR attempts on SIPDC, PtPDC, or PgPDC showed successful amplification. These being the only constructs that code for phenylalanine decarboxylase, the enzyme which produces phenylethylamine, we speculate that due to lack of full nisin promoter repression in *E. coli*, toxic levels of phenylethylamine or a deprivation of phenylalanine could arise in *E. coli*, killing the cell ([NICE Expression Handbook](https://www.mobitec.com/media/mobitec/old_content/NICE_Expression_System-Handbook.pdf)).

Two transformation attempts in MC1061 *E. coli*, with assembled plasmids extracted from positive MC1061 colonies and purified yielded no colony growth. After discussing cloning difficulties with the company which provided our team the PNZ8148 plasmid, we were informed of an accidental mutation in the repC region of the plasmid. We hypothesize that this mutation may be the cause of our amplification difficulties and expect more success with the fixed plasmid.

# Cloning in *Lactococcus*

When we purified the plasmid from our *E. coli* liquid cultures, we then electroporated that DNA into *Lactococcus*. However, nothing grew on chloramphenicol plates with both M17 and LB media, even after 5 days at 30 degrees Celsius.

# *Lactococcus* Media

Both LB media and M17 media, a media designed specifically for the *Lactococcus* species, were used to grow *L. lactis*. *L. lactis* grown overnight in LB media had a mean optical density (measured at a wavelength of 600 nm in 1 cm light path) of 1.152, while *L. lactis* grown overnight in M17 media with the same conditions had a mean OD600 of 1.033. We speculate that there is little significant difference in growth efficiency between *L. lactis* bacteria grown in M17 and LB media.

# Smell Tests

In order to characterize the smells of each of the 4 chosen chemicals, we designed smell tests by creating solutions of specific concentrations for each chemical and surveying a group of individuals regarding the intensity of sensation they experienced for specific types of smells. In attempt to replicate the percentage makeup of each chemical as it appears in chocolate in order to calculate the solvent concentrations we would use for the experient, we determined that appropriate solvent concentrations for HDMF (Furaneol), phenylethylamine, pyrazine, and theobromine would be 1 mg/mL, 1 mg/uL, 5 ug/mL, and 10 mg/uL, respectively. We asked each participant to rate the intensity, from a scale of 1-5, of a specific smell in regards to each of the four chemicals. This included the smells sweetness, nuttiness, fishiness, earthiness, bitterness, sourness, fruitiness, caramel scent, roasted scent, saltiness, and vanilla scent. The below graphs show the average responses for each scent between the four chemicals from the survey:

![Graph of rankings of each olfactor for the compounds](https://static.igem.wiki/teams/5272/smell-graphs.png)

The average intensity of smell in between all scents for furaneol was 2.43 from a scale of 1-5, while for phenethylamine it was 1.63, pyrazine 1.61, and theobromine 1.68. While the experiment was designed to measure the resemblance each chemical had to a specific smell, the strength of a chemical’s odor can often increase the intensity of each scent it resembles and bring out scents that otherwise would not have been noticed in a chemical of generally weaker odor. Therefore, because furaneol’s concentration in this experiment resembles its naturally occurring concentration in chocolate, it can be noted that in order to compare more specifically the resemblance of each particular scent between chemicals, further trials should be conducted decreasing the concentration of furaneol to more accurately distinguish between scent resemblance and general chemical odor strength. That furaneol’s concentration is closer to its natural presence in chocolate shows it dominates the smell profile, but adjustments to the concentration could give a clearer picture of the roles played by the other chemicals.

Additionally, we asked participants to give their own short-answer input as to what the chemical smelled like to them. For furaneol, 75% of responses commented on its sweet, caramel scent. 71% of responses regarding phenethylamine commented on its fishy scent. Pyrazine had more varied responses, 20% of which noted its fishy smell, 20% of which noted its caramel smell, 20% of which noted its nutty smell, and 20% of which noted its fruity/raspberry scent. Likewise, theobromine had varied responses, with 36% of participants commenting on its fishy smell, and a variety of comments on its garlic, marinara, vinegar, alcohol, and musky scents.

In characterizing the scents of each of the chemicals, we find that furaneol dominates the smell profile, and reducing the concentration of furaneol in further experiments that involve mixing chemicals in different concentrations will likely be necessary to ensure the right balance of chemicals needed to resemble the chocolate smell. Furthermore, in highlighting the sweet, caramelized scent of furaneol, the fishy scent of phenethylamine, the more varied yet fishy smell of theobromine, and the distinctly varied and complex smell of pyrazine, we are able to create standards to compare to after producing the chemicals via gene expression in *L. lactis*.

# Future Plans

Now informed of the presence of the repC mutation in our original PNZ8148 plasmid, we plan to repeat the assembly of our constructs using the fixed plasmid. The repC mutation could explain many of the difficulties we faced cloning in both *E. coli* and *L. lactis*.

Additionally, we plan to find out what is causing the phenylethylamine pathway to not grow inside of *E. coli*. We plan to do this in two steps: 1) check for lack of promoter repression in *E. coli* by creating a green fluorescent protein pathway and checking whether or not there is fluorescent even without nisin present, and 2) directly transform ligated DNAproducts into *Lactococcus*, thus skipping through *E. coli* cloning.

We also hope to conduct additional experiments in order to better determine the concentrations of mixed chemicals that best produce a chocolate-resembling odor. We hope to perform further smell tests surveying human responses to mixtures of our chemicals with different concentration conditions. As noted in the above description of our smell test, we would also like to repeat the original smell test experiment with the same conditions, but with a lower concentration of furaneol, in order to better characterize scent resemblance regardless of chemical odor intensity.

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