iGEM 2022 — Team uam

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

![](https://static.igem.wiki/teams/4422/wiki/banners/notebook.png)

This notebook is the representation of all the activities we did throughout the season ordered in months. The effort of all the commissions, team members, advisors, collaborators, and instructors is resumed in this document.

{% endblock %}

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

![banner](https://static.igem.wiki/teams/4422/wiki/engineering.png)

* [Design](#design)
* [Build](#build)
* [Test](#test)
* [Learn](#learn)
* [References](#references)

For a synthetic biologist it is fundamental to develop a deep understanding of the cellular mechanisms. Through the improvement of experimental techniques for the manipulation of the cell’s genotype, it has been possible to engineer cells in order to exhibit a new programmed behavior that could be useful to produce molecules of industrial interest. The capability of building complex biological constructs has made possible to solve environmental, health and agricultural problems around the world (Chandran et al., 2008).

## Design

##

### Chassis selection

The chassis selection is a crucial step during the design process. Two of the most common chassis were selected to be analyzed in the context of our project. However, we asked for advice from an expert in metabolic engineering and the production of recombinant proteins. Thus, we added to our list the well-known emergent chassis  Pichia pastoris.

To start with the chassis selection, we identified two fundamental characteristics that had to be accomplished to successfully produce our proteins. First, these peptides are short and come from a eukaryotic organism. Then, the chassis should be capable of folding and assembling eukaryotic proteins. Second, these peptides have disulfide bonds. Then, the chassis should make post-translational modifications correctly.

Due to the characteristics of the chassis Pichia pastoris, this methylotrophic yeast was selected as the host cell of our gene construct. Additionally,  P. pastoris has enormous advantages such as obtaining high recombinant protein production yields.

![](https://static.igem.wiki/teams/4422/wiki/engineering/1.png)

Figure 1. Chassis selection

### Engineering Design

Throughout the design step it is essential to ideate creative solutions to solve the possible problems and disadvantages that could affect the development of the project. Thus, the design step is a constant search for improvement. Six different designs of genetic circuits were proposed at the beginning of the project. We classified these circuits in two types, the ones that use methanol and the methanol free systems. From the analysis it was determined that the more feasible designs were the ones that use methanol.

![](https://static.igem.wiki/teams/4422/wiki/engineering/2.png)

Figure 2. Methanol System vs Free Methanol System

Three of the original genetic circuit designs were selected to perform a second analysis process. Each option proposes the protein production in a different manner. In the first one, two different strains are employed to produce the peptides. In the second one, the production of both peptides happens in the same strain using the same inducible promoters. In the third option, two different promoters will be employed to the protein production, avoiding a possible metabolic overload (Balbas, 2004). From this analysis, the circuit with simultaneous expression was discarded.

![](https://static.igem.wiki/teams/4422/wiki/engineering/3.png)

Figure 3. Number of cells and promoters

The most optimal genetic circuit designs were selected from the previous screening process. The main goal of this project is the development of a system where the expression happens in different cells. Thus, we assemble in silico circuits, design and optimize lab protocols and perform an experimental process to develop this option.

### Genetic circuits description

Two genetic circuits were designed to express each peptide in different strains. However, both have the same general fundamentals and behavior. The pPICZαA plasmid was selected to assemble the two genetic circuits. The protein expression will be regulated by the well-characterized and strong promoter PAOX1. This promoter is induced with methanol and repressed with glycerol, ethanol, and glucose. This promoter contains a Kozak consensus sequence that serves as the initiation site for transcription. After it, an alpha factor secretion signal will target recombinant proteins to the growth medium, simplifying the purification step. After these parts, both circuits can be divided into three sections: a purification section, a fusion protein section, and a detection section.

![](https://static.igem.wiki/teams/4422/wiki/engineering/4.png)

Figure 4. Genetic circuits

### Purification section

This section contains an 8X His tag domain head that will be used in the purification step. This tag was selected because it is unlikely to affect the protein function and has well-established protocols (Young et al., 2012). However, the peptides U1-theraphotoxin-Sp1a and Omega-hexatoxin-Hv2a only have 34 and 45 amino acids (UniProt, 2021) thus the interference of the tag with its function is probable. In order to overcome this possible issue, we proposed two solutions. First, we added a flexible linker of four amino acids (Gly-Gly-Ser-Gly) that allows the tag mobility, decreasing the probability of an interference. Second, in case the linker does not work as expected, the removal of the tag and linker after purification is possible due to the insertion of the Xa factor that once translated will be recognized by the protease (Young et al., 2012).

### Fusion protein section

It contains one of the venom insecticidal peptides OAIP-1 or AcTx-Hv2a fused to a lectin. The snowdrop lectin has been characterized as a transporter of molecules with biological activity throughout the hemolymph of the insects after oral ingestion. In addition, a 3X Ala linker was added in between to ensure flexibility and no affection of the peptide function (Powell, 2020).

Lastly, a c-Myc sequence was added after lectin for further detection and quantification of the protein expression.

![](https://static.igem.wiki/teams/4422/wiki/engineering/5.png)

Figure 5. Purified protein

### Detection section

The Green Fluorescent Protein (GFP) will be employed as a reporter protein. it will confirm the expression of the fusion protein; also, it will help us select high producers and obtain protein expression data for the math model. To achieve coexpression, we added the “self cleaving” peptide T2A before the eGFP sequence. The cleaving mechanism of T2A is the ribosome skipping in the formation of a glycyl-prolyl peptide bond at its C-terminus (Liu, 2017). The eGFP is not part of the fusion protein, avoiding its possible interference with the venom peptides. Also, the GFP will not be secreted to the growth medium.

In addition, the circuits have two restriction enzymes sites. One for KpnI and one for Cfr421

![](https://static.igem.wiki/teams/4422/wiki/engineering/6.jpeg)

Figure 6. Protein production induced by methanol

### Boolean behavior

A common standardized way to present the behavior of a genetic circuit in synthetic biology is through boolean operators (AND, NOT and OR). These operators use biological inputs such as the presence or absence of a reagent and translated to an algebraic meaning that can 1 or 0 respectively.

In this project the circuits iUAM\_PICZαA\_OAIP and UAM\_PICZαA\_Hv2a behave as a NYPLY gate which is the union between an AND and a NOT gate. Thus, the AND gate must have two 1 in the input to produce a 1 in the output that means the production of heterologous proteins. In order to produce these inputs, glycerol should be absent and methanol present (Fig. 7).

![](https://static.igem.wiki/teams/4422/wiki/engineering/boolean-3.jpeg)

Figure 7. Boolean representation of the genetic circuits

This behavior allows a growth phase with glycerol and an expression phase with methanol, avoiding a metabolic overload and an efficient utilization of carbon sources in each phase (Fig. 8).

![](https://static.igem.wiki/teams/4422/wiki/engineering/boolean1.jpeg)

Figure 8. Protein production with methanol induction

## Build

##

### Experimental overview

The build step of the design cycle is a great opportunity to put hands-on work. Therefore, this could be one of the most challenging phases of the project development. The  [results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) obtained from the experimental phase are highly important for the next steps. The data obtained during this phase could be fundamental to propose new improvements.

An experimental process was designed and proposed to assemble the gene constructs. We aim to obtain a strain of the yeast Pichia pastoris capable of executing the instructructions programmed in the expression cassette that will be integrated into the genome of this microorganism.

![](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/1-molecular-biology-overview.png)

Figure 9. Experimental overview

## Test

##

The test phase helped us to understand the overlook points in our design and improve them:

1. During the test phase we noticed that restriction enzymes needed to exchange the fragments inside the circuit were expensive and unaffordable. Thus, we could not use them.
2. In addition, the Gibson Assembly does not recommend using primers with His tag sequences. In our case, one of our primers had an His tag sequence; these could be the cause of the little amount of transformant colonies. To learn more about it visit the [results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) link.
3. Although the test helped us identify the points a and b to improve, we were able to appreciate our achievements. One of them was the use of eGFP, which helped us identify the expressing colonies.

## Learn

##

Overall, these are key points to improve in further designs:

* Avoid His tag sequences in Gibson Assembly or add extra pair bases at the beginning .
* Exchange the restriction enzyme sites for a more common pair (KpnI and XhoI).

Additionally, these are key points that should be conserved in further designs:

* The eGFP is a good tool to identify the expressing colonies.

Thus, the optimized final circuit is:

![](https://static.igem.wiki/teams/4422/wiki/engineering/boolean-2.jpeg)

Figure 10. Optimized circuit

## References

##

* Chandran, D., Copeland, W., Sleight, S., & Sauro, H. (2008). Mathematical modeling and synthetic biology. Drug Discovery Today: Disease Models, 5(4), 299–309. https://doi.org/10.1016/j.ddmod.2009.07.002
* Balbás, P., & Lorence, A. (2004). Recombinant Gene Expression: Reviews And Protocols, 2/E (Second Edition). Balbas, Paulina, Lorence, Argelia (Eds.).
* Young, C. L., Britton, Z. T., & Robinson, A. S. (2012). Recombinant protein expression and purification: a comprehensive review of affinity tags and microbial applications. Biotechnology journal, 7(5), 620–634. https://doi.org/10.1002/biot.201100155
* UniProt. (2021). U1-theraphotoxin-Sp1a precursor - Selenotypus plumipes (Australian featherleg tarantula). UniProtKB - K7N5K9 (TX1\_SELPU). Recuperado 18 de octubre de 2021, de https://www.uniprot.org/uniprot/K7N5K9
* UniProt. (2021). Omega-hexatoxin-Hv2a - Hadronyche versuta (Blue mountains funnel-web spider). UniProtKB - P82852 (TOT2A\_HADVE). Recuperado 18 de octubre de 2021, de https://www.uniprot.org/uniprot/P82852
* Powell, M. E., Bradish, H. M., Cao, M., Makinson, R., Brown, A. P., Gatehouse, J. A., & Fitches, E. C. (2020). Demonstrating the potential of a novel spider venom-based biopesticide for target-specific control of the small hive beetle, a serious pest of the European honeybee. Journal of Pest Science, 93(1), 391–402. https://doi.org/10.1007/s10340-019-01143-3

{% endblock %}

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

![](https://static.igem.wiki/teams/4422/wiki/experiments-buena.png)

## Molecular biology

##

### Click the figures to preview the protocols

[![](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/2-pcr-of-gibson-fragments.png)](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/1-pcr-for-gibson-assembly.pdf)

[![](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/6-sequential-restriction-digest.png)](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/vector-linearization-for-gibson-assembly-benchling.pdf)

[![](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/1-dna-purification-from-an-agarose-gel.png)](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/dna-purification-from-an-agarose-gel-benchling.pdf)

[![](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/3-lipase-assay-protocol.png)](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/gibson-assembly.pdf)

[![](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/2-bacterial-transformation.png)](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/e-coli-transformation-heat-shock-benchling.pdf)

[![](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/4-preparation-of-plasmid-dna-by-alkaline-lysis-midipreparation.png)](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/preparation-of-plasmid-dna-by-alkaline-lysis-with-sds-minipreparation-benchling.pdf)

[![](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/3-pichia-pastoris-transformation-by-electroporation-1.png)](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/pichia-transformation.pdf)

[![](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/3-visualization-of-gfp-expression.png)](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/visualization-of-gfp-expression-benchling.pdf)

[![](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/5-pichia-pastoris-genomic-dna-purification.png)](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/pichia-pastoris-genomic-dna-purification-benchling.pdf)

[![](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/5-expression.png)](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/expression.pdf)

[![](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/6-precipitation.png)](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/precipitation-and-purification-of-proteins.pdf)

[![](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/4-western-blot.png)](https://static.igem.wiki/teams/4422/wiki/protocols/molecular-biology/western-blot.pdf)

## Math Modelling

##

### Click the figures to preview the protocols

[![](https://static.igem.wiki/teams/4422/wiki/protocols/math-modeling/calibration-curve-with-e-coli.png)](https://static.igem.wiki/teams/4422/wiki/protocols/math-modeling/calibration-curve-with-escherichia-coli.pdf)

## Safety & Security

##

### Click the figures to preview the protocols

[![](https://static.igem.wiki/teams/4422/wiki/protocols/safety/2.png)](https://static.igem.wiki/teams/4422/wiki/protocols/safety/proof-of-concept-benchling.pdf)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

![banner](https://static.igem.wiki/teams/4422/wiki/banners/results.png)

## Overview

##

* Assemble two different circuits iUAM\_PICZɒA\_OAIP and iUAM\_PICZɒA\_Hv2 to produce two spider venom peptides
* Clear confirmation of the genetic circuit constructions using two techniques PCR and restriction enzymes
* Test of a new affordable expression medium (YPD with 1% glycerol) compared with the commonly used expression medium (BMMY)
* Use a simple screening method using eGFP
* Proof of concept of both peptides

Additionally, we added advice that could help future iGEMers generations. These advice are the collection of learnings from our instructors, advisors and personal thoughts.

## Fragments amplification

##

In order to create the constructions iUAM\_PICZɒA\_OAIP and iUAM\_PICZɒA\_Hv2a, the fragments Fragment A\_OAIP (1302 bp) and Fragment B\_Hv2a (1347 bp) were synthesized. Both fragments contained similar parts, except for the spider venom peptide, which was specific for each one (Fig. 1). As it is shown, the fragment A\_OAIP contained the spider venom peptide U1-theraphotoxin-Sp1a (OAIP-1) from Selenotypus plumipes while the fragment B\_Hv2a contained the fragment Omega-hexatoxin-Hv2a (AcTx-Hv2) from Hadronyche versuta.

![](https://static.igem.wiki/teams/4422/wiki/results/1-lipase-assay-protocol.png)

Figure 1. Synthesized fragments

In addition, two primers were synthesized for a further Gibson Assembly; thus, these primers amplify the fragments and add 20 nucleotides which overlap with the vector pPICZɒA. Although the expected annealing temperature was close to 55 °C, a temperature gradient was performed to confirm it, using the polymerase Phusion High -Fidelity DNA Polymerase.

![](https://static.igem.wiki/teams/4422/wiki/results/2-pcr.png)

Figure 2. Temperature gradient

Surprisingly, the temperature gradient (Fig. 2) shows that at 55°C a higher specificity and good yields are achieved, leading to the following PCR protocol (Table 1).

Table 1. PCR optimized protocol

![](https://static.igem.wiki/teams/4422/wiki/results/3-pcrfingib.png)

Although this optimization improved the PCR results, a nonspecific amplification occurred; then, it was necessary to do an electrophoresis gel and purify the fragments band.

#### Advise Alert

* Flanking the synthesized fragments with two different restriction enzymes can be useful to perform a restriction enzyme cloning method, in case the Gibson Assembly fails.
* During the fragment purification, one of the critical decisions is the final elution volume. In our case, the kit Wizard® SV Gel and PCR Clean-Up System Technical Bulletin recommended 50 µL per purification column. However, better results in quantification and Gibson Assembly were obtained using 40 µL per purification column.
* The use of Taq polymerases not only risks the fidelity sequence, but also adds a poly(A) tail that could interfere with the Gibson Assembly method. Thus, it is highly recommended not to use them to amplify Gibson fragments.

## Restriction digestion

##

pPICZα A was selected as the expression vector to assemble our two fragments. To perform the assembly protocol it was needed to carry out some previous steps. The vector was amplified using the E. coli DH5α strain and it was purified by running a DNA extraction protocol with a MidiPrep commercial kit from Promega.

Due to the coexpression of eGFP and pesticide proteins, it was necessary to eliminate the His-tag from the original pPICZα A. This change in the fragments order made impossible the use of two restriction enzyme sites from the multiple cloning site (Figure 3). Luckly, the restriction enzyme AgeI was present after de His-tag and was neither present in the fragments nor other places in the vector. The second restriction site was EcoRI which is inside the multiple cloning site.

![](https://static.igem.wiki/teams/4422/wiki/results/4-digest.png)

Figure 3. pPICZαA digestion

In order to perform a double digestion, it was necessary to execute them separately since these enzymes do not share a buffer where they exhibit an activity higher than 50% as it is shown in table 2 (specifically for NEB enzymes).

Table 2. Buffers for enzyme digestion

![](https://static.igem.wiki/teams/4422/wiki/results/5-buffer.png)

Although the quick protocol suggested 15 minutes at 37 °C as enough time to perform each digestion, they were carried overnight at room temperature, obtaining optimal results (Fig. 4) and avoiding the presence of circular vector after digestion. In this case, the length of the complete vector is 3593, the expected length of the linearized vector was 3405 bp, and the length of the liberated fragment is 188 bp.

![](https://static.igem.wiki/teams/4422/wiki/results/6-digest-chida.png)

Figure 4. pPICZαA vector digestion

It is important to mention that after the first digestion a purification step was done. Then, after the second one, an electrophoresis gel was necessary to purify the linearized vector band.

#### Advise Alert

* To linearize the vector, selecting two restriction enzymes that are present only once in the vector is necessary to avoid more cuts that could make the vector unsuitable for further Gibson techniques.
* To linearize the vector, selecting two restriction enzymes that are not present in the fragments allows the confirmation of the construction when the Gibson Assembly method is performed, since this method does not leave scars in the junctions, conserving the restriction sites.
* When linearizing the vector with restriction enzymes, it is important to make sure that there are no lefts of circular vector, since it could be detrimental for further steps.

In figure 5, an electrophoresis gel of pPICZɒA with EcoRI and AgeI is showed; however, the size of vector is higher than the expected (3405 bp). This could be due to a poor restriction enzyme digest.

![](https://static.igem.wiki/teams/4422/wiki/results/7-digest-fea.png)

Figure 5. pPICZɒA poor enzyme digestion

* During the purification of linearized vector, one of the critical decisions is the final elution volume. In our case, the kit Wizard® SV Gel and PCR Clean-Up System Technical Bulletin recommended 50 µL per purification column. However,  better results in quantification and Gibson Assembly were obtained using 35 µL per purification column.

## Fragment and linearized vector quantification

##

Before performing the Gibson assembly, it is necessary to quantify the concentration of fragments and linearized vector to obtain the volumes that contain the optimum amount of DNA. In addition, it is important to know the purity of the DNA, using the 260/230 and 260/280 ratios.

The quantification of the fragments and linearized vector pPICZɒA was performed using a ThermoFisher NanoDrop One/One-c (Table 3). In each case the 260/230 and 260/280 ratios were optimum, between 2.0-2.2 and 1.-2.0, respectively.

Table 3. Fragment and linearized vector quantification

![](https://static.igem.wiki/teams/4422/wiki/results/8-quant.png)

## Gibson Assembly

##

Due to their multiple advantages, such as no need for specific restriction sites and no scars between fragments, Gibson Assembly was the selected method to assemble our genetic circuit. Using this method, we were able to have complete control over the fragments that were assembled into the expression vector. Thus, the insertion of additional unwanted sequences was prevented.

The assembly protocol was performed using the Gibson Cloning Kit from New England Biolabs. This protocol recommends a total of 0.02–0.5 pmols of DNA fragments when 1 or 2 fragments are being assembled into a vector. Therefore, the number of pmols of each fragment was calculated to increase the efficiency of the reaction.

![](https://static.igem.wiki/teams/4422/wiki/results/9-copy-of-lipase-assay-protocol.png)

Figure 6. Gibson assembly quantities

The reactions were set up on ice and they were incubated in a thermocycler at 50 °C for 30 minutes. The gene constructs obtained from the assembly are shown in the following illustration.

![](https://static.igem.wiki/teams/4422/wiki/results/10-vectors.png)

Figure 7. Genetic circuits

Then, a sample of 5 ul of the Gibson Assembly reaction was analyzed through an agarose gel electrophoresis.

![](https://static.igem.wiki/teams/4422/wiki/results/13-electro.png)

Figure 8. Gibson Assembly reaction

The agarose gel analysis shows a 1500 base pairs band of length that corresponds to the DNA fragments. The band between 3000 and 4000 base pairs corresponds to the empty plasmid. Finally, the bands observed above 5000 base pairs correspond to the gene construct where the plasmid isoforms can be observed (Fig. 8).

#### Advise Alert

* The Gibson Assembly Cloning Kit from NEB recommends an incubation time of 15 minutes when 2-3 fragments are being assembled. However, in orden to increase the assembly efficiencies we recommend using extended incubation times. During our experiments time was extended up to 30 minutes to assemble two fragments.
* It is not recommended to use overlaps containing His-tag repeats. His-tag sequences should be flanked on both sides with at least two nucleotides.

### E. coli transformation

5-alpha Competent E. coli cells were transformed with 2 µl of the reaction sample following the Gibson Assembly Transformation Protocol from NEB. Next, 100 µl of the cells were plated onto LB medium plates with Phleomycin at a concentration of 20 ug/ml. The plates were incubated overnight at 37 °C.

![](https://static.igem.wiki/teams/4422/wiki/results/12-petri.png)

Figure 9. Transformants

Five transformants were obtained for the iUAM\_PICZαA\_OAIP construct and three for the iUAM\_PICZαA\_Hv2a construct. All the colonies were picked and purified on fresh LB-Phleomycin plates. Once the colonies were isolated in new LB plates, a 5 ml liquid culture was inoculated to perform a MiniPrep protocol. The purified DNA was used to run the confirmation protocols

## Construction confirmation

##

### Restriction analysis

In orden to confirm the correct assembly of the gene circuit two restriction enzymes were performed. Due to the capability of the Gibson method to conserve the original sequence without modifications the EcoRl and Agel restriction sites allowed the confirmation of the gene constructs iUAM\_PICZαA\_OAIP and iUAM\_PICZαA\_Hv2a. Additionally, A second restriction assay was performed using the enzyme Hindlll in both constructs.

As shown in figure 10 the restriction assay with the enzymes EcoRl and Agel two DNA bands can be observed in well number five. One near to the 5000 base pairs length and another in 1500 base pairs of length. This result was predicted using the restriction enzyme tool from Benchling. Similarly, the restriction digest performed with the enzyme Hindlll shows consistency with the computational simulation.

![](https://static.igem.wiki/teams/4422/wiki/results/figura10.png)

Figure 10. Confirmation by restriction analysis

### PCR confirmation

Similarly, the transformants were confirmed by PCR. The primers used amplify the Gibson fragments; thus, the length should correspond to 1302 bp for the construction iUAM\_PICZɒA\_OAIP since the amplified fragment is Fragment A\_OAIP and 1347 bp for the construction iUAM\_PICZɒA\_Hv2a since the amplified fragment is Fragment B\_Hv2a. As it is shown in the figure 11, all the tested transformed colonies amplified the cloned fragment, while the last sample containing the vector PICZɒA purified from  Escherichia coli did not amplified, validating that the construction was present in the transformed colonies.

![](https://static.igem.wiki/teams/4422/wiki/results/14-copy-of-digest-chida.png)

Figure 11. Confirmation by PCR

Advise Alert

* Confirmation is one of the most important steps in science. When it results as expected you should feel proud, you did great. However, when it turns out different you should stay calm and remember that science is always about learning.

The best way to understand which steps are causing problems is double checking your previous data and creating hypotheses to probe. Professor Barrios-Gonzales (our PI) told us that when we fail we can discover and learn new things that are valuable. We should not feel ashamed, failing is part of making science.

## Pichia pastoris Transformation

##

To start with the transformation protocol of Pichia pastoris a MidiPrep protocol was performed. It allowed us to obtain a stock of purified DNA of the gene constructions iUAM\_PICZαA\_OAIP and iUAM\_PICZαA\_Hv2a

The linearization of the expression vector is recommended to promote the integration of the genetic cassette into the yeast genome. Therefore, the restriction enzyme Sacl was used to linearize the plasmids. It is fundamental to ensure the complete digestion of the DNA considering that it has been reported that the linearization increases the transformation efficiency.

The transformation was performed using the pPICZ A, B, and C manual from invitrogen. Electroporated cells were plated into YPDS plates containing 50 ug/ml of phleomycin and the plates were incubated 48 hours at 30 ºC.

![](https://static.igem.wiki/teams/4422/wiki/results/pichia-pastoris.png)

Figure 12.Pichia pastoris transformants

In this step 10 colonies from each plate were picked and purified on fresh YPD plates containing 50 ug/ul of phleomycin.

## Transformants confirmation with eGFP detection

##

In order to easily screen the transformants and identify the expressing colonies, our construction co-express eGFP. We screened 25 Pichia pastoris colonies of each construction, obtaining 5 transformants in total: 3 transformants with the iUAM\_PICZαA\_OAIP construction and 2 transformants with the iUAM\_PICZαA\_Hv2a construction. In the figure 13, the comparison between the expressing colonies and no expressing colonies is shown.

![](https://static.igem.wiki/teams/4422/wiki/results/13-pichia-pastoris-egfp-fluorecenc.png)

Figure 13. Pichia pastoris transformants screening

Advise Alert

* The suggested protocol to express proteins in Pichia pastoris with methanol uses the medium BMGY and BMMY. These mediums use reagents such as biotin that can be expensive. However, optimal results can be obtained, using YPD with 2% of glycerol and YPD with 1% of methanol.
* Analyzing multiple colonies is necessary to select expressing colonies. Although these can take time, you can use agar plates and screen multiple colonies.

## Proof of concept

##

To verify the efficacy of Spidicide-CX over a commercial pesticide named Malathion, we designed a proof of concept. It consisted of feeding groups of larvae and adult insects with different treatments every six hours. And during that, we will observe the mortality of the insects and do an ANOVA and Tukey tests to compare the data recollected. Due to the lack of insect samples and of time, we modified the initial protocol. These new changes consisted in reducing the treatments and recollected data for the first 18 hours. Despite the preliminary data, we can observe favorable results because it shows that our biopesticide has a similar effect to the chemical pesticide Malathion. You can check the results in the section dedicated to the  [proof of concept](%7B%7Burl_for%28%27pages%27%2Cpage%3D%27proof-of-concept%27%29%7D%7D).

{% endblock %}