iGEM 2020 — Team MSP-Maastricht

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:MSP-Maastricht/Notebook

LabBook

#### [![](https://static.igem.org/mediawiki/2020/4/49/T--MSP-Maastricht--Image2.png)](https://2020.igem.org/Team%3AMSP-Maastricht)

Project

[Description](https://2020.igem.org/Team%3AMSP-Maastricht/Description)
[Design](https://2020.igem.org/Team%3AMSP-Maastricht/Design)
[Experiments](https://2020.igem.org/Team%3AMSP-Maastricht/Experiments)
[Parts](https://2020.igem.org/Team%3AMSP-Maastricht/Parts)
[LabBook](https://2020.igem.org/Team%3AMSP-Maastricht/Notebook)
[Safety](https://2020.igem.org/Team%3AMSP-Maastricht/Safety)

Results

[Results](https://2020.igem.org/Team%3AMSP-Maastricht/Results)
[Engineering Success](https://2020.igem.org/Team%3AMSP-Maastricht/Engineering)
[Contribution](https://2020.igem.org/Team%3AMSP-Maastricht/Contribution)
[Implementation](https://2020.igem.org/Team%3AMSP-Maastricht/Implementation)

Human Practices

[Human Practices](https://2020.igem.org/Team%3AMSP-Maastricht/Human_Practices)
[Science Communication](https://2020.igem.org/Team%3AMSP-Maastricht/Education)

Collabs

[Collaborations](https://2020.igem.org/Team%3AMSP-Maastricht/Collaborations)
[Partnership](https://2020.igem.org/Team%3AMSP-Maastricht/Partnership)

Team

[Team](https://2020.igem.org/Team%3AMSP-Maastricht/Team)
[Attributions](https://2020.igem.org/Team%3AMSP-Maastricht/Attributions)
[Judging Form](https://igem.org/2020_Judging_Form?id=3476)

## Project ![](https://static.igem.org/mediawiki/2020/3/39/T--MSP-Maastricht--Image.png)

### LAB-BOOK

![](https://static.igem.org/mediawiki/2020/3/32/T--MSP-Maastricht--Image126.png)

![](https://static.igem.org/mediawiki/2020/f/f1/T--MSP-Maastricht--Image120.png)

#### The experiments performed in the lab are listed day by day in the pdf below. Lab work started in September and went on until the WIKI freeze. See the [Experiments page](https://2020.igem.org/Team%3AMSP-Maastricht/Experiments) for more information about our protocols.

## DESIGN

![](https://static.igem.org/mediawiki/2020/0/03/T--MSP-Maastricht--Image118.png)

#### [Learn more](https://2020.igem.org/Team%3AMSP-Maastricht/Design)

## JOURNAL

![](https://static.igem.org/mediawiki/2020/3/3e/T--MSP-Maastricht--Image115.png)

#### [Learn more](https://2020.igem.org/Team%3AMSP-Maastricht/Education)

## COLLABS

![](https://static.igem.org/mediawiki/2020/6/69/T--MSP-Maastricht--Image116.png)

#### [Learn more](https://2020.igem.org/Team%3AMSP-Maastricht/Collaborations)

![](https://static.igem.org/mediawiki/2020/3/37/T--MSP-Maastricht--F4.png)
![](https://static.igem.org/mediawiki/2020/d/de/T--MSP-Maastricht--F1.png)
![](https://static.igem.org/mediawiki/2020/7/78/T--MSP-Maastricht--F2.png)
![](https://static.igem.org/mediawiki/2020/c/c6/T--MSP-Maastricht--F3.png)
![](https://static.igem.org/mediawiki/2020/3/3b/T--MSP-Maastricht--F5.png)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:MSP-Maastricht/Engineering

Model

#### [![](https://static.igem.org/mediawiki/2020/4/49/T--MSP-Maastricht--Image2.png)](https://2020.igem.org/Team%3AMSP-Maastricht)

Project

[Description](https://2020.igem.org/Team%3AMSP-Maastricht/Description)
[Design](https://2020.igem.org/Team%3AMSP-Maastricht/Design)
[Experiments](https://2020.igem.org/Team%3AMSP-Maastricht/Experiments)
[Parts](https://2020.igem.org/Team%3AMSP-Maastricht/Parts)
[LabBook](https://2020.igem.org/Team%3AMSP-Maastricht/Notebook)
[Safety](https://2020.igem.org/Team%3AMSP-Maastricht/Safety)

Results

[Results](https://2020.igem.org/Team%3AMSP-Maastricht/Results)
[Engineering Success](https://2020.igem.org/Team%3AMSP-Maastricht/Engineering)
[Contribution](https://2020.igem.org/Team%3AMSP-Maastricht/Contribution)
[Implementation](https://2020.igem.org/Team%3AMSP-Maastricht/Implementation)

Human Practices

[Human Practices](https://2020.igem.org/Team%3AMSP-Maastricht/Human_Practices)
[Science Communication](https://2020.igem.org/Team%3AMSP-Maastricht/Education)

Collabs

[Collaborations](https://2020.igem.org/Team%3AMSP-Maastricht/Collaborations)
[Partnership](https://2020.igem.org/Team%3AMSP-Maastricht/Partnership)

Team

[Team](https://2020.igem.org/Team%3AMSP-Maastricht/Team)
[Attributions](https://2020.igem.org/Team%3AMSP-Maastricht/Attributions)
[Judging Form](https://igem.org/2020_Judging_Form?id=3476)

## Results ![](https://static.igem.org/mediawiki/2020/3/39/T--MSP-Maastricht--Image.png)

### ENGINEERING

![](https://static.igem.org/mediawiki/2020/b/b2/T--MSP-Maastricht--Image127.png)

### RESEARCH

#### From the moment of choosing to fight against the oak processionary caterpillar and consulting with the OPC expert of the shortcoming of the current control methods, we knew we had to utilise recent scientific advances to make an effective and specific pesticide. Whilst first looking for potential mechanisms to achieve this task, we looked at many different options, such as nanoparticles, however, we zeroed on RNA interference (RNAi) in the end. There has been a growing interest in the application of RNAi in pest control and in the development of biological pesticides [(Niu et al., 2018).](https://2020.igem.org/Team%3AMSP-Maastricht/Attributions) RNAi is already widely applied to control gene expression, and refers to a mechanism of gene silencing, where translation of target mRNA is inhibited by homologous, double-stranded siRNAs [(Petrova, Zenkova & Chernolovskaya, 2013).](https://2020.igem.org/Team%3AMSP-Maastricht/Attributions) There has been plenty of promising research for the use of RNAi as a new pest control approach in protecting food crops [(Fishilevich et al., 2016; Palli, 2014).](https://2020.igem.org/Team%3AMSP-Maastricht/Attributions) It appeared to us that another potential and significant target for the implementation of RNAi is the control of invasive species and nature conservation.

### DESIGN

#### Our idea was to create a bacterium which would produce siRNA molecules, targeting specific and essential genes of the OPC. This would lead to inhibition of the growth of the caterpillars and consequently decrease in the out-of-control populations, providing an improved control method. The first crucial step in our approach, and specifically in designing our siRNA, was the identification of unique target genes. Since the OPC genome has not yet been sequenced completely, we searched different articles to find possible targets. We used NCBI sequence data for BLAST search of barcode genes to find unique regions.

![](https://static.igem.org/mediawiki/2020/9/99/T--MSP-Maastricht--EST.png)

#### Unfortunately, due to the ongoing Covid-19 pandemic and strict regulations to stop its spread, we had no access to the lab and no way to validate our targets. Hence, we started looking at possible models to use to determine lethality of silencing the identified target genes. The challenge we faced was the limited data on the OPC itself and the lack of possible models to determine the effects of these genes in the OPC. To overcome this took a different approach to get to a model.

### METABOLIC MODEL

#### After extensive research, we found several tools, which allows one to build a metabolic model of an organism using either protein or RNA expression by linking this data to the corresponding reaction fluxes from the chosen framework. To determine the effect of silencing a gene, a metabolic model for the OPC using the IMAT algorithm was created [(Zur, Ruppin & Shlomi, 2010).](https://2020.igem.org/Team%3AMSP-Maastricht/Attributions) This model was built with the COBRA toolbox and works with expression data of one cell in an organism, as the expression of a gene or protein is directly linked to the flux of the associated reactions. The model uses the Gurobi solver to perform flux balance analysis and single gene deletion analysis, with which it can help identify the effects of a gene silencing. To check whether the silencing of the chosen gene has any effect, a fake BIOMASS reaction was added into the model, since it requires materials for the cell to survive and duplicate. The model was then set to optimize for the BIOMASS reaction. When the flux of the reaction is significantly lowered, this is an indication of harming the cell. We then encountered another problem - no arthropod frameworks existed, and we had to take a step back and modify approach.

![](https://static.igem.org/mediawiki/2020/2/29/T--MSP-Maastricht--Image41.png)
![](https://static.igem.org/mediawiki/2020/7/74/T--MSP-Maastricht--Image49.png)

![](https://static.igem.org/mediawiki/2020/8/82/T--MSP-Maastricht--Image42.png)

#### When we re-examining our targets, we noticed that our targets were highly evolutionary conserved in terms of their functions and pathways [(Nomiyama & Yoshie, 2014; Teo, Möhrlen, Plickert, Müller, & Frank, 2006).](https://2020.igem.org/Team%3AMSP-Maastricht/Attributions) This gave us an idea to instead use gene analogues with an existing framework. We deduced that since our targets are well conserved, they should remain functionally intact using mammalian analogues of a drosophila melanogaster gene expression data set. For this we chose the human framework Recon3D, as this one was the most complete and detailed available [(Brunk et al, 2018).](https://2020.igem.org/Team%3AMSP-Maastricht/Attributions) Next, we translated our OPC RNA expression data into their respective human analogues before mapping them to the framework. This created the metabolic model that was as close to an approximation of the OPC with the data available. The model did not directly model the interaction of the siRNA but the expected effect of a gene knockout, and using this model we were able to determine the lethality of all but one possible target genes.

### TARGETS AND SIRNAS

#### With the help of our models we focused our research and prepared to start work in the lab. We learned which genes would have the desired effect if knocked out. In total 4 genes with the most promising effect were identified, and these became our priority in planning the future experiments in the lab. These include the gene for the subunit 2 of the allergenic protein Tha p2 that can be found in the urticating setae of the caterpillars [(Berardi, Battisti & Negrisolo, 2015);](https://2020.igem.org/Team%3AMSP-Maastricht/Attributions) Pro2 photolyase gene that is involved in proline metabolism; and EF1a gene for elongation factor-1 alpha which takes part in the protein synthesis elongation phase. As well as, Wg gene for wingless protein, that is thought to be involved in numerous processes through development [(Simonato et al., 2013).](https://2020.igem.org/Team%3AMSP-Maastricht/Attributions)

![](https://static.igem.org/mediawiki/2020/4/4b/T--MSP-Maastricht--ESTd.png)

#### In the design of the siRNAs, 21 nucleotide sequences were identified within the genes according to siRNA design guidelines [(Thermo Fisher Scientific, n.d.)](https://2020.igem.org/Team%3AMSP-Maastricht/Attributions) which include 30-50% GC content, absence of stretches with 4 or more A’s or T’s in a row, and TT nucleotide beginning. These siRNA sequences were selected at different target sites for the same gene to potentially increase the effectiveness of RNA interference. These short sequences were run through the NCBI BLAST algorithm again in order to assess their specificity. Finally only siRNA that were most specific to OPC were chosen.

![](https://static.igem.org/mediawiki/2020/2/2b/T--MSP-Maastricht--Image35.png)

#### *Figure 1. BLAST-N search for one of our target genes (potential specific targets marked as features)*

### RESEARCH INTO DESIGNING THE PLASMIDS AND BACTERIA

#### After obtaining the promising target genes and designing siRNAs for them, the next step was to design the bacteria to be used to produce and deliver these siRNAs. To design our "Oak Shield" strain of bacteria, we first researched different papers which use bacterial mediated RNAi for specifically pest control. Since several studies showed success using the HT115(DE3) *E. coli* strain and the L4440 plasmid, we decided to use this strain and plasmid to express our siRNAs [(Ganbaatar et al., 2017; Giesbrecht et al., 2020; Zhang et al., 2019).](https://2020.igem.org/Team%3AMSP-Maastricht/Attributions) The *E. coli* strain HT115(DE3) was selected because it is RNAse III deficient, therefore our RNA's are not degraded by the bacteria. Furthermore, HT115(DE3) expresses T7-polymerase which allows for efficient expression of dsRNA products. We wanted the bacteria to precisely express the siRNAs we have designed and selected. However, since the plasmid does not contain a transcription termination signal we decided to insert a terminator sequence into our expression cassette. We feared that prolonged transcription of the siRNA coding region would lead to nonspecific and ineffective products. Literature research confirmed that a termination signal significantly increases the yield of correctly sized and effective shRNAs. We designed our final expression cassette using the L4440 plasmid as a template [(Sturm, Saskoi, Tibor, Weinhardt, & Vellai, 2018).](https://2020.igem.org/Team%3AMSP-Maastricht/Attributions) We decided to keep the T7 promoters of the plasmid but replaced the MCS with EcoRI & BamHI restriction sites (to clone in the shRNA coding DNA-oligo) followed by a T7 terminator sequence. This fragment was ordered as a gBlock and cloned into L4440. We created 14 different siRNAs for 4 different genes with adapters for EcoRI and BamHI cloning, each siRNA-oligo was inserted into the L4440-expression cassette plasmid. The resulting plasmid can be used universally to clone in any desired shRNA sequence. This way we do not need to order each siRNA as a separate gBlock containing the whole expression cassette. The inserts were ordered as single stranded oligonucleotides that after annealing have the necessary overhangs for EcoRI and BamHI, therefore the shRNA DNA constructs can be inserted in the vector when it is cut with the same restriction enzymes (Figure 2).

![](https://static.igem.org/mediawiki/2020/8/8c/T--MSP-Maastricht--Image384.png)

#### *Figure 2. The expression cassette is cloned into L4440 linearized with Pci1 & NgoMIV using gibson assembly. A shRNA coding DNA-sequence can then be inserted into the vector using BamH1 and EcoR1.*

### PARTS

#### In total we designed 14 shRNAs to target essential genes in the OPC. In addition we designed the bacterial expression cassette for these shRNAs. Learn more about our designed parts [here.](https://2020.igem.org/Team%3AMSP-Maastricht/Parts)

![](https://static.igem.org/mediawiki/2020/9/93/T--MSP-Maastricht--Image3840.png)

### LABWORK

#### When we finally got permission to enter the lab, we had plenty of work to do in an extremely limited timeframe. First task was to validate the target sequences. For this, a series of experiments were conducted. DNA was first extracted from early stage OPCs, followed by a PCR using primers designed for the target genes. This lets us identify if the targeted regions are indeed present. Gel electrophoresis was used to separate our DNA fragments (from the PCR) based on their size to see the success of our PCR amplification of the target fragments. Afterwards, these fragments were isolated and further amplified in order to obtain a sufficient concentration for them to be sequenced by Macrogen. This was necessary to ensure that the target sequences are exactly the same as the ones that were found on databases. Only this way, we can ensure that the siRNAs can specifically and efficiently bind to their selected target. In total, 11 siRNA target regions were confirmed by the Macrogen sequencing with 100% identity, 1 region was confirmed with 86% identity to the reference sequence, and 2 were not found. This turned out to be because one of the primer sets for Tha p2 gene did not produce any results.

![](https://static.igem.org/mediawiki/2020/8/87/T--MSP-Maastricht--4798.png)

#### The next step was the plasmid assembly and testing of the production of the shRNAs by the bacteria. The assembly was done in several steps of digestion and annealing. In the end we had 6 different plasmids+inserts in the DH5α bacteria that were successful, as there were visible colonies on the plates. As some did not grow, we would have required more time to repeat the vector+insert assembly in order to obtain all 14 inserts.

![](https://static.igem.org/mediawiki/2020/0/05/T--MSP-Maastricht--4748.png)

#### *Figure 3. Antibiotic selection plate of our final Oakshield strain*

#### Afterwards, the successful plasmids were further transfected into HT115(DE3) *E. coli*, from which 4 were successful, as colonies could be seen on the plates. For these 4, IPTG induction was done to produce the siRNAs.

### FUTURE TESTS

#### Unfortunately due to the pandemic we were not able to do all lab work we wanted. 1 ½ months are not enough to correctly test if all our designed parts are successfully engineered and implemented. However, despite the limited timeframe in which we were able to go the labs, we were managed to construct the basic expression cassette vector and were able to insert 6 shRNA sequences. These final constructs still need to be confirmed by sequencing. As the next step, we would test if the shRNAs are produced by the IPTG induction in the HT115(DE3) cells. This would be done by extracting the RNA content of the bacteria and running a gel to see if the desired shRNAs are being produced. If this test yielded expected results, we would move on to conduct tests with the caterpillars. In case of unexpected results, we would need to do more research on whether there are, for example, problems with our expression cassette. Another subsequent step would be to test how the HT115(DE3) *E. coli*, with the inserts, survive sprayed on the oak branches. This would provide us key information about wether we would need to further modify the bacteria to survive sufficient time on the oak branches.

## DESIGN

![](https://static.igem.org/mediawiki/2020/0/03/T--MSP-Maastricht--Image118.png)

#### [Learn more](https://2020.igem.org/Team%3AMSP-Maastricht/Design)

## PARTS

![](https://static.igem.org/mediawiki/2020/8/83/T--MSP-Maastricht--Image121.png)

#### [Learn more](https://2020.igem.org/Team%3AMSP-Maastricht/Parts)

## LAB BOOK

![](https://static.igem.org/mediawiki/2020/f/f1/T--MSP-Maastricht--Image120.png)

#### [Learn more](https://2020.igem.org/Team%3AMSP-Maastricht/Notebook)

![](https://static.igem.org/mediawiki/2020/3/37/T--MSP-Maastricht--F4.png)
![](https://static.igem.org/mediawiki/2020/d/de/T--MSP-Maastricht--F1.png)
![](https://static.igem.org/mediawiki/2020/7/78/T--MSP-Maastricht--F2.png)
![](https://static.igem.org/mediawiki/2020/c/c6/T--MSP-Maastricht--F3.png)
![](https://static.igem.org/mediawiki/2020/3/3b/T--MSP-Maastricht--F5.png)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:MSP-Maastricht/Experiments

Experiments

#### [![](https://static.igem.org/mediawiki/2020/4/49/T--MSP-Maastricht--Image2.png)](https://2020.igem.org/Team%3AMSP-Maastricht)

Project

[Description](https://2020.igem.org/Team%3AMSP-Maastricht/Description)
[Design](https://2020.igem.org/Team%3AMSP-Maastricht/Design)
[Experiments](https://2020.igem.org/Team%3AMSP-Maastricht/Experiments)
[Parts](https://2020.igem.org/Team%3AMSP-Maastricht/Parts)
[LabBook](https://2020.igem.org/Team%3AMSP-Maastricht/Notebook)
[Safety](https://2020.igem.org/Team%3AMSP-Maastricht/Safety)

Results

[Results](https://2020.igem.org/Team%3AMSP-Maastricht/Results)
[Engineering Success](https://2020.igem.org/Team%3AMSP-Maastricht/Engineering)
[Contribution](https://2020.igem.org/Team%3AMSP-Maastricht/Contribution)
[Implementation](https://2020.igem.org/Team%3AMSP-Maastricht/Implementation)

Human Practices

[Human Practices](https://2020.igem.org/Team%3AMSP-Maastricht/Human_Practices)
[Science Communication](https://2020.igem.org/Team%3AMSP-Maastricht/Education)

Collabs

[Collaborations](https://2020.igem.org/Team%3AMSP-Maastricht/Collaborations)
[Partnership](https://2020.igem.org/Team%3AMSP-Maastricht/Partnership)

Team

[Team](https://2020.igem.org/Team%3AMSP-Maastricht/Team)
[Attributions](https://2020.igem.org/Team%3AMSP-Maastricht/Attributions)
[Judging Form](https://igem.org/2020_Judging_Form?id=3476)

## Project ![](https://static.igem.org/mediawiki/2020/3/39/T--MSP-Maastricht--Image.png)

### EXPERIMENTS

![](https://static.igem.org/mediawiki/2020/2/2d/T--MSP-Maastricht--Image12.png)

## DNA EXTRACTION AND POLYMERASE CHAIN REACTION (PCR)

#### We obtained early stage OPC DNA by first freezing the samples in liquid nitrogen and grinding them followed by genomic DNA extraction using the HotSHOT protocol which leaves the samples ready to be added to the PCR-reaction. The concentration of our extracts was determined using a Nanodrop Spectrophotometer. Primers were designed for the genes of interest in a way that the resulting amplicons contain the regions of the genes we are trying to target with our siRNAs. This enables us identify if the target gene regions are indeed present by sequencing the amplicons. PCR was performed using Invitrogen™ Platinum™ SuperFi™ PCR Master Mix or Phusion® High-Fidelity PCR Master Mix.

![](https://static.igem.org/mediawiki/2020/f/f8/T--MSP-Maastricht--Image50.png)
![](https://static.igem.org/mediawiki/2020/e/ef/T--MSP-Maastricht--Image51.png)

![](https://static.igem.org/mediawiki/2020/3/3e/T--MSP-Maastricht--Image52.png)
![](https://static.igem.org/mediawiki/2020/2/26/T--MSP-Maastricht--Image53.png)

## GEL ELECTROPHORESIS

#### Gel electrophoresis was used to separate and visualize our DNA fragments (obtained from the PCR) based on their size to see the success of amplification, as a first proof of the presence of the target fragments. In general, gel electrophoresis is conducted by injecting DNA sample of interest into wells and then using an electric current to pull the DNA through the gel. DNA fragments have a negative charge hence they will always be pulled towards the positive end of the electric current. The fragments are separated on the basis of their length and are compared to a reference (ladder). The gel electrophoresis protocols used were taken from the creators of the product; Invitrogen™ TrackIt™ 1 Kb Plus Ladder (Pub. No. MAN0000843).

## SEQUENCING

#### The OPC target genes amplified by PCR, were isolated and further amplified in a second PCR round in order to obtain a sufficient fragment concentration to be sequenced by Macrogen. This is necessary to ensure that the target sequences are exactly the same as the ones that were found on databases. Only this way, we can ensure that the siRNAs can specifically and efficiently bind to their selected target.

![](https://static.igem.org/mediawiki/2020/7/71/T--MSP-Maastricht--Image54.png)

![](https://static.igem.org/mediawiki/2020/8/86/T--MSP-Maastricht--Image55.png)
![](https://static.igem.org/mediawiki/2020/a/ac/T--MSP-Maastricht--EP1.png)

## PLASMID ASSEMBLY

#### GIBSON ASSEMBLY L4440 plasmids were isolated with the QIAprep® Spin Miniprep Kit from Qiagen. Afterwards, L4440 was linearized with PciI and NgoMIV in CutSmart® Buffer or NEBuffer™. The linearized plasmid was isolated from an agarose gel using QIAprep® Spin Miniprep Kit. Following this step, the expression cassette gBlock was added to the linearized plasmid in a 2:1 ratio and assembled using NEBuilder HiFi DNA Assembly Reaction Protocol. The resulting vector was transfected into high copy number DH5α cells. pUC19 plasmid was transfected to the cells as a positive control. Cells were grown to an exponential phase and plated onto ampicillin containing selection plates and incubated overnight. Finally, single colonies were picked and grown in overnight cultures. PLACING THE INSERTS The shRNA coding DNA-sequences were ordered as ssDNA oligonucleotides from IDT. The single strands were designed to form EcoRI and BamHI overhangs complementary to the ones created in L4440 by digest. The oligos were annealed in IDT oligonucleotide annealing buffer at 94°C for 3 min and then slowly cooled down to room temperature in order to ensure annealing despite potential secondary structures formed by the hairpin coding DNA. CREATING THE FINAL STRAIN The L4440 plasmids containing the expression cassette were isolated with the QIAprep® Spin Miniprep Kit from Qiagen. The plasmids were digested with the restriction enzymes EcoRI and BamHI in CutSmart® Buffer. Ligation protocol was used to insert the shRNA coding DNA-sequences into the plasmid. First, the plasmids were transfected into high copy number DH5α cells and spread on ampicillin containing selection plates. Afterwards, from successfully grown colonies, liquid cultures were made and the plasmids with the final construct were isolated. These plasmids were transfected into the final HT115(DE3) strain and spread on ampicillin+tetracycline containing selection plates.

## INDUCTION AND DETECTION OF shRNAs

#### shRNA production was induced by addition of IPTG (0.4mM) to induce the expression of our shRNAs. This procedure was followed by a total RNA extraction of the cells.

![](https://static.igem.org/mediawiki/2020/d/d5/T--MSP-Maastricht--Imagejfgrjc.png)

![](https://static.igem.org/mediawiki/2020/3/38/T--MSP-Maastricht--Image2535.png)

## HATCHING CATERPILLARS

#### To properly test the function of the shRNAs they must be tested on the target. In this case that would require OPCs that are in their earliest stages, which is when the pesticides would be sprayed on the leaves and subsequently kill them. For this we required live, young caterpillars that we could observe consume the shRNAs in our laboratory. The hatching protocol consisted of very few steps to properly maintain the climate conditions required for natural hatching of the eggs. The main device used was a climate chamber; in this machine we were able to mimic the conditions required for natural hatching of the eggds, such as temperature, humidity, and hours of light/dark exposure. We found that the optimal temperature was 18°C with approximately 12 hours of light in a day. The humidity was not a factor in the OPC egg hatching hence it was kept at room humidity. In further testing, we are planning to hatch additional caterpillars and equip the climate control chamber with oak branches sprayed with OakShield to test the influence of our pesticide on the caterpillars when ingested.

## PROTOCOLS

## DESIGN

![](https://static.igem.org/mediawiki/2020/0/03/T--MSP-Maastricht--Image118.png)

#### [Learn more](https://2020.igem.org/Team%3AMSP-Maastricht/Design)

## ENGINEERING

![](https://static.igem.org/mediawiki/2020/c/c5/T--MSP-Maastricht--Image122.png)

#### [Learn more](https://2020.igem.org/Team%3AMSP-Maastricht/Engineering)

## LAB BOOK

![](https://static.igem.org/mediawiki/2020/f/f1/T--MSP-Maastricht--Image120.png)

#### [Learn more](https://2020.igem.org/Team%3AMSP-Maastricht/Notebook)

![](https://static.igem.org/mediawiki/2020/3/37/T--MSP-Maastricht--F4.png)
![](https://static.igem.org/mediawiki/2020/d/de/T--MSP-Maastricht--F1.png)
![](https://static.igem.org/mediawiki/2020/7/78/T--MSP-Maastricht--F2.png)
![](https://static.igem.org/mediawiki/2020/c/c6/T--MSP-Maastricht--F3.png)
![](https://static.igem.org/mediawiki/2020/3/3b/T--MSP-Maastricht--F5.png)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:MSP-Maastricht/Results

Results

#### [![](https://static.igem.org/mediawiki/2020/4/49/T--MSP-Maastricht--Image2.png)](https://2020.igem.org/Team%3AMSP-Maastricht)

Project

[Description](https://2020.igem.org/Team%3AMSP-Maastricht/Description)
[Design](https://2020.igem.org/Team%3AMSP-Maastricht/Design)
[Experiments](https://2020.igem.org/Team%3AMSP-Maastricht/Experiments)
[Parts](https://2020.igem.org/Team%3AMSP-Maastricht/Parts)
[LabBook](https://2020.igem.org/Team%3AMSP-Maastricht/Notebook)
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Results

[Results](https://2020.igem.org/Team%3AMSP-Maastricht/Results)
[Engineering Success](https://2020.igem.org/Team%3AMSP-Maastricht/Engineering)
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[Human Practices](https://2020.igem.org/Team%3AMSP-Maastricht/Human_Practices)
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[Collaborations](https://2020.igem.org/Team%3AMSP-Maastricht/Collaborations)
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## Results ![](https://static.igem.org/mediawiki/2020/3/39/T--MSP-Maastricht--Image.png)

### DRY-LAB | MODEL | WET-LAB |

![](https://static.igem.org/mediawiki/2020/1/1d/T--MSP-Maastricht--Image30.png)

## DRY LAB RESULTS

[

](https://static.igem.org/mediawiki/2020/8/8b/T--MSP-Maastricht--Videosudusl.mp4)

### TARGET GENES

#### We as the MSP-Maastricht team have identified four main OPC target genes. These include, the gene for the subunit 2 of the allergenic protein Tha p2 that can be found in the urticating setae of the caterpillars [(Berardi, Battisti & Negrisolo, 2015)](https://2020.igem.org/Team%3AMSP-Maastricht/Attributions); Pro2 photolyase gene that is involved in proline metabolism; the EF-1a gene for elongation factor-1 alpha which takes part in the protein synthesis elongation phase; as well as, Wg gene for wingless protein, that is thought to be involved in numerous processes of the caterpillar's development [(Simonato et al., 2013).](https://2020.igem.org/Team%3AMSP-Maastricht/Attributions)

### siRNA + BLAST

#### Based on the designing guidelines and BLAST research for the specificity, the following sequences within target genes were identified (Table 1).

![](https://static.igem.org/mediawiki/2020/e/e8/T--MSP-Maastricht--Table.png)

#### *Table 1. For Tha p2, 5 DNA sequences were identified as target sites for the siRNA. 2 DNA sequences were identified for Wg and 5 for the Pro2 partial gene. For EF-1a gene 2 DNA sequences were found.*

### shRNA

#### Based on the obtained siRNA the following DNA sequences for the shRNA were designed.

![](https://static.igem.org/mediawiki/2020/a/a2/T--MSP-Maastricht--Table2.png)

#### *Table 2. For Tha p2, 5 DNA sequences were identified as target sites for the siRNA. 2 DNA sequences were identified for Wg and 5 for the Pro2 partial gene. For EF-1a gene 2 DNA sequences were found.*

### PLASMID DESIGN

#### T7-polymerase is prone to read-through transcription which is why we decided to include a termination signal in order to increase the yield of effective product RNAs produced by our strain [(Sturm, Saskoi, Tibor, Weinhardt, & Vellai, 2018).](https://2020.igem.org/Team%3AMSP-Maastricht/Attributions) Furthermore because the shRNA sequences were not possible to produce as dsOligos (because of their secondary structure) we decided to go with a universal insert (inserted with Gibson assembly) to which we can add any shRNA by using restriction enzymes. Finally, this design allows us to add multiple of the expression cassettes (consisting of promotor, shRNA and terminator) together into one construct, so we can later on create an “oakshield” strain expressing all the promising shRNAs of different targets.

![](https://static.igem.org/mediawiki/2020/e/e4/T--MSP-Maastricht--Plasmidf.png)

## MODEL RESULTS

![](https://static.igem.org/mediawiki/2020/9/9b/T--MSP-Maastricht--Image44.png)
![](https://static.igem.org/mediawiki/2020/7/79/T--MSP-Maastricht--Image43.png)

## DYNAMICAL SYSTEMS

#### From the model it is found that when 3 steady states exists, at least one of the steady states is unstable and, when environmental conditions change in just the right way, a stable point can merge with an unstable one and become a single half stable point before disappearing, thus leaving only one steady state. This explains how the population in an area can suddenly explode. Another interesting observation when not parameterising the model is that due to the small effect of predation (β), (R) will always be relatively large. This implies that the system will be more sensitive to changes in (Q). One of these factors is (α), in which a reduction of alpha increases (Q). However, since reducing (α) causes an increase in (R), it means that it cancels itself out. The only factor remaining then is (K’), the carrying capacity. It can be concluded that the biggest factor in determining the population is not predation or death rate, but the maximum carrying capacity of the ecosystem. This demonstrates that the population, if left alone, would keep increasing until the ecosystem collapses. Furthermore, it implies that the best approach to combat the OPC would be to reduce the general carrying capacity of the caterpillar instead of trying to directly combat the caterpillar. For more in-depth explanation of the model visit our [contribution page.](https://2020.igem.org/Team%3AMSP-Maastricht/Contribution)

## METABOLIC MODEL

#### A model containing 5407 reactions, 3526 metabolites, and 2248 genes was created. In this model 23 genes had a BIOMASS reduction greater than 10%, which is considered lethal [(Brunk et al., 2018).](https://2020.igem.org/Team%3AMSP-Maastricht/Attributions). 2 out of our 4 proposed genes were in that list: Pro2 and Acyl-CoA desaturase were classified as lethal, with a reduction of 16% and 13% in biomass production respectively. The WG gene was not lethal as there seemed to be many backup genes and alternative pathways. The model was not able to test the EF-1a since it was not programmed to take into account transcription.

![](https://static.igem.org/mediawiki/2020/8/8d/T--MSP-Maastricht--Image447.png)

## WET LAB RESULTS

### Transfections

#### The final constructed plasmids with our siRNA inserts were first cloned into DH5α high copy number cells and subsequently into HT115(DE3) cells. Both times pUC19 plasmids were used as positive control and showed colony growth on the plates, indicating a successful transfection process. The final HT115(DE3) cells capable of siRNA expression were plated on to ampicillin and tetracycline containing selection plates. To confirm the success of our transfection and construction method, we decided to first only transfect one siRNA per gene (Pro2\_A, Wg\_A, EF1a\_A, Tha p2\_A). Colonies grew and overnight cultures were prepared from single colonies for further analysis (Figure 1-4).

### Polymerase Chain Reaction

#### Touchdown PCRs were run on OPC DNA-extract samples resulting in amplicons of our desired target genes. Specific amplicons of the correct lengths were produced (Pro 2=651 bp, Wg= 164 bp, EF1a=541 bp, Tha p2= 269 bp). The bands of the Wg and Tha p2 were shorter than expected and after multiple PCRs and changes of primers, the bands did not become more intense. Therefore, we decided to sequence the two most promising bands and prepared these genes for sequencing, together with the other amplicons.

### Sequencing

#### The PCR amplicons of our target genes were sent to Macrogen for targeted sequencing (Figure 5): The Pro2 amplicons were almost 100% identical to our reference sequence and the amplicon spanned all 5 siRNA target regions (Pro2\_A, B, C, D, E siRNAs) with 100% identity. The EF1a amplicons were almost 100% identical to our reference sequence and the amplicon spanned both of the 2 siRNA target regions with 100% (EF1a\_A) and 86% (EF1a\_B) identity. The Wg amplicon only yielded results from the reverse primers sequencing. However, the short amplicon produced spans includes both siRNA target regions with 100% identity for both of the 2 siRNA target regions (Wg\_A) and (Wg\_B). The Tha p2 amplicon was split into two amplicons spanning the whole gene but only one primer produced sequencing results. Thus, only 3 out of 5 potential siRNA target regions were included in the produced amplicon (Tha p2\_C, D, E siRNAs) However, all of these 3 siRNA target regions were 100% identical to our reference sequence.

### Confirmation of Successful Gibson Assembly of Empty Expression Vector

#### To confirm that our expression cassette was indeed inserted into the L4440 Plasmid, we performed a restriction digest with the two enzymes PciI and NgoMIV on the plasmid isolated from the bacterial strain used for transfections. Since the Gibson assembly removed the the PciI & NgoMIV restriction sites, the empty siRNA expression vector should not be cut, while the empty L4440 vector would be cut. This would leave us with the predicted bands on the gel. Our gel perfectly matches the predicted gel, confirming that the Gibson assembly of our siRNA expression vector was successful (Figure 6). Unfortunately, our final HT115(DE3) *E. coli* strain did not seem to produce shRNAs after IPTG induction. To confirm that our siRNA inserts were assembled into our vector, we performed a restriction digest with EcoRI & BamHI to remove and detect our shRNA inserts. However, the predicted bands were not seen on the gel. This could be possibly due to low shRNA insert concentration and/or low ligation efficiency. However, we will sequence the produced plasmids to confirm the shRNA insert presence in the vector and repeat the insertion step if necessary (Figure 7).

### Testing the Lifetime of Our Bacteria on Oak Trees

#### To determine the efficacy of our bacteria we wanted to test how long our bacterial strain could survive on the oak trees. To do this we collected oak branches and sprayed these with our HT115(DE3) *E. coli* strain. SRK hygiene monitoring swabs were used to swab our branches every 24 hours and 500 μL of isotonic solution were plated on to tetracycline selection plates. Spraying was performed twice, once with medium containing tetracycline and once with regular LB medium. The bacteria sprayed in LB could not be detected after 48 hours. However, the bacteria with tetracycline medium survived on the branches for 4 days. Of course, the tetracycline would have an effect on the microbiome of oak trees (Figure 8-9).

![](https://static.igem.org/mediawiki/2020/0/05/T--MSP-Maastricht--4748.png)

#### Figure 1. Selection plates with EF1a\_A insert in HT115(DE3)*E. coli* strain.

![](https://static.igem.org/mediawiki/2020/c/c5/T--MSP-Maastricht--47484.png)

#### Figure 2. Selection plates with Pro2\_A insert in HT115(DE3)*E. coli* strain.

![](https://static.igem.org/mediawiki/2020/b/b1/T--MSP-Maastricht--4484.png)

#### Figure 3. Selection plates with The p2\_A insert in HT115(DE3)*E. coli* strain.

![](https://static.igem.org/mediawiki/2020/a/a3/T--MSP-Maastricht--44284.png)

#### Figure 4. Selection plates with Wg\_A insert in HT115(DE3)*E. coli* strain.

![](https://static.igem.org/mediawiki/2020/2/29/T--MSP-Maastricht--fdhdicgeg.png)

#### Figure 5. Schematic overview of siRNA target sequences in Wg, Pro2, EF1a and The p2 genes.

![](https://static.igem.org/mediawiki/2020/d/da/T--MSP-Maastricht--48934.png)

#### Figure 6. Predicted and obtained gels for Gibson assembly confirmation. Well 1: plasmid with expression cassette. Well 2: uncut plasmid without expression cassette.

![](https://static.igem.org/mediawiki/2020/f/f6/T--MSP-Maastricht--imagesafetyf7.png)

#### Figure 7. Gel for shRNA DNA insertion, containing plasmids with EF1a\_A, Pro2\_A, The p2\_A and Wg\_A.

![](https://static.igem.org/mediawiki/2020/d/dd/T--MSP-Maastricht--imagesafety7.png)

#### Figure 8. Oak branch testing set up for spraying with HT115(DE3) cells.

![](https://static.igem.org/mediawiki/2020/1/13/T--MSP-Maastricht--imagesafety8.png)

#### Figure 9. Spray bottle used in branch testing, filled with HT115(DE3) in LB medium.

## DESIGN

![](https://static.igem.org/mediawiki/2020/0/03/T--MSP-Maastricht--Image118.png)

#### [Learn more](https://2020.igem.org/Team%3AMSP-Maastricht/Design)

## ENGINEERING

![](https://static.igem.org/mediawiki/2020/c/c5/T--MSP-Maastricht--Image122.png)

#### [Learn more](https://2020.igem.org/Team%3AMSP-Maastricht/Engineering)

## LAB BOOK

![](https://static.igem.org/mediawiki/2020/f/f1/T--MSP-Maastricht--Image120.png)

#### [Learn more](https://2020.igem.org/Team%3AMSP-Maastricht/Notebook)

![](https://static.igem.org/mediawiki/2020/3/37/T--MSP-Maastricht--F4.png)
![](https://static.igem.org/mediawiki/2020/d/de/T--MSP-Maastricht--F1.png)
![](https://static.igem.org/mediawiki/2020/7/78/T--MSP-Maastricht--F2.png)
![](https://static.igem.org/mediawiki/2020/c/c6/T--MSP-Maastricht--F3.png)
![](https://static.igem.org/mediawiki/2020/3/3b/T--MSP-Maastricht--F5.png)