iGEM 2024 — Team heidelberg

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{% 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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# Notebook

### **A Detailed Record of Our Project**

Explore our wet lab notebooks, where you will find comprehensive PDFs documenting every experiment from our iGEM project. Each notebook provides a detailed record of the protocols, results, and insights that shaped our project.

[Scroll down](#CasStaples)

* [CRISPR/Cas Staples](#CasStaples)
* [Staple Functionalization](#StapleFunctionalization)
* [Basic Staples](#ReadoutSystems)
* [Delivery System](#DeliverySystem)
* [Back to the top](#top)

## CRISPR/Cas Staples

### Fusion Cas

### CRISPR/Cas Staples

### Enhancer Hijacking

## Staple Functionalization

### Cathepsin B-Cleavable Linker

### In Vitro Intein Trans-Splicing

## Basic Staples

### Simple Staples

### Mini Staples

## Delivery System

### Conjugation

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
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# Engineering Cycle

### **Wet Lab**

Discover our step-by-step **development process of CRISPR/Cas-based protein staples** for engineering the 3D genome architecture. See how we build upon the commonly used CRISPR gene editing tool to create easily programmable, highly specific, bivalent DNA binding protein complexes, that can bring any DNA elements of choice into spatial proximity.

### **Dry Lab**

Learn how we developed DaVinci, a comprehensive PICasSO modeling platform, by iterating through **multiple engineering cycles.** Find out how we overcame software constraints and computational limitations and discovered workarounds to **adjust hyperparameters precisely**.

### **Human Practices**

Explore our implementation of a science-based, sustainable, and holistic approach to integrated human practices. Discover how we expanded the possibilities of synthetic biology education, inspiring and empowering the next generation of young scientists. Learn about our journey from **identifying the specific needs in scientific education** to developing a refined and impactful educational program.

[Scroll down](#wetlab1)

### Wet Lab

[∞](#wetlab1)
[∞](#wetlab2)
[∞](#wetlab3)
[∞](#wetlab1)
[∞](#wetlab2)
[∞](#wetlab3)

### Dry Lab

[∞](#drylab1)
[∞](#drylab2)
[∞](#drylab3)

### Human Practices

[∞](#hp1)
[∞](#hp2)
[∞](#hp3)

##### Click on any cycle icon for fast navigation!

##### Fusion guide RNAs

##### Fusion Cas Proteins

##### Cas staples

##### Cathepsin B

##### FRET Measurement

##### Delivery System

##### Structure Prediction

##### MD-Simulations

##### Wet Lab Integration

##### Survey

##### Summer School

##### School Workshops

## Fusion guide RNAs

## Iteration 1/5

### Design

Building on insights from Iteration 1 of the fusion Cas engineering cycle and findings from Kweon *et al.* (2017), fusion guide RNAs (fgRNAs) were designed by combining the sgRNA from SpCas9 with the crRNA from Cas12a. The goal of the first iteration is to replicate the results presented in the literature and demonstrate the editing activity of both proteins using fgRNA. The genes **VEGFA** and **FANCF** were selected as targets for Cas12a and Cas9, each target was tested with each Cas protein. Editing efficiency will be analyzed with the T7 Endonuclease I (T7EI) assay. Controls will include crRNAs and sgRNAs as positive controls, and non-targeting guides as negative controls.
An entry vector was designed to facilitate efficient cloning of different spacer sequences. The entry vector includes a U6 promoter, the Cas12a scaffold, a bacterial promoter driving **ccdB** expression, and the SpCas9 scaffold. Successful spacer integration leads to the removal of the **ccdB** gene, allowing bacterial growth to be used as an indicator for cloning success.

Kweon, J., Jang, A.-H., Kim, D.-e., Yang, J. W., Yoon, M., Rim Shin, H., Kim, J.-S., and Kim, Y. (2017). Fusion guide RNAs for orthogonal gene manipulation with Cas9 and Cpf1. Nature Communications 8, doi:10.1038/s41467-017-01650-w.

### Build

An existing vector containing the U6 promoter and the MbCas12a scaffold was selected as the basis for entry vector cloning. The vector and a ccdB-SpCas9 scaffold construct were PCR amplified and fitting overhangs for SapI were introduced. Golden gate assembly with Esp3I was used to create the final plasmid. The transformation was carried out in the ccdB-resistant XL1 Blue *E. Coli*  strain.
Desired spacer sequences can be ordered as oligos, annealed, and cloned in via golden gate with SapI.

![](https://static.igem.wiki/teams/5237/wetlab-results/entry-vector.svg)

**Figure 1: Concept of the entry vector.** The entry vector would have a ccdB in between the two scaffolds. SapI sites can be utilized for Golden Gate assembly cloning in an oligo anneal with fitting overhangs. On the right site the assembled gene fragment is shown.

### Test

The T7EI assay showed more bands than expected for both tested loci, potentially due to nonspecific primer binding. However, clear editing activity was observed (highlighted with an arrow).

![](https://static.igem.wiki/teams/5237/engineering/engin-ba/gel-image-bad-primers.png)

**Figure 2: First test of fgRNA in a T7EI assay.** T7EI samples were run on a 2% agarose gel in 1x TBE buffer dyed with gel red. The top gel was used for the FANCF locus and the bottom gel for VEGFA locus targeting.
Editing is indicated by an extra band compared to the negative control indicated by the arrow.

### Learn

Overall low editing activity was observed. Additionally, the PCR conditions require further optimization to improve specificity and reduce nonspecific amplification. Together these results were not sufficient to unambiguously prove editing activity.

# Fusion guide RNAs

## Iteration 2/5

### Design

To minimize possible off-target amplification, different annealing temperatures were tested ranging from 60 °C up to 72 °C. In parallel, a new set of primers was designed to use for the assay.

### Build

New primers for *VEGFA* and *FANCF* were designed using the NCBI primer BLAST tool to ensure high target fidelity.

### Test

The old primers, tested across different annealing temperatures, did not show a notable improvement compared to the previous test. However, the new primers performed better, revealing editing for all samples tested.
No significant difference in editing efficiency was observed between AsCas12a and MbCas12a. The fgRNA constructs showed overall lower editing activity compared to sgRNA controls.

![](https://static.igem.wiki/teams/5237/engineering/engin-ba/gel-image-fancf.png)
A

![](https://static.igem.wiki/teams/5237/engineering/engin-ba/gel-image-vegfa-pcr.png)
B

![](https://static.igem.wiki/teams/5237/wetlab-results/results-ascas-2.svg)
C

**Figure 3: Primer evaluation for further T7EI testing.** A and B, different primer pairs for VEGFA and FANCF tested at different temperatures run on a 2 % agarose gel in 1x TBE. Gel image was taken before the T7 endonuclease I digestion step. N, P and S mark the negative control, positive control and a sample, respectively. Contrast was increased to check for byproducts.
C, after T7EI digestion, samples were run on a 2% agarose gel in 1x TBE buffer dyed with gel red (gel image not shown). Editing rate for the T7EI assay, calculated with ImageJ from the gel image after T7EI digestion, is plotted. The schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below show which parts are in each sample.

### Learn

The newly designed primers produced improved results, allowing for a more accurate assessment of editing activity. No notable difference in editing efficiency between AsCas12a and MbCas12a was observed. Given the insights gained from the fusion Cas engineering cycle, we chose to only continue with MbCas12a for future experiments.

# Fusion guide RNAs

## Iteration 3/5

### Design

Building upon the findings from the fusion Cas engineering cycle, we aimed to combine the fgRNA with the fusion Cas protein to assess their combined editing activity. For this purpose, we adapted the previously optimized T7 Endonuclease I (T7EI) assay, using the established primer pair.

### Build

Since no prior T7EI assay had been conducted with the fusion Cas, we carefully adjusted the transfection ratios. As the fusion Cas combines the two previously tested Cas proteins into one construct, we therefore used the cumulative transfection amounts of the individual Cas proteins.

### Test

The assay shows no editing for the non-targeting negative control, and strong editing for the single Cas protein positive control. The co-transfected single Cas proteins display comparable editing activity with the positive control. The fusion Cas proteins exhibited editing activity in combination with fgRNA, albeit lower than compared to sgRNAs. Notably, better editing efficiency could be shown for VEGFA compared to FANCF.

![](https://static.igem.wiki/teams/5237/engineering/engin-ba/results2-fancf-vegfa.png)
A

![](https://static.igem.wiki/teams/5237/engineering/engin-ba/results2-vegfa-fancf.png)
B

![](https://static.igem.wiki/teams/5237/engineering/engin-ba/results2-vegfa-ccr5.png)
C

**Figure 4: Editing rates for fusion guide RNAs with fusion Cas proteins.** A - C, after T7EI digestion samples were run on a 2% agarose gel in 1x TBE buffer dyed with gel red (gel image not shown). Editing rate for the T7EI assay, calculated with ImageJ from the gel image after T7EI digestion, is plotted. The schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below show which parts are in each sample. Cas proteins linked by a dash ("–") were fused to each other. Replicates are marked as individual dots.

### Learn

The T7EI assay confirmed fusion Cas activity for both targets in combination with fgRNA, demonstrating the feasibility of using fusion Cas proteins and fgRNAs in our system. The results from this and previous iterations, along with the fusion Cas engineering cycle, establish a strong foundation for developing the Cas staples. However, simultaneous binding of both Cas proteins to the fgRNA can not be shown with this type of assay. To address this, alternative experiments need to be conducted.

# Fusion guide RNAs

## Iteration 4/5

### Design

A proximity assay based on CRISPR activation (CRISPRa) was developed to assess simultaneous binding of dSpCas9 and dMbCas12a. The fgRNA was designed with a tetO target site for dSpCas9 and a non-targeting spacer for dMbCas12a, which was fused to the transcriptional activator VP64. In the assay, a minimal promoter and firefly luciferase gene were positioned adjacent to the tetO binding site. This design ensures that luciferase expression is only induced when both dSpCas9 and dMbCas12a bind to the fgRNA simultaneously. A non-targeting spacer and dSpCas9-VP64 were included as negative and positive controls, respectively.

![](https://static.igem.wiki/teams/5237/figures-corrected/luciferase-assay-dcas12avp64-dcas9binds-correct.svg)
A

![](https://static.igem.wiki/teams/5237/figures-corrected/luciferase-assay-dcas12a-dcas9vp64binds-correct.svg)
B

**Figure 5: Design to test simultaneous binding of Cas9 and Cas12a to the same fgRNA.** A shows our model for a successful sample. For the sample, the fgRNA consists of a non-targeting spacer for Cas12a and a TRE targeting spacer for dCas9 resulting in dCas9 binding. dCas-RNA complex formation of the dCas12a with the fgRNA already complexed by the dCas9 would result in activation of the Luciferase gene.
In B we also depict the positive control, dCas9 being fused to VP64, and the negative control, both spacers of the fgRNA being non-targeting.

### Build

The VP64 gene was PCR amplified and inserted into an already existing plasmid containing dMbCas12a. The final plasmid was then assembled with golden gate assembly. Multiple fgRNA constructs with varying linker lengths were designed and cloned in as annealed oligos in the previously described entry vector.

### Test

The positive and negative controls produced the expected outcomes. However, no binding of dSpCas9 or recruitment of dMbCas12-VP64 could be demonstrated across all linker types tested. Data normalization using Renilla luciferase was not possible due to a planning error made during the assay preparation.

![](https://static.igem.wiki/teams/5237/engineering/engin-ba/results-luci1.png)

**Figure 6: Testing simultaneous complexing of the fgRNA with Cas9 and Cas12a.** Firefly luminescence intensity measured 48 h after transfection. On the x-axis the negative control (empty spacer), positive control (sgRNA, no linker, 5 nt linker, 10 nt linker) and the sample ( no linker, 5 nt linker, 10 nt linker). The symbols above show the composition of each sample.
Data is depicted as the mean +/- SD (n=3).
No statistical analysis was performed.

### Learn

Despite the lack of data normalization, incorrect evaluation is unlikely, as both controls performed as expected. Further literature research was conducted to explore potential reasons for the assay outcome and no published data supporting the functionality of a dMbCas12a-VP64 construct could be found. Additionally, incorrect transfection ratios may have prevented efficient Cas protein binding to the fgRNA, potentially contributing to the lack of observed activity. Based on these findings, the assay design was critically re-evaluated and revised.

# Fusion guide RNAs

## Iteration 5/5

### Design

Based on previous learnings, the assay was re-designed. dSpCas9 was fused to VP64 and dMbCas12a was used with a targeting spacer to position dSpCas9 near the minimal promoter and firefly luciferase.

![](https://static.igem.wiki/teams/5237/figures-corrected/luciferase-assay-dcas12abinds-dcas9vp64-correct.svg)

**Figure 7: Improved design to test simultaneous binding of Cas9 and Cas12a to the same fgRNA.** Composition changed for the following iteration of the assay compared to figure 5. For the sample the fgRNA consists of a non-targeting spacer for dCas9 and a TRE targeting spacer for dCas12a resulting in dCas12a binding. dCas-RNA complex formation of the Cas9 with the fgRNA already complexed by the dCas9 would result in activation of the Luciferase gene by the VP64 fused to the dCas9.

### Build

Since both dCas12a and dCas9-VP64 plasmids were already available, no additional cloning was required for these components. The modified fgRNAs, containing a dMbCas12a spacer targeting tetO and a non-targeting dCas9 spacer, were cloned into the entry vector via a Golden Gate reaction. Different fgRNA concentrations were transfected to explore varying fgRNA to protein ratios.

### Test

Similar to the previous outcome, the controls worked as expected.
No change in firefly expression could be shown with all tested fgRNA constructs.

![](https://static.igem.wiki/teams/5237/engineering/engin-ba/results-luci3.png)

**Figure 8: Testing simultaneous complexing of the fgRNA with an improved design.** Firefly luminescence intensity measured 48 h after transfection. Normalized against renilla luminescence. On the x-axis the negative control (empty spacer), positive control (sgRNA, CG-linker) and the sample (GC-linker bottom strand, CG-linker top strand, GC-linker bottom strand 1 ng, CG-linker top strand 1 ng). The symbols above show the composition of each sample.
Data is depicted as the mean +/- SD (n=3).
No statistical analysis was performed.

### Learn

The adjustments made in this iteration did not lead to improved assay outcomes. Simultaneous binding of both Cas proteins with the fgRNA could still not be confirmed. Future experiments may require the development of new assay systems and further optimization such as adjustments to the linker lengths.

# Fusion Cas proteins

## Iteration 1/6

### Design

Our idea was to utilize catalytically inactive Cas (dCas) proteins to bring two DNA strands into close proximity. We decided to use dSpCas9 and different dCas12a orthologs (MbSas12a, AsCas12a, and LbCas12a), for further testing. As a start, we focused on well established editing assays, assuming that editing capabilities are positively correlated with DNA binding capabilities. Editing capabilities were tested with a T7 Endonuclease I (T7EI) assay.

### Build

For the T7EI, we transfected a SpCas9 targeting CCR5 and each Cas12a ortholog targeting Runx1, Dnmt1 and FANCF. The Cas proteins were constitutively expressed by a CMV promoter and the sgRNA/crRNA were expressed by a U6 promoter. Transfection ratios were chosen as established. Cells were lysed 72 hours post-transfection, and regions of approximately 1000 base pairs around the cut sites were amplified by PCR for analysis. T7EI recognizes and cleaves mismatches generated by the DNA repair machinery, resulting in two DNA fragments of roughly equal size. These fragments were visualized on an agarose gel, indicating whether the target sites were cleaved. Quantification of editing efficiency was performed using the ImageJ software for image analysis.

### Test

The results of the T7EI assay are depicted in the figure below. SpCas9 worked, as expected, the best. The Cas12a orthologs displayed lower editing capabilities overall, but demonstrated detectable activity at the RUNX1 locus. Among the Cas12a variants, AsCas12a showed the strongest activity, followed by MbCas12a and LbCas12a. Due to a PCR error, no band was visible for the negative control of RUNX1. However, since all negative controls originated from the same lysate, it is valid to compare this result with the other RUNX1 controls. Unfortunately, editing at the FANCF locus could not be confirmed due to cloning errors during the preparation of constructs.

![](https://static.igem.wiki/teams/5237/engineering/fuscas-engi-fig1-1.svg)
![](https://static.igem.wiki/teams/5237/engineering/fuscas-engi-fig1-2.svg)

**Figure 1: Preliminary T7 Endonuclease I testing of Cas12a orthologs.** T7EI samples were run on a 2% agarose gel in 1x TBE buffer dyed with gel red. SpCas9 targeting CCR5 functions as a benchmark for proper editing. For the Cas12a orthologs, LbCas12a, AsCas12a and MbCas12a, the three loci RUNX1, DNMT1 and FANCF were targeted. Editing is indicated by an extra band compared to the negative control.

Also due to a cloning mistake, we were only able to test editing for the FANCF locus with the LbCas12a and the AsCas12a, but not with the MbCas12a.
As one can see, the editing for Dnmt1 and FANCF was non-existent, suggesting that either the chosen site for editing was inaccessible or the spacer being expressed in the crRNA was not able to properly bind to the Cas12a.

### Learn

Following the T7EI assay results, we identified AsCas12a and MbCas12a as the most promising candidates for further development, while LbCas12a was excluded due to its poor performance. To improve the accuracy and speed of our proximity measurements, we decided to switch to an alternative assay, with faster turn-around time and better quantification. Future iterations will focus on developing new assays and successfully implementing the fusion Cas.

# Fusion Cas proteins

## Iteration 2/6

### Design

After extensive literature research, we identified this as a promising assay for quantifying editing efficiency. This assay measures the luminescence produced by Firefly luciferase, which decreases in proportion to editing efficiency at the target site. To account for variations in cell count and transfection efficiency, the luminescence is normalized to Renilla luciferase, which acts as an internal control. We hope this assay provides a straightforward and efficient approach for measuring editing activity in a high-throughput 96-well plate format.

### Build

We tested SpCas9, AsCas12a, and MbCas12a using sgRNA/crRNA constructs targeting the Firefly luciferase gene. To optimize the system, two variations of the reporter construct were designed: one where both luciferase genes were located on the same plasmid, and an alternative version with the Firefly and Renilla luciferase genes on separate plasmids.

### Test

The results from the Dual-Luciferase assay were consistent with the general trend observed in the previous iteration, with SpCas9 showing the highest editing efficiency. However, this assay provided qualitative resolution in distinguishing between the Cas12a orthologs. Contrary to the previous assumption, MbCas12a exhibited higher editing efficiency than AsCas12a. The one-plasmid system, with both luciferases on the same construct, showed significantly reduced editing efficiency compared to the two plasmid system (p=0.0002) but still provided a reliable distinction for the SpCas9. Strongest difference between the two reporter systems was visible when comparing SpCas9 editing. Both showed very little editing for AsCas12a but no significant differences. For MbCas12a, we can see better editing than AsCas12a with both reporters.

![](https://static.igem.wiki/teams/5237/engineering/fuscas-engi-fig2-v2.svg)

**Figure 2: Assessment of a suitable reporter system for future dual luciferase assays.** Firefly luminescence intensity measured 48 h after transfection. Normalized against renilla luminescence. On the x-axis, the samples Cas9, AsCas12a and MbCas12a are grouped by assay tested.
Data is depicted as the fold change calculated from the non-targeting negative control over the targeting sample displayed as the mean +/- SD (n=3).
Statistical analysis was performed using 2way ANOVA with Tukey’s multiple comparisons test, only significant differences within a group are shown. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001.

### Learn

The Dual-Luciferase assay successfully confirmed higher editing activity for SpCas9. Interestingly, this iteration revealed that MbCas12a outperformed AsCas12a, a finding that contradicts the previous iteration’s results. Because of this discrepancy, we continued testing both Cas12a orthologs.
The assay provided an easy way to efficiently test out different constructs and transfection concentration, while still providing useful and quantitative data. Based on the overall stronger editing, the two plasmid system will be adopted for future assays.

# Fusion Cas proteins

## Iteration 3/6

### Design

Building on our promising results and further literature research (Kweon *et al.* (2017)), we fused together MbCas12a to SpCas9 and AsCas12a to SpCas9. These new Cas staple could provide one way to create functional staple, capable of bringing two DNA loci into close proximity. To ensure efficient nuclear delivery and expression, the Cas staples were modified with N-terminal (SV40 NLS) and C-terminal (nucleoplasmin NLS) nuclear localization signals.

Kweon, J., Jang, A.-H., Kim, D.-e., Yang, J. W., Yoon, M., Rim Shin, H., Kim, J.-S., and Kim, Y. (2017). Fusion guide RNAs for orthogonal gene manipulation with Cas9 and Cpf1. Nature Communications 8, doi:10.1038/s41467-017-01650-w.

### Build

The constructs were created by PCR amplifying the Vector of the previously tested SpCas9 construct. The PCR product included the plasmid backbone, CMV promoter, and both NLS sequences. The insert, containing the other Cas proteins, was also amplified from the previously tested plasmid.
Golden Gate assembly was used to construct the final plasmids, utilizing overhangs with enzyme recognition sites introduced during PCR.
The assembled plasmids were verified through sequencing and subsequently transfected into HEK293T cells for functional testing using the Dual-Luciferase Assay.

### Test

The Dual-Luciferase Assay was used to compare the editing efficiency of the fused Cas proteins with each other and their co-transfected single counterparts. In each construct, only one Cas protein targeted the Firefly luciferase gene, allowing for a direct comparison of editing efficiency.
For the single co-transfected Cas proteins, the SpCas9 showed, as expected, the highest editing efficiency, shown by low relative luminescence units. Contradicting the T7EI, but being in-line with the previous dual luciferase, MbCas12a showed better editing efficiency compared to AsCas12a (p=0.005). The Fusion Cas proteins exhibited less editing efficiency compared to the single counterparts. Of the MbCas12a-SpCas9 fusion, SpCas9 showed higher efficiency. For the Cas12s in the fusion Cas, both proteins exhibit low editing activity, with no significant difference.

![](https://static.igem.wiki/teams/5237/engineering/fuscas-engi-fig3-v2.svg)

**Figure 3: Comparison of AsCas12a and MbCas12a with a dual luciferase assay.** Firefly luminescence intensity measured 48 h after transfection. Normalized against renilla luminescence. On the x-axis, the samples Cas9 + AsCas12a , Cas9 + MbCas12a, AsCas12a and MbCas12a are grouped by fused proteins. Co-transfected contains single Cas proteins, in contrast to the fusion Cas having the same cas proteins covalently bound to each other.
Data is depicted as the mean +/- SD (n=3).
Statistical analysis was performed using 2way ANOVA with Tukey’s multiple comparisons test. For better clarity, only significant differences within a group between the same Cas proteins are shown. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001

### Learn

This iteration confirmed that our selected Cas fusions show editing. Based on the qualitative comparison to the single Cas proteins, improvements can still be made. Based on the results, we chose to omit the AsCas12a-SpCas9 fusion construct and only focus on MbCas12a-SpCas9 for further development. This decision was done on the basis of future plans, further comparing single co-transfected Cas proteins with fusion Cas proteins. We believe both to be suitable for stapling but further tests are needed beforehand.

# Fusion Cas proteins

## Iteration 4/6

### Design

After successfully showing editing activity of each part of the Cas staple, we aimed to test simultaneous editing activity.
We adapted the previously established Dual-Luciferase assay, where MbCas12a continued to target the Firefly luciferase gene, and SpCas9 was now designed to target the N-terminus of Renilla luciferase. This modification enabled simultaneous monitoring of editing at both targets.
For normalization, GFP was constitutively expressed as a new baseline. However, to ensure that GFP fluorescence would not interfere with the luminescence readout, a parallel test without GFP was conducted to serve as a control.

### Build

To introduce the SpCas9 target site, a well-characterized protospacer was cloned at the N-terminus of the Renilla luciferase gene. Since RenLuc would no longer be used as a baseline for normalization, the transfection ratio required adjustment. We matched the RenLuc plasmid amount to that used for Firefly luciferase (FFLuc) in previous experiments and similarly matched the GFP plasmid amount, which now served as the new baseline. Corresponding adjustments were made to the Cas protein and guide RNA amounts. For the test without GFP, additional staple plasmid was transfected, matching the amount of missing GFP.

### Test

The EGFP as baseline led to great variability between measurements. Sadly the negative controls for the co-transfected single Cas proteins did not yield the expected results. Therefore no significance could be calculated. However the fusion Cas negative controls worked great. Overall the results indicate significant editing for MbCas12a in the fusion Cas (p=0.0073) and for the SpCas9 in the fusion Cas in the single sample (p < 0.0001) and both editing (p < 0.0001). Unfortunately the MbCas12a had no significant editing in both editing samples.

![](https://static.igem.wiki/teams/5237/engineering/fuscas-engi-fig4.svg)

**Figure 4: Double cut dual luciferase assay with EGFP baseline for simultaneous editing assessment.** On the left Firefly luminescence intensity is plotted and on the right renilla luminescence intensity is plotted, both measured 48 h after transfection. Normalized against EGFP fluorescence. On the x-axis the samples non targeting (nt), SpCas9, MbCas12a and Cas9 and Cas12a editing at the same time (both) , are grouped by single and fused proteins. Co-transformed contains single Cas proteins, in contrast to the fusion Cas having the same cas proteins covalently bound to each other. Firefly editing is showing Cas12a editing while lower renilla counts are caused by Cas9 editing.
Data is depicted as the mean +/- SD (n=3).
Statistical analysis was performed using 2way ANOVA with Sidaks’s multiple comparisons test. For better clarity, only significant differences within a group between the negative control and editing Cas proteins are shown. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001

Retransfecting the same assay lacking GFP showed similar results as the previous test. The control of the co-transfected Cas proteins’, once again did not work as expected. Although some data indicated successful editing, it lacked consistency. In contrast, the fusion Cas construct exhibited reliable controls and demonstrated highly significant editing at both targets, both individually and simultaneously (for all p < 0.0001), reinforcing the effectiveness of the Cas fusion strategy in this assay.
Taking a qualitative look at the co-transfected single Cas proteins is strongly suggesting high editing rates for both MbCas12a and SpCas9 in the single and simultaneous samples.

![](https://static.igem.wiki/teams/5237/engineering/fuscas-engi-fig5.svg
            )

**Figure 5: Double cut dual luciferase assay without EGFP baseline for simultaneous editing assessment.** On the left Firefly luminescence intensity is plotted and on the right renilla luminescence intensity is plotted, both measured 48 h after transfection. Normalized against EGFP fluorescence. On the x-axis the samples non targeting (nt), SpCas9, MbCas12a and Cas9 and Cas12a editing at the same time (both) , are grouped by single and fused proteins. Co-transformed contains single Cas proteins, in contrast to the fusion Cas having the same cas proteins covalently bound to each other. Firefly editing is showing Cas12a editing while lower renilla counts are caused by Cas9 editing.
Data is depicted as the mean +/- SD (n=3).
Statistical analysis was performed using 2way ANOVA with Sidaks’s multiple comparisons test. For better clarity, only significant differences within a group between the negative control and editing Cas proteins are shown. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001

### Learn

Although the assay did not yield the expected results, we could still draw valuable conclusions out of it. Most importantly the Cas proteins can be fused together while retaining functionality, though a quantitative comparison to the individual Cas proteins is not yet feasible without proper controls.
Interestingly the EGFP was not the sole problem, co-transfection of multiple Cas proteins appears to prove challenging with a proper negative control.
Another observation we thought important, was reduced growth rate in cells harboring the fusion Cas protein. This could be due to the large construct size which may stress the cells. It is apparent when comparing the relative luminescence units (RLU) between groups, for the with fusion Cas transfected cells to have lower RLU in general because of metabolic burden expressing this large protein fusion.

# Fusion Cas proteins

## Iteration 5/6

### Design

To mitigate the apparent problems and further improve editing, we sought to create a stable cell line for continuous fusion Cas expression. To ensure successful genomic integration and stable expression, the open reading frame was expanded with a P2A peptide and puromycin resistance gene (PuroR) downstream of the fusion Cas protein. The P2A peptide facilitates ribosomal skipping, allowing both the Cas fusion protein and PuroR to be expressed from the same mRNA while being translated as separate proteins.

### Build

The plasmid harboring the fusion Cas protein and a second plasmid containing the P2A-Puromycin resistance sequence were PCR-amplified and fitting overhangs for subsequent Golden Gate assembly were introduced. After verifying the correct assembly via sequencing, the final plasmid construct was transfected into HEK293T cells. Selection pressure was applied by addition of puromycin. As a negative control the fusion Cas plasmid lacking the selection gene was transfected.

The creation of the stable cell line proceeded as expected. After initial growth in 6-well plates, the cells were successfully transferred to T75 flasks for continued expansion. Due to a noticeably reduced growth rate, a weekly splitting schedule was implemented.

### Test

The stable cell line was used to repeat both the standard dual luciferase assay (iteration 3) and the modified version for simultaneous editing (iteration 4).
While the data exhibited high variability, some trends could still be discerned for the editing to work in the co-transfected Cas12a (p=0.0018). As for the stable cell line small changes in luminescence intensity with no significance still allow a qualitative assessment of the data. Overall the luciferase expression, and changes thereof, are smaller than compared to the co-transfected samples.

![](https://static.igem.wiki/teams/5237/engineering/fuscas-engi-fig6.svg)

**Figure 6: Single cut dual luciferase assay repeating preliminary tests.** Firefly luminescence intensity measured 48 h after transfection. Normalized against renilla luminescence. On the x-axis the samples nt, Cas9 and Cas12a are grouped by single and fused proteins. Co-transformed contains single Cas proteins, in contrast to the fusion Cas having the same Cas proteins covalently bound to each other.
Data is depicted as the mean +/- SD (n=3).
Statistical analysis was performed using 2way ANOVA with Tukey’s multiple comparisons test. Only significant differences within a group are shown. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001

The modified luciferase assay, designed to measure simultaneous cutting efficiency, provided more consistent results. The co-transfection of SpCas9 (p < 0.0001) and MbCas12a (p < 0.0001) demonstrated highly significant editing for both Cas proteins, both individually and simultaneously (for both Cas proteins p < 0.0001). The stable cell lines did not show significant editing activity, with only minor changes in expression levels allowing just vague qualitative interpretation.

![](https://static.igem.wiki/teams/5237/engineering/fuscas-engi-fig7.svg)

**Figure 7: Double cut dual luciferase assay testing fusion Cas in stable cell line.** On the left Firefly luminescence intensity is plotted and on the right renilla luminescence intensity is plotted, both measured 48 h after transfection. Normalized against EGFP fluorescence. On the x-axis the samples non targeting (nt), SpCas9, MbCas12a and Cas9 and Cas12a editing at the same time (both) , are grouped by single and fused proteins. Co-transformed contains single Cas proteins, in contrast to the fusion Cas having the same cas proteins covalently bound to each other. Firefly editing is showing Cas12a editing while lower renilla counts are caused by Cas9 editing.
Data is depicted as the mean +/- SD (n=3).
Statistical analysis was performed using 2way ANOVA with Tukey’s multiple comparisons test. For better clarity, only significant differences within a group between the negative control and editing Cas proteins are shown. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001

### Learn

Further optimization is required to determine the ideal ratios for the fusion Cas constructs. Integration of the fusion Cas proteins into the genome resulted in lower expression levels, which may not be sufficient to achieve significant editing activity. This iteration all the negative controls for the co-transfected Cas proteins worked beautifully, finally allowing for proper significance testing. These can now function as a proper comparison for the fusion Cas.

# Fusion Cas proteins

## Iteration 6/6

### Design

To further optimize the dual luciferase assay careful consideration went into the transfection plan and plasmid ratios needed.
Based on literature information we factored in differences in maturation time and degradation. With these adjustments we hope to improve the clarity of the assays output.

### Build

To ensure a balanced production of Cas protein and guide RNA, we calculated the necessary gene copy numbers to achieve a 1:1 ratio of protein to gRNA. One gene copy was equated to one plasmid, with nanogram amounts of plasmid DNA representing the respective copy numbers for transfection. Due to differing plasmid sizes, we adjusted the nanogram quantities to maintain proportional representation and scaled them down to the maximum possible nanogram amount for transfection.
Since the fusion Cas was stably expressed in the generated cell line, we also had to scale the transfected amount based on the cell line generation.
Notably, the transfected plasmid per cell in the 6-well plates was half of what we would typically use in a 96-well plate, so all subsequent calculations were adjusted accordingly to maintain consistent expression across experiments.

### Test

The data for the co-transfected Cas proteins showed efficient editing for both Cas9 (p=0.0003) and Cas12a (p=0.0005) to almost complete depletion. The stable cell line did not show such promising results, with only minor changes for both cas enzymes, with strong variation within replicates.

![](https://static.igem.wiki/teams/5237/engineering/fuscas-engi-fig8.svg)

**Figure 8: Single cut dual luciferase assay improvement of preliminary tests.** Firefly luminescence intensity measured 48 h after transfection. Normalized against renilla luminescence. On the x-axis the samples nt, Cas9 and Cas12a are grouped by single and fused proteins. Co-transformed contains single Cas proteins, in contrast to the fusion Cas having the same Cas proteins covalently bound to each other.
Data is depicted as the mean +/- SD (n=3).
Statistical analysis was performed using 2way ANOVA with Tukey’s multiple comparisons test. Only significant differences within a group are shown. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001

The modified dual luciferase assay revealed similar outcomes. The co-transfection showed strong editing efficiency for both Cas12a and Cas9, individually (Cas12a p=0.0044; Cas9 p < 0.0001) and simultaneously (Cas12a p=0.0048; Cas9 p < 0.0001). The stable cell line did not show significant editing activity for MbCas12a separately and simultaneous, but significant editing for SpCas9 (seperate p = 0.0049; simultaneous p = 0.002) is shown. Again trends are visible for MbCas12a which will be further discussed in the learning from this iteration.

![](https://static.igem.wiki/teams/5237/engineering/fuscas-engi-fig9.svg)

**Figure 9: Double cut dual luciferase assay further testing fusion Cas in stable cell line.** On the left Firefly luminescence intensity is plotted and on the right renilla luminescence intensity is plotted, both measured 48 h after transfection. Normalized against EGFP fluorescence. On the x-axis the samples non targeting (nt), SpCas9, MbCas12a and Cas9 and Cas12a editing at the same time (both) , are grouped by single and fused proteins. Co-transformed contains single Cas proteins, in contrast to the fusion Cas having the same cas proteins covalently bound to each other. Firefly editing is showing Cas12a editing while lower renilla counts are caused by Cas9 editing.
Data is depicted as the mean +/- SD (n=3).
Statistical analysis was performed using 2way ANOVA with Tukey’s multiple comparisons test. For better clarity, only significant differences within a group between the negative control and editing Cas proteins are shown. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001

### Learn

The Fusion Cas stable cell line did not function as expected. We suspect that prolonged exposure to selection pressure resulted in the loss of integrated gene copies due to genomic repair mechanisms. This could explain the lower-than-expected expression and editing efficiency observed in the stable cell line. Consequently, the calculated plasmid ratios for the Fusion Cas system need further tweaking. Assessing the data from figure 8 this becomes apparent taking a qualitative approach for data evaluation.
Nonetheless this line of experiments gave us an in-depth understanding on different assay systems and the Cas proteins accepting fusion proteins. We used these learnings and combined them with our fusion gRNA learnings to create functional protein staples with catalytically inactive (dCas) versions.

# Cas staples

## Iteration 1/2

### Design

Building on previous experience of engineering fusion gRNAs (fgRNAs) and fusion Cas systems, the aim of this engineering cycle was to design protein staples capable of bringing specific DNA sequences into close proximity. As the literature on this topic is sparse, all reasonable construct combinations of sgRNA, fgRNA, single Cas and fusion Cas were tested.
The first assay we designed to demonstrate such proximity was based on a fluorophore quencher pair. In this system, a fluorophore and a quencher are covalently bound at opposite ends of a double-stranded DNA (dsDNA) sequence. When the Cas staple brings the two ends of the DNA strand together, the quencher should be in close proximity to the fluorophore and thus reduce fluorescence intensity measurably.
Careful consideration was given to the design of a suitable fluorophore-quencher pair. Some considerations included thermal stability to allow incorporation into larger DNA fragments by PCR amplification. The quenching strength and availability as ssDNA modifications were also considered. The fluorophore quencher pair of choice was 6-FAM and Iowa Black QG.

![](https://static.igem.wiki/teams/5237/figures-corrected/fluorophor-quencher-dcas12a-dcas9-correct.svg)

**Figure 1: Design and theory behind fluorophore quencher assay.**Depicted on the left is the dsDNA fragment amplified by PCR with primers containing the fluorophore 6-FAM and the quencher Iowa Black FQ. Introducing the Cas staple, fgRNA with single dCas proteins or sgRNAs with fusion dCas or fgRNA with fusion dCas, as shown on the right, to this dsDNA piece would, as we theorize, bring the fluorophore and quencher into close proximity resulting in a reduction of fluorescence.

### Build

A catalytically inactive version of the fusion Cas construct was created through site-directed mutagenesis PCR, followed by Gibson assembly. The DNA fragment used in the assay was derived from a plasmid containing previously validated protospacers. This fragment was amplified by PCR using primers modified with a fluorophore (6-FAM) or a quencher (Iowa Black QG) attached to the 5’ OH group. Special care was taken during the assembly process to maintain mild reaction conditions and avoid exposure to light to preserve the integrity of the fluorophore-quencher pair.

### Test

HEK293T cells were co-transfected with various Cas and gRNA constructs along with the linear dsDNA fragment. After 8 hours, the media was replaced to remove any untransfected fragments, and fluorescence intensity was measured every 4 hours. At the 24-hour mark, measurements were taken every 12 hours, continuing until the 48-hour mark. A steady decline in fluorescence intensity was observed across all samples, indicating no evidence of successful DNA stapling.

![](https://static.igem.wiki/teams/5237/engineering/casstp-engi-fig2.svg)

**Figure 2: Fluorophore quencher assay for proving proximity induction of DNA by Cas staples.** 6-FAM fluorescence measured in time intervals after a media change 8h after transfection. After 48h the measurement was stopped. As controls on dsDNA fragment without a quencher is used, while the negative control consists of single Cas proteins with sgRNAs. Samples range from the parts of the Cas staple missing(fgRNA, Cas9 + fgRNA, Cas12a + fgRNA) to all parts of the Cas staple present ( 0nt fgRNA, 20nt fgRNA, 30nt fgRNA, 40nt fgRNA).
Data is depicted as the mean (n=3).
Statistical analysis was not performed.

### Learn

The assay proved hard to optimize and therefore was not pursued further. Alternative assays were taken into account, with the focus to find a suitable system closer to naturally occurring mechanisms.

# Cas staples

## Iteration 2/2

### Design

One promising application of our tool could be enhancer hijacking. To simulate this, we modified the already existing VP64 activated dual luciferase assay. Instead of directly fusing the VP64 to one of the Cas proteins, a VP64-Gal4 fusion protein was designed. Two plasmids will be stapled together, one of the plasmids has the dCasd12a target site next to the Gal4 target site (UAS), and another plasmid with the dCas9 target site next to the minimal promoter and firefly luciferase gene.
We hypothesized that a linker between the spacer sequences would be essential for simultaneous binding of both Cas proteins to the fgRNA. AlphaFold3 predictions suggested a minimum linker length of 20 nt to facilitate this interaction. Consequently, fgRNAs with linker lengths of 20 nt, 30 nt, and 40 nt were designed for testing in the wet lab.
The amounts transfected were calculated as already mentioned in the fusion Cas engineering cycle, while employing an extra factor having fgRNA and the Cas proteins be transfected in a 1:2 ratio.

![](https://static.igem.wiki/teams/5237/figures-corrected/simulated-enhancer-hijacking-gal4-vp64-luciferase-correct2.svg)

**Figure 3: Design and theory behind simulated enhancer hijacking assay.** Depicted on the left is the reporter plasmid and the Gal4 binding plasmid stapled together by Cas staples. On the right a closer look at a Cas staple stapling the two plasmids together with the Gal4-VP64 binding and acting as a transactivator driving luciferase expression forward is shown.

### Build

The Gal4 binding system was constructed by modifying an existing plasmid containing 5 UAS sites. Oct1 binding sites were introduced by annealing three oligo pairs, which were subsequently ligated into the digested vector.
New fgRNAs targeting the Oct1 and tetO sites were synthesized as DNA oligos. Following annealing, these oligos were cloned into the entry vector using Golden Gate assembly to create the desired constructs for further testing.
As described in the fusion Cas engineering cycle, special care was taken to calculate transfected plasmid amounts. This included adjusting for expression levels and decay rates, ensuring the intended 1:2 ratio of fgRNA to Cas proteins.

### Test

Firefly luciferase luminescence intensities were normalized to Renilla luciferase to account for variations in cell count. For the fgRNAs with single Cas proteins, the 0 nt linker exhibited no difference in fluorescence compared to the control. A clear trend emerged, showing increasing luminescence with longer linker lengths, with significant differences observed at linker lengths of 30 nt and 40 nt (p = 0.0185 and p = 0.0011, respectively).
The fusion Cas constructs showed no change in luminescence for the sgRNA and 0 nt linker. Interestingly, fgRNAs with 20 nt and 30 nt linkers outperformed those with a 40 nt linker, differing from the results seen with the single Cas proteins.

![](https://static.igem.wiki/teams/5237/engineering/engin-ba/tech-rep-fgrna.svg)
A

![](https://static.igem.wiki/teams/5237/engineering/engin-ba/tech-rep-fuscas.svg)
B

**Figure 4: Dual luciferase assay on simulated enhancer hijacking.** Firefly luminescence intensity measured 48 h after transfection. Normalized against renilla luminescence. A shows stapling with single Cas proteins and B with fusion Cas. On the x-axis in red the negative control is followed by the samples consisting of fgRNAs with the different linker lengths 0 nt, 20 nt, 30 nt and 40 nt. Whereas for the fusion Cas one extra sample with the sgRNAs was measured.
Data is depicted as the mean +/- SD (n=3).

### Learn

This assay successfully demonstrated that a fgRNA can be bound by two dCas proteins simultaneously and staple together two separate DNA fragments, resulting in transactivation for both fgRNA-single dCas and fgRNA-fusion dCas constructs. For the fgRNA-single dCas system, a steady increase in luminescence was observed with increasing linker lengths, suggesting improved Cas binding. For future experiments, longer linker lengths may be tested to further optimize the construct. The fgRNA-fusion dCas system, however, exhibited overall lower activity compared to single Cas constructs. The significant drop in activity at the 40 nt linker length indicates potential structural constraints in fgRNA binding caused by the peptide linker. In future iterations, altering peptide linkers could further enhance activity and improve system performance. The experiment will be repeated in biological replicates to acquire more significant data.

# Cathepsin B

## Iteration 1/3

### Design

We designed a fluorescence-based readout system to test if different peptide linkers could be cleaved by human cathepsin B *in vivo*. This would allow for the selective activation of our PICasSO system in tumor microenvironments overexpressing cathepsin B.

### Build

Our fluorescence readout system was comprised of a plasmid encoding mCherry with a Gal4 upstream activating sequence (UAS) and a fusion protein that consists of the DNA-binding domain (DBD) of Gal4, one of the five different peptide linkers (GFLG, FFRG, FRRL, VA, FK), and the transactivator domain VP64. Binding of Gal4-DBD-Linker-VP64 to the UAS would induce overexpression of mCherry. Consequently, cathepsin B cleavage of the peptide linker would reduce the expression of mCherry.

### Test

We transfected our genetic constructs into HEK293T cells. The fluorescence intensity of mCherry was measured 48 hours after transfection. Our initial tests did not result in the unambiguous identification of a cathepsin B-cleavable peptide linker (see **Fig. 1**). For all linkers, we did not observe a large decrease in fluorescence intensity between the negative control and test conditions. In some conditions, the fluorescence intensity even increased between the negative control and test conditions.

![](https://static.igem.wiki/teams/5237/wetlab-results/catb-results/catb-fluorescent-readout-no-dox-w.svg)

**Figure 1: Fluorescence Readout After 48 Hours for Five Different Peptide Linkers and Three Different Conditions.** The fluorescence intensity for mCherry was measured for five different linkers. The negative control was not transfected with the plasmid encoding cathepsin B. The fluorescence intensity of the negative control was set to one. Two different test conditions were investigated, in which either 30 ng or 60 ng of the plasmid encoding cathepsin B were transfected.

### Learn

Since cathepsin B is a lysosomal protease that is normally only active in the lysosome and the extracellular environment but not in the cytosol, we decided to change the native amino acid sequence of cathepsin B. Upon further investigation of the lysosomal maturation process of cathepsin B, we chose to express a truncated and mutated version of cathepsin B.

# Cathepsin B

## Iteration 2/3

### Design

We truncated the native cathepsin B amino acid sequence N-terminally by the first twenty amino acids. This would delete a signal peptide that normally facilitates translation of cathepsin B in the rough endoplasmic reticulum ensuring translation of cathepsin B in the cytosol. Additionally, we introduced three point mutations (D22A, H110A, R116A) in the amino acid sequence of cathepsin B. This has been shown to increase the activity of cathepsin B at higher pH values by disrupting the interactions of an occluding loop with the substrate binding pocket of cathepsin B.

### Build

We used overlap extension PCR to clone our truncated and mutated version of cathepsin B based on our original nucleotide sequence of cathepsin B. Additionally, we used the same plasmid backbone to overexpress our new construct in HEK293T cells.

### Test

We performed the same fluorescence readout assay in HEK293T cells that we also used for full-length cathepsin B. The fluorescence intensity of mCherry was measured 48 hours after transfection. However, we observed no decrease in fluorescence between our negative control and test conditions, indicating that Gal4-DBD-Linker-VP64 was not cleaved (see **Fig. 2**). Additionally, we performed a western blot, where the bands for the truncated and mutated version of cathepsin B were barely visible or absent altogether, indicating lower protein expression compared to the wild type (see **Fig. 3**). Another key insight from this experiment was that this version of cathepsin B was not activated by cleavage in the cell, as no additional protein bands were observed.

![](https://static.igem.wiki/teams/5237/wetlab-results/catb-results/catb-fluorescent-readout-mutated-and-truncated-w.svg)

**Figure 2: Fluorescence Readout for the Truncated and Mutated Version of Cathepsin B.**

![](https://static.igem.wiki/teams/5237/wetlab-results/catb-wb-w.svg)

**Figure 3: Western Blot of Two Versions of Cathepsin B With and Without Doxorubicin.** From left to right: protein ladder, wild-type (wt) cathepsin B with (+) and without (-) doxorubicin, truncated and mutated version of cathepsin B with (+) and without (-) doxorubicin.

### Learn

Since our truncated and mutated version of cathepsin B did not seem to be active in the cytosol, we decided to go back to wild-type cathepsin B. Therefore, we focused on improving the activity of wild-type cathepsin B in the cytosol.

# Cathepsin B

## Iteration 3/3

### Design

Instead of changing the amino acid sequence of cathepsin B, we decided to induce lysosomal escape of cathepsin B by treating the transfected cells with the cytostaticum doxorubicin.

### Build

After consulting the literature, we decided to treat the HEK293T cells with 500 nM doxorubicin 24 hours after transfection.

### Test

We used the same fluorescence readout assay that we had used in our initial testing (see **Fig. 4**). 24 hours after transfection, the cells were treated with 500 nM doxorubicin. The fluorescence intensity of mCherry was measured 48 hours after transfection. We observed a significant decrease in fluorescence for the GFLG linker between the negative control and the two test conditions.This indicates successful cathepsin B cleavage of the linker *in vivo*.

![](https://static.igem.wiki/teams/5237/wetlab-results/catb-results/catb-fluorescent-readout-final-results-w.svg)

**Figure 4: Fluorescence Readout After 48 Hours for Five Different Peptide Linkers and Three Different Conditions.** The fluorescence intensity for mCherry was measured for five different linkers. The negative control was not transfected with the plasmid encoding cathepsin B. The fluorescence intensity of the negative control was set to one. Two different test conditions were investigated, in which either 30 ng or 60 ng of the plasmid encoding cathepsin B were transfected.

### Learn

Through these tests, we identified one peptide linker (GFLG) that was consistently cleaved in the cytosol by cathepsin B. This enables us to functionalize our PICasSO system to be selectively active in microenvironments, such as cancerous tissue, that overexpress cathepsin B.

# FRET Measurement

## Iteration 1/4

### Design

To create a robust FRET measurement system, we first needed a simple staple to bring two DNA strands into proximity
and
identify suitable FRET pairs for efficient measurement. Our design process began with extensive literature research
to
select well-characterized DNA-binding proteins and fluorescent pairs that were proven to work in E. coli without
compromising binding efficiency when fused. Among the potential candidates—Oct1-DBD, Gal4-DBD, tetR, and
NFKB-DBD—Oct1-DBD was the only naturally monomeric protein. We hypothesized that ths monomeric nature is important
to
ensure stoichiometric binding at staple target sites.

We selected Oct1-DBD as one DNA-binding protein due to its proven strong expression and binding affinity in *E.
coli.* Our
second protein was an engineered version of the well characterized Tetracycline Repressor (tetR) that forms a
functional
monomer. This scTetR is a fusion of two tetR proteins with a flexible linker which was described to maintain the
same
DNA-binding affinity and specificity as wild-type tetR.

For the FRET pairs, we established the following key criteria: small size, monomeric, minimal photobleaching, high
fluorescence and quantum yield, and emission outside the blue and near-infrared spectra to reduce cell damage and
background fluorescence. After reviewing literature and using FPbase to predict FRET efficiency, we chose mNeonGreen
and mScarlet-I for their superior performance in FRET applications both reported in literature and calculated with
FPbase.

### Build

We constructed a two-plasmid system: an expression plasmid and a folding plasmid. The expression plasmid contained
the following required proteins: scTetR-Oct1 fusion (staple), scTetR-mScarlet-I (FRET acceptor), and Oct1-mNeonGreen
(FRET donor), additionally the vector had 15 repeats of the tet response element (TRE) to ensure efficient tetR
binding. The folding plasmid was designed with 15 repeats of the Oct1 binding motif. To ensure compatibility,
special consideration went into deciding the origin of replications (ori), we selected a high-copy pMB1 origin for
the expression plasmid and a medium-copy p15A origin for the folding plasmid.

To maintain balanced expression and minimize metabolic burden, we designed a polycistronic expression cassette under
the T7 promoter. This allowed the transcription of one mRNA encoding all three proteins, each with its own ribosome
binding site ([BBa\_B0030](https://parts.igem.org/Part%3ABBa_B0030)). To facilitate future modifications, we
included restriction sites between the ORFs and at
strategic locations. Gene synthesis was used to generate the inserts, which were cloned into the backbones using
Gibson assembly.

As controls, we constructed a positive control by fusing mScarlet-I and mNeonGreen to observe direct FRET
efficiency,
and a negative control by using a folding plasmid lacking the Oct1 target sequence.

### Test

Fluorescence intensity for mNeonGreen, mScarlet-I, and FRET was measured 18 hours post-incubation with varying IPTG
concentrations (0.8 mM to 0.0125 mM).

![engineering-sist-initial-test](https://static.igem.wiki/teams/5237/engineering/engineering-sist-first-assay.svg)

**Figure 1: Fluorescence measurement of first simple staple construct.**Fluorescent measurement, normalized to cell count, of mNeonGreen (ex. 490 nm, em. 530 nm), mScarlet-I (ex. 560
nm, em. 600 nm), and FRET (ex. 490 nm, em. 600 nm) in *E. coli*, 18 h
after induction with different IPTG concentrations. Data is presented as mean ± SD.

The positive control exhibited strong fluorescence for both proteins and FRET, indicating that the system was
functional in principle. However, the constructs expressing the staple and FRET-pairs didn’t exhibit any
fluorescence for all three measurements, indicating an issue with protein expression

### Learn

The lack of fluorescence in the polycistronic constructs suggested problems in transcription or translation possibly
due to the metabolic burden of expressing large, complex constructs. This hypothesis is also supported by the
positive control where increasing fluorescence could be measured with decreasing IPTG concentrations, reinforcing
the idea that overexpression might be causing stress and inhibiting efficient protein production.
Importantly this initial testing showed that the FRET pairs are compatible resulting in measurable fluorescence
readout, motivating us to further pursue this line of experiments.

# FRET Measurement

## Iteration 2/4

### Design

To address the potential issue of overexpression causing metabolic burden in our cells, we decided to test the
impact of reducing transcriptional activity using a mutated T7 polymerase with lower activity. This approach aimed
to reduce cell stress, but should still produce sufficient protein for fluorescent measurement. Since the mutated T7
was on a plasmid with the same origin of replication as the folding plasmid, we opted to co-express it only with the
expression plasmid, using BL21 cells that lacked the genomic T7 copy.

### Build

The mutated T7 polymerase was on a plasmid with the same ori as the folding plasmid, making it incompatible for
co-expression with the full system. As a result, we co-expressed the mutant T7 polymerase only with the expression
plasmid in BL21 cells that lack a genomic T7 copy, focusing solely on evaluating expression levels.

### Test

Similar to the first iteration, we measured fluorescence intensity for mNeonGreen, mScarlet-I, and FRET after
inducing the constructs with IPTG. Fluorescence was successfully detected for mNeonGreen in both ori variants,
indicating stable expression. However, mScarlet-I was still not expressed in the staple constructs, suggesting an
issue specific to this protein.

![engineering-sist-mut-T7-test](https://static.igem.wiki/teams/5237/engineering/engineering-sist-mut-t7.svg)

Figure 2: Fluorescent measurement of expression plasmid with mutant T7 polymerase.
Fluorescent measurement, normalized to cell count, of mNeonGreen (ex. 490 nm, em. 530 nm), mScarlet-I (ex. 560
nm, em. 600 nm), and FRET (ex. 490 nm, em. 600 nm) in *E. coli*, 18 h
after induction with different IPTG concentrations. Data is presented as mean ± SD.

### Learn

Fluorescence could be detected for mNeonGreen, indicating successful expression, but mScarlet-I still failed to show
any signal. This suggests that the problem with scTetR-mScarlet-I is not solely due to the high expression levels or
transcriptional activity, as reducing transcription did not resolve the issue. Based on the location of
scTetR-mScarlet-I in the polycistronic operon (before Oct1-mNeonGreen), the issue seems to be related to translation
or protein folding rather than transcription. Further investigations into protein stability or folding will be
required to address this.

# FRET Measurement

## Iteration 3/4

### Design

In the previous iteration, reducing transcriptional activity through a mutated T7 polymerase did not resolve the
issue of low ScTetR-mScarlet-I expression. This led us to hypothesize that the burden of a two plasmid system (one
of them being high copy) and expression of multiple proteins, is still too high. Therefore, in this iteration, we
aimed to reduce the metabolic load by lowering the copy number of the expression plasmid. To achieve this, we
designed two alternative constructs: For the first, the *rop* gene was introduced into the plasmid backbone.
The Rop protein is known to regulate plasmid replication and should reduce the plasmid count to approximately 15-20
copies per cell. For the second modification the pMB1 ori was replaced with the low copy pSC101 ori. This should
reduce the copy number to 5-10 copies per cell.

### Build

To test the impact of lowering plasmid copy number, we cloned the rop protein into the expression plasmid via
restriction and ligation cloning, and similarly replaced the pMB1 origin with pSC101 using PCR amplification and
cloning. After transformation into Top10, colonies were picked and plasmid purified. Correct assembly was verified
by whole plasmid sequencing.

### Test

The co-transformation of the modified expression plasmids with the folding plasmid in BL21 did not work. Next we
first transformed the expression plasmid, which worked and could be propagated stably. These cells were then made
competent following our protocol and used to transform the folding plasmid. The transformation worked but cells did
not show stable growth in selection media. These experiments were repeated multiple times to rule out possible
mistakes.

### Learn

The co-transformation of the modified expression plasmids with the folding plasmid consistently failed, and stable
growth in selection media was not achieved. These issues suggest that lowering plasmid copy number, either through
the rop gene or the pSC101 origin, may have negatively impacted plasmid stability or protein expression. Despite
successful individual transformations, co-expressing both plasmids may have led to incompatible replication dynamics
or insufficient protein levels due to reduced copy numbers. Given the repeated failures and time constraints, we
discontinued this line of experiments.

# FRET Measurement

## Iteration 4/4

### Design

The previous iterations showed that expression, especially of ScTetR was not possible. To improve expression and
reduce cellular burden, we replaced ScTetR with the standard tetR for both the staple construct and FRET acceptor.
This substitution likely induces homo-dimerization for tetR binding, which was deemed acceptable.

### Build

## Build

The modified operon was assembled by restriction and ligation cloning.

### Test

The modified constructs were again tested with different IPTG concentrations. This experiment can be found on our
results page. In short this final version was successful to induce proximity between the folding and expression
plasmid and measure it through the FRET interaction.

### Learn

The FRET assay successfully demonstrated induced proximity between the folding and expression plasmid, resulting
increase in FRET interaction and fluorescent measurement. Moving forward, refining the measurement setup will be
necessary to achieve quantitative assessment of successful stapling and proximity. Moving forward, applying more
advanced FRET analysis techniques, such as those described by
Hochreiter *et al.* (2019) and
Coullomb *et al.* (2020), would allow for precise quantification
of protein interactions, stoichiometry, and affinity. Future iterations could implement acceptor photobleaching or
time-resolved fluorescence measurements to further enhance the sensitivity and accuracy of our system, facilitating
real-time analysis of DNA-protein interactions.

Coullomb, A., Bidan, C. M., Qian, C., Wehnekamp, F., Oddou, C., Albigès-Rizo, C., Lamb, D. C. & Dupont, A. (2020). QuanTI-FRET: a framework for quantitative FRET measurements in living cells. Scientific Reports, 10(1). https://doi.org/10.1038/s41598-020-62924-w

Hochreiter, B., Kunze, M., Moser, B. & Schmid, J. A. (2019). Advanced FRET normalization allows quantitative analysis of protein interactions including stoichiometries and relative affinities in living cells. Scientific Reports, 9(1). https://doi.org/10.1038/s41598-019-44650-0

# Delivery System

## Iteration 1/1

### Design

For constructing our pNeae2\_7D12 plasmid, we first decided to follow a classical restriction-ligation cloning strategy as was done by Pinero-Lambea *et al.*, (2015). We introduced the 5’ and 3’ overhangs for SfiI and NotI recognition sites into the 7D12-nanobody gene block (DSg3). An *in silico* simulation of the agarose gel after double restriction by SfiI and NotI indicated that we should observe one band of 5801 bp and a second, smaller band of 31 bp, which would not be visible on a 1% agarose gel.

Piñero-Lambea, Carlos, *et al.* "Programming controlled adhesion of E. coli to target surfaces, cells, and tumors with synthetic adhesins." ACS synthetic biology 4.4 (2015): 463-473.

### Build

We first performed PCR amplification of DSg3. Following the restriction digest of the pNeae2 backbone and DSg3 with SfiI and NotI, we analyzed the digestion products by gel electrophoresis (1% agarose). We observed two bands: one above 5000 bp and another at around 3000 bp. While we hypothesized that the upper band was the expected one, we could not explain the existence of the lower band. As the reaction conditions were not optimal for both restriction enzymes, it is reasonable to assume that the smaller product resulted from undesired star activity of one of the enzymes. We chose to continue the cloning procedure with the larger band, having our expected size. After gel extraction, we went on to ligate the digestion products overnight at 16 ℃ using T4 DNA ligase. The next day, the ligation product was transformed into chemically competent E. coli cells, plated on LB agar supplemented with chloramphenicol, and incubated at 37 ℃.

![](https://static.igem.wiki/teams/5237/engineering/eng-cycle-delivery-system-1.png)

**Figure 1: Gel electrophoresis analysis of double restricted 7D12 nanobody and pNeae2 backbone.** The first lane represents the DNA ladder, the second lane represents the 7D12 nanobody gene block restricted with SfiI and NotI. Lanes 3-6 represent four samples of the pNeae2 backbone digested with SfiI and NotI.

### Test

Several colonies grew on the selective LB agar plate and we assumed that the cloning was successful. To solidify our assumption, we ran a test digest with EcoRI. In case of the backbone without the nanobody insert, we expected only one cut site, corresponding to only one band of the size 5832 bp, and in case of a successful insertion, we would observe two bands: 4050 bp and 2148 bp. The test digest clearly showed a single band, which meant that the backbone had religated despite the incompatible overhangs.

![](https://static.igem.wiki/teams/5237/engineering/eng-cycle-delivery-system-2.png)

**Figure 1: Gel electrophoresis analysis of the test digest of pNeae2\_7D12 plasmid.** The digest was performed with the EcoRI restriction enzyme. The first lane represents the DNA ladder, lanes 2-7 represent six samples of the cloned pNeae2\_7D12 and the third lane represents the negative control, which was the pNeae2 backbone.

### Learn

Initially we expected to observe a single band after digestion of the pNeae2 backbone. This expectation laid on the assumption that the digest had been fully completed. If undigested parts of the backbone remained, this would result in a second band. However, the size of this band would be 5832 bp and thus indistinguishable from the expected band of 5801 bp. Consequently, excising only the digested backbone and leaving behind the incompletely digested (e.g. digested by only one of the enzymes) plasmid would be nearly impossible. Therefore, the growth of single colonies on the selective plate after the transformation might have been due to retransformation of the circularized backbone without the needed insert. In conclusion, we decided to change the cloning strategy.

# Structure Prediction

## Iteration 1/3

### Design

To investigate DNA-DNA interactions properly, we developed DaVinci - an in silico clone that models the entirety of our PICasSO system. Accurate modeling of the biological components required us to understand the 3D structure of our staples, identify relevant parts to the model, examine the behavior of our structures, and their interactions with one another, as well as understand the system’s limitations. To better understand and visualize our staple structures, we chose AlphaFold2, as it was the current state-of-the art software and because of its ease of use.

### Build

To efficiently predict the staple protein structures, we familiarized ourselves with supercomputing infrastructure and gained access to the BwForCluster Helix. Utilizing the AF2 module on the cluster, we predicted protein multimers (Jumper *et al.* 2021).

Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., et al. (2021). Highly accurate protein structure prediction with AlphaFold. Nature, 596(7873). https://doi.org/10.1038/s41586-021-03819-2

### Test

Although the predictions ran successfully and provided the protein structures, AlphaFold2 does not model nucleic acids and cofactors. These elements are important components in our system, rendering the predictions incomplete.

### Learn

For a full structure prediction including DNA, RNA and cofactors, where both Cas proteins bind the same gRNA, we need an atomistic structure prediction model to suit our needs.

# Structure Prediction

## Iteration 2/3

### Design

To predict protein structures alongside nucleic acids and cofactors, we opted for RosettaFold All-Atom.

### Build

We ran RosettaFold predictions on the BwForCluster Helix, using the open-source RosettaFold AllAtom (RFAA) package (Baek *et al.* 2023).

Baek, M., McHugh, R., Anishchenko, I., Jiang, H., Baker, D., & DiMaio, F. (2023). Accurate prediction of protein–nucleic acid complexes using RoseTTAFoldNA. Nature Methods, 21(1). https://doi.org/10.1038/s41592-023-02086-5

### Test

The predictions ran unsuccessfully, as RFAA ran out of any memory allocation we provided on our HPC setup, not simulating our big system of interest containing complex proteins, cofactors, DNA and RNA.

### Learn

To accurately and successfully make our predictions, we need functional all-atom software that can handle complex systems of large size.

# Structure Prediction

## Iteration 3/3

### Design

We predicted the protein structures along with cofactors and nucleic acids using the newly released all-atom software AlphaFold3 (Abramson *et al.*, 2024).

J., Adler, J., Dunger, J., Evans, R., Green, T., Pritzel, A., et al. (2024). Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature, 630(8016), 493–500. https://doi.org/10.1038/s41586-024-07487-w

### Build

The protein structures were predicted using the online interface of Alphafold3.

### Test

The protein structures ran successfully, providing the protein structures. We then tested the accuracy of predictions against available crystal structures and ranked them using AlphaFolds internal metrics, as well as evaluating them externally using metrics during CAPRI and a continuous implementation of their combination: DockQ metrics (Janin et al., 2003; Sankar Basu & Björn Wallner, 2016).

Janin, J., Henrick, K., Moult, J., Eyck, L. T., Sternberg, M. J. E., Vajda, S., Vakser, I., & Wodak, S. J. (2003). CAPRI: A critical assessment of predicted interactions. Proteins: Structure, Function, and Bioinformatics, 52(1), 2–9. https://doi.org/10.1002/prot.10381

Basu, S., & Wallner, B. (2016). DockQ: A quality measure for protein-protein docking models. PLOS ONE, 11(8). https://doi.org/10.1371/journal.pone.0161879

### Learn

We are much more interested in the actual contact between the proteins and RNA / DNA / Protein-interactions, rather than a truly accurate description of the global minimum of the energy landscape. And going one step further, as our predictions are static snapshots in time, not considering the systems behavior over time, we need to expand our approach to model the dynamic behavior of our system.

# MD-Simulations

## Iteration 1/5

### Design

Being interested in the effects of our PICasSO System on the genome, we aimed for a model simulating long-range DNA interactions induced by our Cas staples.
Since we experimentally demonstrated enhancer hijacking using two plasmids, we decided to use these exact plasmids to represent the genome structure in our simulations. We set out to model the stapling dynamics involved in using a synthetic enhancer.

### Build

As both plasmids used for the enhancer hijacking assays consist of almost 20 thousand nucleotides in total, we opted for the coarse-grained molecular dynamics approach using oxDNA (Ouldridge *et al.*, 2011). This software is a simulation tool specifically designed for nucleic acid modeling, which focuses on interactions and mechanical properties. We then simulated a linear DNA strand and streamlined the simulation process with customized scripts.

Ouldridge, T. E., Louis, A. A., & Doye, J. P. K. (2011). Structural, mechanical, and thermodynamic properties of a coarse-grained DNA model. J. Chem. Phys, 134(8), 85101. https://doi.org/10.1063/1.3552946

### Test

The simulation of linear DNA strands at plasmid lengths was successful. However, simulating plasmids requires circular DNA. Although oxDNA offers a functionality to generate circular starting structures, we encountered issues when attempting to use it—the generation of the circular structure did not work.

### Learn

We concluded that coarse-grained models are suitable for modeling plasmids, with oxDNA providing an optimal balance between computational efficiency and necessary detail.
Given that the integrated function to generate circular starting structures did not work, we recognized the need to start simulations from linear strands that are circularized during the simulation process. After consulting experts in the field, we recognized the necessity to develop a workaround to bring the ends of the linear DNA together, effectively forming a circular plasmid within the simulation.

# MD-Simulations

## Iteration 2/5

### Design

In order to model plasmids, the next goal is the circularization of plasmids.

### Build

We utilized mutual traps—a harmonic force that pulls the particle of interest toward a reference particle—to pull the two ends of the DNA strands together, effectively circularizing the plasmid within the oxDNA simulation environment.

### Test

The linear strands where circularized within one simulation - trying to bring the strand ends together at once. We achieved this through employing mutual traps.

### Learn

Through this process, we deepened our understanding of mutual traps and force interactions in oxDNA. Circularizing the DNA strands proved to be particularly challenging. In order to bring the DNA strands into proximity without causing them to break, we applied unnaturally weak forces and set the radius parameter r0 to 20nm. Since oxDNA does not model proteins, we can employ mutual traps to emulate the forces the staples exert on the DNA. Our next critical objective is to accurately calculate these forces in subsequent iterations.

# MD-Simulations

## Iteration 3/5

### Design

To simulate the physical movements of molecules over time and their respective force fields with higher fidelity, we decided to use GROMACS, an all-atom molecular dynamics simulation software.

### Build

We initiated simulations of the Cas protein in an aqueous environment and familiarized ourselves with the GROMACS setup in a high performance cluster setting. We simulated simple characterization parts from the wet lab project, starting with the Mini staples, followed by the Simple staples, and finally the dual Cas staple.

### Test

The Mini staples, Simple staples, and dual Cas staples were successfully simulated in GROMACS. However, the simulations proved to be inefficient, achieving a runtime of only 1–3 nanoseconds per day, not harnessing the resources of our HPC setup to full extend.

### Learn

We observed that the GROMACS simulations proved to be very inefficient, highlighting the necessity of optimizing the parameters to achieve better runtimes.

# MD-Simulations

## Iteration 4/5

### Design

The goal for this iteration was to optimize the runtime of all-atom simulations in GROMACS.

### Build

To optimize the runtime, we modulated hardware configurations and process-specific calculation distribution in GROMACS. On the BwForCluster Helix HPC system we implemented strategies for GPU acceleration and multi-node scalability *(Massively Improved Multi-Node NVIDIA GPU Scalability with GROMACS, 2023).*

<https://developer.nvidia.com/blog/massively-improved-multi-node-nvidia-gpu-scalability-with-gromacs/>

### Test

We tested our optimizations using benchmark sets from the Grubmüller group.(Carsten Kutzner *et al.*, 2019) By finetuning simulation parameters, we achieved a maximum speed of 76.82 ns/day.

Kutzner, C., Páll, S., Fechner, M., Esztermann, A., Groot, B. L. d., & Grubmüller, H. (2019). More bang for your buck: Improved use of GPU nodes for GROMACS 2018. Journal of Computational Chemistry, 40(27). https://doi.org/10.1002/jcc.26011

### Learn

Through this iteration, we gained experience in implementing multithreading and GPU acceleration, which proved to improve efficiency significantly for particle-particle interactions and update groups. We maximized parallel processing capabilities across compute nodes by using Open MPI. These optimizations greatly improved our runtimes, making the following steps much more time efficient.

# MD-Simulations

## Iteration 5/5

### Design

This iteration's objective was to simulate the forces of the interaction between the Cas protein and the DNA, which will be integrated into the oxDNA plasmid simulations, for simulation of the long-range effects of the PICasSO system.

### Build

We set up simulations in GROMACS to model the forces between the Cas and DNA. Due to the simulations creating file sizes too big to be computed with efficient RAM storage of data, the process was parallelized across multiple computing cores.
The initial simulations spanned 100 picoseconds, which proved to be too short for effective force analysis, and needed to be expanded to a timeframe of 10 nanoseconds to obtain statistically invariant data. However, this created a storage issue, where in order to not reach capacity, we selectively saved reported data points. We continued to optimize storage usage by working with the compressed .xtc trajectory file formats instead of .trr files, since the .trr files proved to be too big for efficient handling.

### Test

By implementing these optimizations, we successfully ran the extended simulations while keeping file sizes manageable. The parallelization improved computation times, and using .xtc files reduced storage requirements. The MDAnalysis package proved effective for processing and analyzing the simulation data, allowing us to extract valuable insights into the Cas-DNA interactions.

### Learn

Through this iteration, we gained insight into data management and storage optimization in large-scale molecular dynamics simulations.

# Wet Lab Integration

## Iteration 1/2

### Design

This iteration's goal was to input structures from AlphaFold 3 to GROMACS.

### Build

We built the structures with AlphaFold 3 and exported the .cif files through pymol to .pdb. The .pdb was then input into GROMACS.

### Test

We ran gmx pdb2gro, this run however yielded errors.

### Learn

We discovered that AlphaFold 3 initiates 5’ redundant phosphate groups without function. These phosphate groups are excluded from the AMBER force fields by an unwritten naming convention. Thus we created a script to specifically remove these falsely assigned atom groups, which then yielded the desired result.

# Wet Lab Integration

## Iteration 2/2

### Design

The main objective was inputting the representative forces from the GROMACS simulations into oxDNA to simulate one staple.

### Build

We calculated the average force for a nucleotide with the origin at the center of mass and projecting them in the direction of the corresponding base pair. These forces were then averaged over the simulation time. We simulated the system for 100 picoseconds.

### Test

We plotted the variability of the positions and thus assessed if our sample size follows a normal distribution. We found that it does not.

### Learn

We learned that we need to simulate over longer periods of time to achieve ergodicity in our system. We then expanded the simulation time to 10 ns.

# Survey

## Iteration 1/4

### Design

In the initial phase, our objective was to investigate the **relationship between general scientific knowledge and attitudes
toward genetically modified organisms (GMOs)**. We hypothesized that individuals with a higher level of scientific knowledge would
display a more positive attitude toward GMOs. This hypothesis was grounded in the notion that increased scientific literacy could lead
to a **better understanding of the safety**, utility, and potential risks associated with GMOs. Consequently, our survey aimed to explore how a participant's
grasp of basic scientific concepts influenced their perception of GMOs.

![](https://static.igem.wiki/teams/5237/engineering/human-practices/iteration1.svg)

### Build

We began by constructing a questionnaire including  **35 questions** , divided into two sections: **20 questions assessing general scientific knowledge** and
**15 questions concerning attitudes toward GMOs**. The questions were designed to capture various levels of understanding and opinion, utilizing a combination
of **open questions, multiple-choice questions, and Likert-scale questions** (ranging from strongly disagree to strongly agree in 5 steps). Specifically, we assessed
the participants' understanding of key principles in biology and environmental science, as well as their perceptions of safety, environmental impact, and
ethical concerns. The development of our questionnaire was based on a **review of relevant literature** and best practices from previous studies on public
understanding of science and technology

### Test

To ensure the robustness of our survey design, we interviewed the **Chair of Psychological Methodology and Diagnostics at Ludwig-Maximilians-University Munich**,
Prof. Dr. Markus Bühner. Together, we reviewed the content and structure of our survey and refined the questions to ensure they were **scientifically valid** and
easy to interpret. Prof. Dr. Bühner's expertise in survey design and methodology provided invaluable feedback, improving the reliability and comparability of
responses from our survey.

### Learn

The feedback from our expert consultation led to **several key changes in our design**. First, we were advised to **eliminate open questions**, as they would be difficult
to compare and quantify across a large sample group. Open responses often present challenges in standardizing interpretations and may introduce biases in analysis.
Additionally, Mr. Bühner suggested limiting the **total number of questions to 30**, as a more concise survey would likely improve response rates and participant engagement.
Finally, he recommended **conducting a pre-survey** to define and balance the difficulty of the scientific knowledge questions. The target difficulty range of a question should
be defined as **20% to 80% correct answers**. He also advised us to gather responses from at least **20 participants** for this pre-survey to analyze the distribution of difficulty effectively.

# Survey

## Iteration 2/4

### Design

In response to the feedback received during the first iteration, we **designed a pre-survey** focused on evaluating the difficulty of the scientific knowledge questions. The primary goal of this phase was
to ensure that the questions effectively **distinguished between varying levels of knowledge** while maintaining a reasonable level of challenge. Drawing from previous studies and literature,
we revised the content of the questions to reflect key scientific principles that were accessible yet discerning. Analyzing the pre-survey responses would allow us to **fine-tune the questions** and ensure
that they would yield a balanced assessment of scientific knowledge.

![](https://static.igem.wiki/teams/5237/engineering/human-practices/iteration2.svg)

### Build

We designed the pre-survey to include **30 true/false questions** targeting general scientific knowledge. The questions were derived from school textbooks to ensure they reflected an appropriate level of complexity.
We developed a **web-based format** for the pre-survey to allow easy access and completion by participants.

### Test

The pre-survey was distributed **online in April 2024** to a randomly selected group of 20 participants. After collecting the responses, we analyzed them to determine the distribution of correct answers for each question.

### Learn

The analysis revealed that **15 of the 30 questions met the desired difficulty range**, with 20% to 80% of participants answering them correctly and were retained for the final survey. Additionally,
several questions were identified as being confusing or ambiguous in their wording, leading to varied interpretations. These were reworded to **improve clarity** and ensure participants could easily understand and respond accurately.
This learning phase underscored the importance of question clarity in surveys assessing knowledge.

# Survey

## Iteration 3/4

### Design

Having refined the scientific knowledge questions, we proceeded to design the **main survey**, which aimed to test the original hypothesis. The survey consists of two sections: the first assessed **general scientific knowledge**, and the second focused on **participants'
attitudes toward GMOs**. To contextualize these responses, we also included demographic questions related to the participant's **age, gender, and geographic location**.

![](https://static.igem.wiki/teams/5237/engineering/human-practices/iteration3.svg)

### Build

Using the **online** platform LimeSurvey, we developed the main survey, which was distributed via social media channels and personal networks to maximize participation and ensure a **diverse sample of respondents**

### Test

The survey was distributed across **Germany** from **June to September 2024**. As our questionnaire was distributed online participants from various regions could engage with the content at their convenience. In total, **823 valid
responses** were collected, with participants representing nearly **all German states** except Schleswig-Holstein. The broad geographic representation provided a rich dataset for analysis, allowing us to explore potential regional
variations in scientific knowledge and attitudes toward GMOs.

### Learn

The distribution of responses indicated that our outreach efforts engaged with a **diverse audience** across Germany. This phase also reinforced the utility of web-based surveys in **reaching a wide audience**, though it underscored the importance of
ensuring representative participation from all regions.

# Survey

## Iteration 4/4

### Design

With the **data collection complete**, we focused on analyzing the relationship between scientific knowledge and attitudes toward GMOs. We employed **correlation analysis** to examine the association between the Knowledge and Attitude Score.

![](https://static.igem.wiki/teams/5237/engineering/human-practices/iteration4.svg)

### Build

Participants were classified into **three knowledge groups** based on their responses to the 15 knowledge questions. A point was awarded for each correct response, with a **maximum possible score of 15**. Participants with scores between 11 and 15 were
classified as having **High Knowledge**, those scoring between 6 and 10 as **Moderate Knowledge**, and those scoring between 0 and 5 as **Low Knowledge**. This classification was based on equally dividing the range of scores to ensure an objective grouping.
The attitude toward GMOs was assessed using a **Likert scale**, with 6 negative and 9 positive statements. For negative questions, disagreement indicated a positive attitude toward GMOs, while for positive questions, agreement was indicative of a positive attitude.
The responses were scored accordingly, to create a composite Attitude Score for each participant.

### Test

Correlation analyses were conducted to explore the relationship between participants' **Knowledge Scores and their attitudes toward GMOs**. These analyses allowed us to test whether there was a **statistically significant association** between the two variables, thereby addressing our initial hypothesis.

### Learn

The results indicated a **negative correlation (r= -0.62, p-value =0.000)** between Knowledge Score and Attitude Score, suggesting that individuals with **lower scientific knowledge tend to hold more negative attitudes toward GMOs**. This finding **confirmed our original hypothesis**,
demonstrating the critical role of **scientific education** in shaping public opinion on controversial topics like GMOs. The insights gained from this analysis can inform educational initiatives into context, emphasizing the importance of fostering scientific literacy to promote
informed decision-making.

# Summer School

## Iteration 1/3

### Design

We designed a **laboratory workshop** on CRISPR Cas technology, aimed at introducing **high school students** to fundamental molecular biology principles as part of our Human Practices and educational outreach. The goal was to create a unique opportunity for high school students to get **hands-on experience**
in **state-of-the-art scientific methods** and to foster fascination for the life sciences.

### Build

To develop the workshop, we took inspiration from **university-level lab courses** and adapted and simplified protocols to tailor them specifically to high school students. The resulting script was the foundation for our high school workshop and can be found on the wiki.

### Test

Before implementation, we assessed the feasibility of conducting the workshop with younger students by **reviewing the experimental steps and materials**. We ensured that we had all the necessary reagents and evaluated whether our team had the expertise to **guide the students** through the practical experiments.

### Learn

From this initial review, we recognized the need to **adjust the script**, as many background concepts and details that might seem obvious to advanced university students are likely completely new to high school students. We therefore introduced a more **comprehensive theoretical section**, providing additional foundational insights.
Moreover, we ordered all required reagents and **translated the script** from english into german. The language was further simplified and adapted to be more **student-friendly**, making the experimental procedures easier to follow for younger students.

# Summer School

## Iteration 2/3

### Design

In collaboration with the **DKFZ Life Science Lab**, we implemented the workshop over **two weekends**, using the revised and improved script. This workshop formed a core part of our Summer School, designed to provide students with hands-on experience in molecular biology techniques.

### Build

We planned the logistics for the weekends, assigning students to different groups and determining how many **supervisors from the iGEM Heidelberg team** were needed for effective instruction. We ensured that all resources, including materials and personnel, were adequately prepared.

### Test

The first implementation of the Summer School workshop took place in **July 2024**, with a group of 4 high school students. To assess the success of the event and **gather feedback for future improvements**, we encouraged all participants to fill out our feedback form.

### Learn

Several important learning points emerged. Firstly, some reagents were missing, which created challenges during the experiments. Secondly, we realized a **solution for questions asked in the script was missing**, and some procedural details were not clearly outlined in the script.
Although we were able to improvise successfully, it became apparent that certain concepts **required more clarification**. Consequently, we revised the script once more, ensuring that these issues were resolved before the **next iteration of the Summer School**.

# Summer School

## Iteration 3/3

### Design

We used the **updated script**, now incorporating the lessons learned from the previous session. We also double-checked that all required reagents and materials were available to avoid any disruptions.

### Build

All improvements were **implemented** and the materials and reagents were prepared for the second workshop.

### Test

We repeated the workshop, this time with a **larger group of participants**, and continued to refine our approach based on the gathered input. Again, feedback forms allowed us to measure the success of the changes for a final evaluation.

### Learn

In summary, we can conclude that the students enjoyed the **hands-on approach** to learning about laboratory work in the life sciences, appreciating the opportunity to engage with material that would otherwise be **inaccessible due to financial limitations**.
They also valued the fact that the **workshop leaders were not much older than themselves**, which helped create a more relatable and approachable learning environment. Additionally, students responded positively to the **step-by-step explanations**, which clarified why each experimental step was necessary.
Based on the positive feedback, we decided to **offer the Summer School workshop again next year in May** and to make the workshop **materials available online** on our wiki for wider adoption by school teachers and other universities. This ensures that more students and teachers can benefit from our efforts and that the **knowledge gained can be spread beyond our iGEM project**.

# School Workshops

## Iteration 1/3

### Design

In the initial phase of developing our high school workshop program, we brainstormed about the **main topic**, considering what would be the most valuable and interesting for **11th and 12-grade students**. We decided that an introduction
to synthetic biology with important considerations regarding **safety** and the basics of lab methods should be followed by a **glimpse into the reality** of a research project, taking our iGEM project as an example.

### Build

We first implemented **introductory questions** concerning the definition of synthetic biology to define a starting point and engage with the participants. We continued with a **reflection on safety and ethical questions** raised by genetic engineering, promoting awareness of the importance of **biosafety**. We selected
basic molecular biology and genetic engineering techniques essential for synthetic biology. We researched the **official high school syllabus** to get an idea of what prior knowledge can be expected, and what theoretical basics we should explain. Following this, we reflected on the development of our iGEM project,
combining personal experiences with the basics of scientific working practices to compile an overview of **how to conduct a research project**. To deepen the understanding of the concepts conveyed so far, we included a **practical group activity** that requires the transfer of the learned concept to a concrete synthetic biology problem.

### Test

To assess the quality of our workshop concept and optimally prepare for the upcoming school workshops, we presented our concepts to **Prof. Armin Baur**, professor of **biology and didactics**. He provided valuable feedback on our concept and also answered our further questions, providing insights on how professional educators approach the development of courses and **educational material**.

### Learn

The interview with **Prof. Armin Baur** provided valuable insights on how to properly structure a school workshop, like combining **frontal lesson elements** with class conversation and **group activities**, and adapting the content to the way students learn most effectively. He then introduced us to the concept of **lesson plans**, which provide an overview of
**planned activities, social interactions, media forms, and time allocation to ensure a well-balanced lesson**. He also advised us to include a section on our project, allowing the students to get to know us and our work. We decided to implement those valuable suggestions to elevate the quality of our concept.

# School Workshops

## Iteration 2/3

### Design

We reviewed Prof. Baur's opinions and feedback received in the interview. Based on his feedback we **improved our concept**.

### Build

We created a **detailed lesson plan** for our workshop, which was essential for **prioritizing lesson elements** and enabled us to assess which parts realistically fit into one workshop. This also aided us in **mindfully structuring the individual units** and varying the **social activities** and the use of media to maintain the student's attention and engagement.
We created a feedback form to further improve our school workshops.

### Test

With our finished workshop concept, we conducted the **first high school workshop** in an 11th-grade biology course at a school in Heidelberg. The workshop was highly successful with students showing **great participation** and exhibiting very creative thinking, yet inspired some changes that would **improve our concept even further**. After the workshop,
we evaluated our program by **taking the feedback** from the students and our impressions of the student's attention and engagement into account.

### Learn

We realized that the first part of the workshop, including the intro to **synthetic biology, biosafety, and theoretical basics**, required less time than anticipated. This led to more available time for the project planning unit and the practical group task, which allowed us to **include a thorough discussion** of the student's ideas. The students' knowledge exceeded our
expectations, leading to a lively and detailed discussion and questions that allowed us to **dive deeper** into the topic, allowing us to respond to the student's interests. This was reflected in the feedback, where students expressed **strong interest** in the unit about how to conduct a research project as well as our own experiences and project ideas.

# School Workshops

## Iteration 3/3

### Design

The experience of the first school workshop helped us to identify which parts of the workshop were **most interesting and beneficial** to the students and improved our understanding of the time requirements of the different workshop units.

### Build

In response to our experiences, we **optimized the time allocation** for the different workshop units, shortening the introductory units in favor of the unit on research project planning session and discussion of our project. Next, we **improved our slides** for the theoretical basics by adding pictures from the lab and structuring them **to be more understandable**.
Moreover, we refined the presentation of our own project, **by adding information and improving the figures**. Finally, we specifically allocated more time for the **group activity** where students had to develop ideas on how to approach the development of a synthetic biology project, as the discussion of this part had proven to be **fruitful and inspiring** in the first iteration.

### Test

We held our improved workshop in **three more classes**, with each workshop providing a valuable learning experience for the participants, and enabling us to enter the next workshop more prepared.

### Learn

Our learnings from the past workshops were strengthened, showing us that students can best profit from our team's workshops if we take the majority of our time to **provide examples** from the reality of research and academic life, as this is **not covered in their regular school lessons**. The interest shown gives us confidence that we were able to inspire many
students and even **encourage them to pursue a scientific career** in the future.

{% 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 %}

# Experiments

### **Protocols**

Access our **library of standardized protocols**, covering essential DNA cloning methods, a variety of assays, and more.

### **Materials & Methods**

Our **precise experimental steps and materials** for cloning and conducting experiments - enabling future iGEMers to effectively build on our work.

### **Sequences**

The sequence overview includes **all the DNA sequences used in our experiments**, including primers and other essential components.

[Scroll down](#Protocols)

* [Protocols](#Protocols)
* [DNA Cloning Methods](#CloningMethods)
* [Protein Expression & Analysis](#ProteinExpressionPurification)
* [Mammalian Cell Protocols](#MammalianCellProtocols)
* [Assays](#Assays)
* [Materials & Methods](#MaterialsMethods)
* [Sequences](#Sequences)
* [References](#References)
* [Back to the top](#top)

## Protocols

### DNA Cloning Methods

**1. Oligo Annealing**

#### Oligo Annealing

The protocol is typically performed with oligos at a stock concentration of 100 µM and will yield 10 µM of annealed oligos, but can also be adjusted accordingly.

1. Pre-heat a heating block to 95 °C
2. Prepare the following oligo mixture in a 1.5 mL eppi
   * 5 µL rCutsmart buffer
   * 5 µL of each oligo to anneal
   * Fill up to 50 µL with nuclease free water (35 µL)
3. Incubate mixture for 5 min at 95 °C
4. Turn of the heating block and leave eppi inside for at least 1 h
   Alternatively the reaction can be done in a PCR cycler and the mixture cooled with 0.5 °C/s
5. Store at -20 °C until further use

**2. Restriction Digest**

#### Restriction Digest

Restriction endonucleases are commonly used to clone DNA fragments into a plasmid backbone. This protocol is based on High-Fidelity restriction endonucleases (New England Biolabs, Ipswich) but can be easily adapted for other endonucleases.

**Table 1: Typical composition of a restriction digest reaction.**| Component | Volume/Amount |
| --- | --- |
| DNA | 1 µg |
| Restriction Endonucleases | 1 µL each |
| rCutSmart Buffer (10X) | 3 µL |
| Nuclease-free water | to 30 µL |

Incubate the reaction mixtures for 1 h at 37 °C.

**3. Golden Gate Assembly**

#### Golden Gate Assembly

Golden Gate Assembly enables the efficient and precise assembly of multiple DNA fragments in a single reaction using type IIS restriction enzymes.

1. Set up assembly reactions in PCR-tubes with 100 ng vector DNA as follows:

**Table 2: Typical composition of a golden gate assembly reaction.**| Component | Volume/Ratio |
| --- | --- |
| Vector:Insert | 1:3 |
| T4 DNA Ligase Buffer (10X) | 2.5 µL |
| T4 DNA Ligase | 0.5 µL |
| Type IIS Restriction Enzyme | 1.5 µL |
| Nuclease-free water | to 25 µL |

2. Place PCR-tubes in thermocycler and run the following program:

**Table 3: Thermocyler program for a golden gate assembly reaction.**| Step | Temperature [°C] | Time [min] | Return to step | # Passes total |
| --- | --- | --- | --- | --- |
| Activation of Restriction Enzyme | 37 | 5 |  | 10 - 30 |
| Activation of Ligase | 16 | 5 | 1 | 10 - 30 |
| Inactivation of Enzyme and Ligase | 65 | 5 |  | 1 |
| Hold | 4 | ∞ |  | 1 |

3. Store at 4 °C until further use.

**4. Gibson Assembly**

#### Gibson Assembly

Gibson assembly is a molecular cloning strategy used to assemble multiple DNA fragments with overlapping overhangs. In contrast to classical restriction cloning, gibson assembly does not require restriction sites for assembly.

1. Design DNA fragments with 15 - 20 bp overhangs (2 - 3 fragments) or 20 - 30 bp overhangs (4 - 6 fragments).
2. Amplify DNA fragments via PCR.- Set up Gibson assembly reaction mix:

   **Table 4: Typical composition of a gibson assembly reaction.**| Component | 2 - 3 Fragment Assembly | 4 - 6 Fragment Assembly |
   | --- | --- | --- |
   | DNA | 0.02 - 0.5 pmol | 0.2 - 1 pmol |
   | Gibson Assembly Master Mix (2X) | 10 µL | 10 µL |
   | Nuclease-free water | to 20 µL | to 20 µL |- Incubate the reaction mix at 50 °C for 15 minutes (2 - 3 fragments) or 60 minutes (4 - 6 fragments).

**5. Ligation**

#### Ligation

A ligation reaction joins a vector and an insert by linking their compatible ends using DNA ligase.

1. Use 50 ng of vector DNA to set up ligation reactions in PCR-tubes as follows:

**Table 5: Typical composition of a ligation reaction.**| Component | Volume/Ratio |
| --- | --- |
| Vector:Insert Ratio | 1:3 |
| T4 DNA Ligase Buffer (10X) | 2 µL |
| T4 DNA Ligase | 1 µL |
| Nuclease-free water | to 20 µL |

2. Place PCR-tubes in the thermocycler and run at 16 °C for 20 minutes. Inactivate DNA ligase at 65 °C for 10 minutes.

**6. KLD Ligation**

#### KLD Ligation

KLD ligation is used to circularize blunt end DNA fragments. The KLD reaction mix contains a kinase, ligase, and DpnI restriction endonuclease.

1. Set up the KLD reaction mixture (10 µL total volume):

**Table 6: Typical composition of a KLD ligation reaction.**| Component | Volume |
| --- | --- |
| Blunt End DNA | 1 µL |
| KLD Reaction Buffer (2X) | 5 µL |
| KLD Enzyme Mix (10X) | 1 µL |
| Nuclease-free water | 3 µL |

2. Mix the reaction mixture by pipetting up and down.
3. Incubate the reaction mixture at room temperature (25 °C) for at least 5 minutes.
4. Use 5 µL of the reaction mixture for the transformation of 50 µL competent cells.

**7. PCR**

#### PCR

PCR (Polymerase Chain Reaction) is a technique used to amplify a template DNA by cycling through denaturation, annealing, and extension phases with a thermostable DNA polymerase.

1. Set up assembly reactions in PCR-tubes for a total volume of 25 µL as follows:

**Table 7: Typical composition of a PCR reaction.**| Component | Volume/Amount |
| --- | --- |
| Template DNA | 10 - 100 ng |
| Forward Primer (10 µM) | 1.25 µL |
| Reverse Primer (10 µM) | 1.25 µL |
| Q5 High-Fidelity DNA Polymerase | 0.5 µL |
| Q5 Reaction Buffer (5X) | 5 µL |
| dNTP Solution Mix (10 mM) | 1 µL |
| Nuclease-free water | to 25 µL |

2. Mix gently and spin down.
3. Place PCR-tubes in thermocycler and run the following program:

**Table 8: Thermocyler program for a PCR.**| Step | Temperature | Time | Return to step | # Passes total |
| --- | --- | --- | --- | --- |
| Initial Denaturation | 98°C | 30 s |  | 1 |
| Denaturation | 98°C | 10 s |  | 25 - 40 |
| Annealing | 60 - 72 °C | 15 s |  | 25 - 40 |
| Extension | 72°C | 30 s/kb | 2 | 25 - 40 |
| Final Extension | 72°C | 5 min |  | 1 |
| Hold | 4°C | ∞ |  | 1 |

4. Store at 4 °C until further use.

**8. Agarose Gel Electrophoresis**

#### Agarose Gel Electrophoresis

Agarose gel electrophoresis is used to separate DNA fragments of different nucleotide lengths from each other.

1. Depending on the nucleotide lengths of the DNA fragments, gels with a different agarose concentration should be used:

**Table 9: Agarose concentrations for different nucleotide lengths.**| Nucleotide Length | Agarose Concentration |
| --- | --- |
| 600 - 50000 bp | < 1% |
| 400 - 8000 bp | 1% |
| 100 - 2000 bp | 2% |
| 25 - 1000 bp | 3% |

2. Add agarose to TAE buffer in the desired agarose concentration and heat in the microwave until the agarose is completely dissolved (be careful to not let the agarose boil over).
3. Add a staining agent to the agarose before pouring the cell (e.g. SYBR Safe).
4. Pour gel and wait for 15 - 30 min until the gel becomes firm.
5. Run gel at 10 volts/cm until the dye front has reached the bottom of the gel.
6. Visualize gel under UV or blue light (depending on the staining agent used).

**9. Competent Cells Preparation & Transformation**

#### Competent Cells

Competent cells are bacteria that are able to take up DNA from their environment. Chemically competent cells are made competent with different salt solutions and can be transformed via heat shock.

##### Making Chemically Competent *E. coli* Cells

1. To make chemically competent *E. coli* cells, inoculate 5 - 10 mL of LB medium with a single *E. coli* colony.
2. Incubate the culture overnight at 37 °C while shaking until an optical density of 0.4 - 0.6 is reached.
3. Transfer the culture in a sterile centrifuge tube and cool down on ice for 10 - 15 min.
4. Centrifuge the culture at 4000 g for 10 min at 4 °C.
5. Discard supernatant by decanting.
6. Resuspend the cell pellet in an equal volume of cold, sterile 100 mM CaCl2 solution.
7. Incubate for 30 min on ice, gently invert the tube every 5 - 10 min.
8. Centrifuge cells at 4000 g for 10 min at 4 °C.
9. Discard supernatant by decanting.
10. Resuspend the cell pellet in 100 - 200 µL of cold, sterile 100 mM CaCl2 solution.
11. Divide cells in aliquots (20 - 100 µL), flash-freeze in liquid nitrogen, and store at - 80 °C.

##### Transformation of Chemically Competent *E. coli* Cells

1. Thaw cells on ice for 15 min.
2. Add 1 - 2 µL of plasmid DNA to the cells and incubate for 30 min on ice. Do not mix.
3. Heat-shock cells at 42 °C for 45 sec.
4. Place on ice for 5 min.
5. Add 1 mL of SOC outgrowth medium and recover cells for 45 min at 37 °C while shaking (Note: Recovery phase can be skipped if ampicillin is used as a selection marker).
6. Centrifuge for 12000 rpm for 1 min.
7. Discard supernatant by decanting and resuspend cell pellet in remaining supernatant..
8. Plate bacteria on LB agar plates containing the appropriate antibiotics.

### Protein Expression & Analysis

**1. Bacterial Protein Expression & Purification**

#### Bacterial Expression

##### Buffers and Reagents

##### LB media

* 10 g/L Tryptone
* 5 g/L Yeast extract
* 10 g/L NaCl

##### DYT-Media

* 16 g/L Tryptone
* 10 g/L Yeast extract
* 5 g/L NaCl

##### Tris Lysis buffer

* 50 mM Tricine-HCl (pH7.8)
* 300 mM NaCl
* 10 mM Imidazol
* 1% Triton X-100 (vol/vol)
* Shortly bevor using add lysozyme to final concentration of 1 mg/mL
* Add protease inhibitor cocktail (alternatively 1 mM EDTA, 1 mM EGTA, 1 mM NaVO4, 1 mM NaF can be
  used aswell)

##### NaP10 buffer

* 50 mM NaH2PO4
* 150 mM NaCl
* 10 mM Imidazol
* Adjust pH to pH 8 (at 25 °C) with NaOH
* For use as a lysis buffer add lysozyme to a final concentration of 1 mg/mL
* Add protease inhibitor cocktail (alternatively 1 mM EDTA, 1 mM EGTA, 1 mM NaVO4, 1 mM NaF)

##### NaP20 buffer

* 50 mM NaH2PO4
* 150 mM NaCl
* 10 mM Imidazol
* Adjust pH to pH 8 (at 25 °C) with NaOH

##### NaP250

* 50 mM NaH2PO4
* 150 mM NaCl
* 250 mM Imidazol
* Adjust pH to pH 8 (at 25 °C) with NaOH

##### Wash buffer - FLAG

* PBS buffer
  + 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4

##### Peptide elution buffer (FLAG-tag)

* PBS
* dilute 2x DYKDDD (3x FLAG-peptide) to final concentration of 1.5 mg/mL

##### Acid elution buffer (FLAG-tag)

* 100 mM glycine
* Adjust to pH 2.8 with HCl

##### Neutralization buffer

* 1 M Tris-HCl (pH 9.5)

##### Protein Expression

##### Day 0 - Overnight culture

1. Prepare 20 mL over night culture (ONC) in LB substituted with 1 % (wt/vol) glucose and appropiate antibiotics
   for selection

##### Day 1 - Recombinant protein expression

1. Inoculate 500 - 2000 mL DYT media 1:100 with ONC and appropiate antibiotics
2. Grow cells at 37 °C until OD600 0.4 - 0.6 is reached
3. Add appropiate inductor for expression

* e.g. 0.1 - 0.4 mM IPTG

4. Incubate culture for ≥ 4 h

* We recommend expression at 32 °C over night

5. Pellet cells at 400 x g for ≥ 30 min at 4 °C

* Pellet can be stored at -80 °C until further use

#### Cell Lysis

1. Dissolve pellet in 2 mL/g lysis buffer, mix well and incubate pellet on ice for 5-15 min

* For His-tag purification we recommend NaP10 supplemented with protease inhibitors and lysozyme
* For FLAG-tag purification we recommend a Tris based lysis buffer

2. Sonicate samples on ice for 10 min at 40 % amplitude with 20 s on and 30 s off
3. Add DNase I (1:1000) and incubate for 15 min on ice
4. Clear lysate by centrifugation at ≥ 20 000 x g for 30 min at 4 °C

**Take fraction:** Fr.1 - Start
5. Filter supernatant with a 0.22 or 0.45 µm pore size and use filtrate for downstream purification & analysis

* **Take fraction:** Fr.2 - Load

#### Purification of FLAG-tagged Proteins

##### Preparation of FLAG-beads

For the purificication we used Pierce™ Anti-DYKDDDDK Affinity Resin (A36801). Adjustments to the protocol might be necessary for beads from different vendors.
Furthermore specific lysis amount and incubation times are dependant on expressed proteins and may need to be optimized for each expression platform
The following protocol is designed for use of 650 µL resin slurry(325 µL settled resin) using a spin column

1. Prepare 8 column volumes (CV) of PBS in a 10 mL spin column and add 650 µL resin slurry to the column.
2. Insert the spin column in a 50 mL collection tube and centrifuge for 1 min at 1000 x g. Discard the flow through
3. Plug the column tip and add 10 CV (3250 µL) of PBS, flick tube to resuspend resin fully.
   Centrifuge column for 1 min at 1000 x g and discard flow through
4. Repeat wash step once for a total of two washes

##### Sample Loading & Washing

1. Plug the column tip and add prepared lysate to the resin. Resuspend the resin fully by flicking the tube or gently vortexing
2. Mix column with an end-over-end mixer for ≥ 20 min (This step can be performed at 4 °C, but incubation of ≥ 1 h is recommended)
3. Remove plug and centrifuge column for 1 min at 1000 x g

* **Take fraction:** Fr.3 - FT

4. Plug the column tip and fully resuspend the resin in 10 CV PBS (3.25 mL) and centrifuge sample for 1 min at 1000 x g
5. Repeat wash step once for a total of 2 washes

* **Take fractions** of the flow through of each washing step (Fr.3 & Fr.4 - Wash 1 & 2)

6. Wash once more with 10 CV purified water

* **Take fraction:** Fr.5 - Wash3

##### Elution

##### Gentle elution protocol

1. Dilute 3x DYKDDDDK peptide in PBS to a final concentration of 1.5 mg/mL
2. Plug the column tip and add 300 µL of prepared elution buffer. Gently voretx to resupend resin. Place the column on a rotator and mix for 5 minutes at room temperature.
3. Remove the plug from the column and insert it into a clean collection tube. Centrifuge for 1 minute at 1,000 x g.
   A second elution step for maximum recovery is recommended

##### Acid elution protocol

1. Plug the column tip and add 2 mL Elution Buffer (pH 2.8). Cap the column and vortex for 3 seconds to resuspend the resin. Remove the plug and insert the column into a clean collection tube, centrifuge for 1 minute at 1000 x g.
   To neutralze the low pH, add 150 µL of neutralization buffer for each 1 mL of elution buffer.
   A second elution step for maximum recovery is recommended.
   Note: Immediatly add PBS buffer to the resin to avoid denaturation of the DYKDDDDK antibody

##### Regenerate resin

1. Wash the resin with 10 bed volumes of PBS
2. Was the resin with 10 bed volumes of elution buffer
3. Wash the resin with 20 bed volumes of PBS
4. Store the resin in PBS at 50 % slurry containing 0.02 % sodium azide at 4 °C

#### Purification of His6-tagged Proteins

##### Column preparation

1. Add 1 mL of resin slurry (contains 500 µL Ni-NTA resin) to a gravity-flow column and let storage buffer drain.
2. Equilibrate the column with two bed volumes of equilibration buffer.

##### Sample Purification

1. Add the prepared protein extract to the resin and collect the flow-through in a tube.
   Recommended: Re-apply the flow-through at least once or plug the column tip and incubate the lysate for ≥ 30 min
   * **Take fraction:** Fr.3 - FT
2. Wash resin with 10 bed volumes NaP10
   * **Take fraction:** Fr.4 - Wash1
3. Wash resin with 10 bed volumes NaP20
   * **Take fraction:** Fr.5 - Wash2
4. Elute with 2x 2.5 bed volumes NaP250
   * **Take fraction:** Fr.6 - Elution1
   * **Take fraction:** Fr.7 - Elution2

##### Regenerate Resin

Ni-NTA resinc an be typically reused multiple times without affecting protein yield or purity. After each use the column should be washed as described to ensure longevity

1. Wash resin with 10 bed volumes MES Buffer
2. Wash resin with 10 bec volumes of ultrapure water
3. Store resin as a 50 % slurry in 20 % ethanol

**2. SDS-Page**

#### Materials

* 30% acrylamide/bis-acrylamide
* 10% APS
* TEMED
* Tris base
* Tris-HCl
* Glycine
* SDS
* SDS sample buffer
* Deionized water (dH2O)

All buffer recipes are provided at the end of the protocol.

##### 1. Gel Casting

In case of using pre-casted gels, skip to the next section.

* Choose an appropriate acrylamide % for the gels depending on the molecular weight of the target protein.
* Prepare separating and stacking gel mixes according to Table 10 and 11 below (all volumes are in ml).

  Do not add APS and TEMED as it will start polymerizing the gels.

**Table 10: Separating Gel (total 10ml).**| MW of target protein (kDa) | 80-200 | 35-100 | 25-60 | 20-40 |
| --- | --- | --- | --- | --- |
| Gel Percentage | 8% | 10% | 12% | 15% |
| dH2O | 2.1 | 1.5 | 0.8 | 0 |
| 30% Acrylamide | 2.7 | 3.3 | 4 | 5 |
| 2x Separating Buffer | 5.0 | 5.0 | 5.0 | 5.0 |
| 10% APS | 0.1 | 0.1 | 0.1 | 0.1 |
| TEMED | 0.01 | 0.01 | 0.01 | 0.01 |

**Table 11: Stacking Gel (4%).**| Stacking Gel (mls) | 4ml | 6ml | 8ml |
| --- | --- | --- | --- |
| dH2O | 1.4 | 2.1 | 2.7 |
| 30% Acrylamide | 0.5 | 0.8 | 1.1 |
| 2x Stacking Buffer | 2.0 | 3.0 | 4.0 |
| 10% APS | 0.04 | 0.06 | 0.08 |
| TEMED | 0.004 | 0.006 | 0.008 |

**Caution:** Work under the fume hood to avoid direct exposure to acrylamide.

* Add APS and TEMED into the the separating gel mix and pour it into assembled glass plates.
* Layer the top with isopropanol to remove any bubbles and even out the surface.
* After the separating gel has solidified (~30 mins), decant off the isopropanol and add the stacking gel mix (with APS and TEMED).
* Place combs and allow the stacking gel to solidify (~15 mins).
* Once solid, the casted gels in glass plates can be stored at 4°C.

##### 2. Sample Preparation

* Preheat the heat block to 95°C.
* Add SDS sample buffer to the samples to yield a final concentration of 1X.
* Vortex and briefly spin down samples.
* Incubate the samples in the heat block for 5 mins at 95°C.
* Spin down the samples and cool to ambient temperature.

##### 3. Sample Loading

* Insert gels into the running chamber. If using one gel, place a gel dummy in the second gel slot.
* Add runnning buffer and ensure there are no leaks.
* Fill the chamber with running buffer and remove combs from the gels carefully with both hands.
* Wash the gel wells with running buffer.
* Pipette samples into the wells:

+ Add 7 µl marker into the first lane.
+ Add 20-25 µl of sample per well (adjust volume based on protein concentration).

##### 4. Running the Gel:

* Place the electric cap on the running chamber and run the gel at 80-120 V for 1.5 hours or until the blue front reaches the end of the gel. TIP: Placing the gel running chamber on ice reduces gel heating and ensures even running of all lanes.

##### 5. Post-Run:

* The gel is now ready for staining (e.g., Coomassie, Silver-stain) or protein transfer to a membrane (Western Blot).

##### 6. Buffer Recipes

**Table 12: 2x Separating Buffer Recipe (makes 1000ml).**| Component | Amount |
| --- | --- |
| Tris-HCl | 90.8g |
| SDS | 2.0g |

Dissolve compounds thoroughly. Adjust pH slowly to pH 8.8 with concentrated HCl, then add dH2O to 1000ml.

**Table 13: 2x Stacking Buffer Recipe (makes 1000ml).**| Component | Amount |
| --- | --- |
| Tris-HCl | 30.35g |
| SDS | 2.0g |

Dissolve compounds thoroughly. Adjust pH slowly to pH 6.8 with concentrated HCl, then add dH2O to 1000ml.

**Table 14: 1x Running Buffer Recipe (makes 1000ml).**| Component | Amount |
| --- | --- |
| Tris-base | 1.51g |
| Glycine | 7.5g |
| SDS | 0.5g |

Dissolve compounds thoroughly, then add dH2O to 1000ml.

**3. Western Blot**

#### Western Blot

Western blotting is used to immobilize proteins that were separated via SDS-PAGE on a membrane making them accessible for immunodetection.

##### Materials

* Transfer Buffer

+ 48 mM TRIS pH 8.3
+ 39 mM glycine
+ 20% methanol

* TBS-T

+ 20 mM TRIS pH 7.5
+ 150 mM NaCl
+ 0.1% Tween 20

* Blocking Solution

+ 3% bovine serum albumin (BSA) in TBS-T

##### Semi-Dry Western Blot

1. Lyse cells with an appropriate lysis buffer.
2. Determine the protein amount of the lysate via Bradford assay or DC Protein assay (when using lysis buffers that contain detergents).
3. Load an SDS gel with 20 - 50 µg of total protein per pocket.
4. Run gel at 100 V for 1 h at 4 °C until the dye has reached the bottom of the gel.
5. Equilibrate gel for 15 min in transfer buffer while shaking.
6. Activate a PVDF membrane in 100% methanol for a few seconds until it gets translucent.
7. Equilibrate the activated membrane in transfer buffer for 2 - 3 minutes.
8. Soak two sheets of extra-thick blotting paper in transfer buffer.
9. Assembly blotting sandwich on blotting station (Cathode | Blotting Paper | Gel | PVDF Membrane | Blotting Paper | Anode).
10. Blot at 10 V for 1 h.
11. Incubate membrane for 1 h at room temperature with blocking solution.
12. Dilute primary antibodies to working solutions in blocking solution and incubate with membrane at 4 °C overnight while shaking.
13. Wash the membrane three times with TBS-T for 10 min each.
14. Dilute secondary antibodies to working solutions in TBS-T and incubate with membrane at room temperature for 1 h while shaking.
15. Wash the membrane three times with TBS-T for 10 min each.
16. Image blot with the appropriate detection method (colorimetric, chemiluminescence, fluorescence).

### Mammalian Cell Protocols

**1. Cell Maintenance**

#### Cell Maintenance

This is a basic protocol for splitting Human Embryonic Kidney 293T (HEK293T) cells to maintain the culture throughout the week, with excess cells reserved for future experiments and plating. Make sure to work in a sterile environment to avoid contamination of the cells.

1. Warm reagents to 37°C and prepare complete DMEM supplemented with 10% FCS and 1% P/S:

**Table 15: Composition of complete DMEM supplemented with 10% FCS and 1% P/S.**| Reagent | Volume [mL] |
| --- | --- |
| Dulbecco's Modified Eagle Medium (DMEM) | 445 |
| Fetal Calf Serum (FCS) | 50 |
| Penicillin/Streptomycin (P/S) | 5 |

2. Take cells out of the incubator and aspirate the old media from the plate or flask.
3. Wash with Dulbecco's Phosphate Buffered Saline (DPBS).
4. Add trypsin and incubate at 37°C until cells start to detach.
5. Add DMEM to neutralize the trypsin.
6. Count cells or decide on the appropriate dilution based on confluence.
7. Dilute with DMEM until the desired concentration is reached.
8. Seed cells onto a new plate or flask, gently shuffle to ensure even dispersal, and return cells to the incubator.

**2. Cell Seeding**

#### Cell Seeding

This is a basic protocol for seeding Human Embryonic Kidney 293T (HEK293T) cells for future experiments, for example transfections. Make sure to work in a sterile environment to avoid contamination of the cells.

1. Collect cells according to the instructions of the Cell Maintenance protocol.
2. Count cells and calculate the appropriate dilution.
3. Dilute with DMEM until the desired concentration is reached.
4. Seed according to the cell culture dish you used:

**Table 16: Cell seeding protocols for different cell culture dishes.**| Type of dish | Seeding density | Cells at confluency | Volume of growth medium [mL] |
| --- | --- | --- | --- |
| 6-well plate | 0.3 x 106 | 1.2 x 106 | 2 - 3 |
| 12-well plate | 0.1 x 106 | 0.5 x 106 | 1 - 2 |
| 96-well plate | 0.01 x 106 | 0.04 x 106 | 0.1 - 0.2 |
| T-75 flask | 2.1 x 106 | 8.4 x 106 | 8 - 15 |
| T-25 flask | 0.7 x 106 | 2.8 x 106 | 3 - 5 |

5. After seeding, gently shuffle to ensure even dispersal, and return cells to the incubator.

**3. Transfection**

#### Transfection

Transfection refers to the uptake of exogenous DNA into eukaryotic cells.

1. Seed cells in an appropriate well plate and incubate at 37 °C and 5% CO2 for 24 h before transfection (cells should reach a confluency of 70 - 90%).
2. Dilute DNA to desired concentrations in OptiMEM medium (Thermo Fisher Scientific, Waltham).
3. Dilute lipofectamine 2000 (Thermo Fisher Scientific, Waltham) transfection reagent in OptiMEM medium. Incubate for 5 min.

**Table 17: Transfection protocol.**| Component | 96-well | 24-well | 6-well |
| --- | --- | --- | --- |
| Final DNA amount per well | 100 ng | 500 ng | 2500 ng |
| Final lipofectamine 2000 reagent per well | 0.2 - 0.5 µL | 1.0 - 2.5 µL | 5.0 - 12.5 µL |

4. Add lipofectamine to DNA solutions and incubate for 30 min.
5. Add DNA-lipid complexes to cells and incubate cells at 37 °C and 5% CO2 for 1 - 3 days.

### Assays

**1. Electrophoretic Mobility Shift Assay (EMSA)**

#### Electrophoretic Mobility Shift Assay (EMSA)

Electrophoretic Mobility Shift Assay (EMSA) is used to study protein-nucleic acid interactions by analyzing the binding of proteins to specific DNA sequences. It detects the formation of protein-nucleic acid complexes based on their slower migration through a polyacrylamide gel compared to free nucleic acids. Based on the results of an EMSA, quantitative values like the apparent dissociation constant Kd can be calculated.

##### Preparation of Samples

* DNA fragments can be ordered as oligos or extracted from cell lysate. In general, DNA fragments 20 - 40 nt in length are used in concentrations under the dissociation constant of the protein-nucleic acid complex.
* Use purified proteins in appropriate buffers under physiological conditions.
* The protein concentration must be optimized to detect specific binding without overwhelming the system with non-specific interactions. Protein concentration should be titrated from a concentration that lies above the dissociation constant to a concentration that lies below the dissociation constant of the protein-nucleic acid complex.
* Equilibrate samples for 30 min at 4 - 20 °C depending on the protein-nucleic acid complex (Note: Non-protein components should be pre-mixed and brought to temperature before adding proteins).

##### Native-PAGE Gel Electrophoresis

1. Pre-run native PAGE gel in running buffer at 200 - 300 V for at least 30 min.
2. Load samples in 1x Native PAGE loading dye (should contain 2 - 5% glycerol).
3. Run gel at 100 V for approximately 30 min.
4. To detect DNA, incubate the gel in running buffer with SYBR Safe (1:10 000 diluted) for 30 min. Image gel with transilluminator.
5. Wash gel twice with ddH2O.
6. To detect proteins, incubate the gel in Coomassie staining solution for 30 min.
7. Destain with Coomassie destain solution.

**2. Förster Resonance Energy Transfer (FRET)**

#### FRET Bacterial Growth Assay

Förster Resonance Energy Transfer (FRET) is a distance-dependent interaction phenomenon between two fluorescent molecules, where energy is transferred from an excited donor fluorophore to a nearby acceptor fluorophore without emission of a photon. The donor is excited by absorbing light at a specific wavelength, and if the acceptor is within close proximity (1-10 nm), the energy is transferred, leading to emission from the acceptor. In FRET experiments, the key measurement is the emission intensity of the acceptor, while the donor's fluorescence is reduced. This process provides valuable information about molecular distances and interactions.

How to measure FRET with a bacterial culture:

1. Prepare an overnight culture by inoculating 4 mL of LB medium supplemented with the appropriate antibiotics with a single bacterial colony from a LB agar plate. Incubate at 37 °C overnight while shaking.
2. Preparation of pre-culture:

* Set up a pre-culture by inoculating 10 mL of LB medium supplemented with the appropriate antibiotics with 100 µL of the overnight culture.
* Incubate pre-culture at 37 °C while shaking until the culture reaches an OD600 of 0.1 (dilute with LB medium supplemented with the appropriate antibiotics if OD600 is too high).

3. Preparation of 96-well plate:

* Pipette 200 µL of LB medium containing 1.6 mM IPTG and the appropriate antibiotics into the first row of a 96-well plate using a multichannel pipette.
* Pipette 100 µL LB medium in the remaining wells.
* Perform a 1:2 dilution series from row A to G:

+ Pipette 100 µL from the wells of the first row into the second row and mix by pipetting up and down.
+ Repeat for the remaining rows but leave out row H.

4. Pipette 100 µL of the bacterial culture in each well (leave out one column and add 100 µL of LB medium instead; avoid bubbles while pipetting).
5. Measure OD600, fluorescence intensity, and FRET fluorescence intensity with a TECAN plate reader.

* Measure the fluorescence intensity of both donor and acceptor fluorophores separately. And for the FRET interaction.
* Calculate the FRET efficiency as a ratio of acceptor emission to donor emission, correcting for background fluorescence.

**3. Dual Luciferase Assay**

#### Dual-Luciferase Assay

The Dual-Luciferase Assay utilises two types of luciferase: Renilla and Firefly. Renilla luciferase serves as an internal control and oxidises the substrate Coelenterazine, emitting light at approximately 480 nm. Firefly luciferase, on the other hand, acts as a reporter of gene expression by catalysing the oxidation of luciferin, which results in light emission at a wavelength of around 550-570 nm. This assay requires two key reagents from the Dual-Glow Luciferase Assay system from Promega: LAR II, which contains luciferin, and the STOP & Glo reagent, which halts the firefly luciferase reaction while introducing the Renilla substrate, Coelenterazine.

##### Materials

* White 96-well plate
* Dual-Luciferase® Reporter Assay System
* MiliQ water
* 70% Ethanol

##### Preparation of LARII reagent

* Thaw buffer in water
* Use a serological pipette, take up 5 mL LARII buffer (Green cap) and resuspend LARII Substrate (powder in an amber glass bottle), transfer to a brown 15mL falcon tube
* Take up another 5mL LARII buffer and repeat
* Wrap falcon in aluminium foil
* Freeze at -80°C or use directly

##### Preparation of STOP&Glow reagent

* Thaw buffer in water
* Transfer 10mL STOP&Glow buffer to a brown 15 mL Falcon tube
* Add 200 µL Stop&Glow reagent (1.5mL reaction tube with blue cap)
* Wrap falcon in aluminium foil
* Freeze at -80°C or use directly (Protect from Light at all times)

##### Execution of the measurement

##### Day 1

* Seed HEK293T cells into a transparent 96-well plate with a density of 12.500 cells/well

##### Day 2

* Transfect cells with the respective transfection plan & incubate ~48h at 37°C & 5% CO2

##### Day 4

* Before lysis, take out LAR II & STOP&Glow Buffers from -80 °C and thaw in water (35 µL/well of each reagent + 2 mL dead volume for priming)
* Prepare passive Lysis Buffer (needs 30 µL/well, dilute from 5x to 1x)
* Discard media from the 96-well plate by a quick swing over the S1-bin (not too hard otherwise HEK cells will be flung out) and then lyse with 30 µL/well passive Lysis Buffer for 30 min at RT & 500 rpm (tape to a thermomixer and set to 25 °C)
* While Lysis takes place, prepare TeCan Reader by washing the injectors twice with water, then twice with air
* When thawed, place the left injector into 15 mL Falcon of LAR II and the right injector into 15 mL Falcon of STOP&Glow
* Start Priming
* Measure with a protocol first dispensing 30 µL of LARII integrating for 8 seconds followed by 30 µL of STOP & Glow, again integrating for 8 seconds
* After Lysis, either transfer 10 µL/well to a new, white 96-well plate or seal with aluminium foil and store at -20°C
* Place the white 96-well plate into the TeCan reader and start the measurement
* After the measurement, remove the plate, backflush the reagents and wash the injectors three times with water, twice with 70% ethanol, then twice with air

**4. qPCR**

#### RT-qPCR

RT-qPCR (Reverse Transcription Quantitative Polymerase Chain Reaction) is a powerful technique used to quantify gene expression levels in various biological samples. It combines reverse transcription of RNA into complementary DNA (cDNA) with PCR, enabling the precise quantification of specific RNA molecules.

1. RNA isolation is performed from tissue or cells using either the miRNeasy Tissue/Cells Advanced Mini Kit or the RNeasy Mini Kit, following the manufacturer’s instructions.
2. Isolated RNA is reverse transcribed into cDNA using the iScript Advanced cDNA Synthesis Kit for RT-qPCR, according to the manufacturer’s protocol.
3. Preparation of cDNA Synthesis Reaction Mix:

**Table 18: Typical composition of a cDNA Synthesis Reaction Mix.**| Component | Volume |
| --- | --- |
| 5x iScript Advanced Reaction Mix | 4 μL |
| iScript Advanced Reverse Transcriptase | 1 μL |
| Isolated RNA (100 fg – 7.5 µg) | variable |
| Nuclease-free water | to 20 μL |

4. Incubate the reaction mix in a thermocycler under the following conditions:

* Reverse transcription: 20 min at 46 °C
* RT inactivation: 1 min at 95 °C
* *(Note: Alternatively, use 30 min at 42°C and 1 min at 95°C.)*

5. After cDNA synthesis, dilute the cDNA based on RNA input:
   * For high RNA input (1.0 – 7.5 µg), dilute cDNA at least 10-fold
   * For low RNA input (< 1 µg), use cDNA directly in qPCR- Preparation of RT-qPCR Reaction Mix (Total Volume: 15 μL):

   **Table 19: Typical composition of a RT-qPCR Reaction Mix .**| Component | Volume |
   | --- | --- |
   | iQ SybrGreen Master Mix | 7.5 μL |
   | Forward primer (300 nM) | 0.5 μL |
   | Reverse primer (300 nM) | 0.5 μL |
   | Diluted cDNA | 6.5 μL |- Mix gently and spin down.
   - Pipette samples onto plate and seal wells with optically transparent film.
   - Run the qPCR using the following program:

   **Table 20: Thermocyler program for a qPCR.**| Step | Temperature | Time | Return to step | # Passes total |
   | --- | --- | --- | --- | --- |
   | Polymerase Activation and DNA Denaturation | 95°C | 30 s |  | 1 |
   | Denaturation | 98°C | 5 –15 s |  | 35 - 40 |
   | Annealing/Extension and Plate Read | 55 – 60 °C | 15 –30 s | 2 | 35 - 40 |
   | Melt-Curve Analysis | 65 – 95 °C with 0.5 °C increments | 2 – 5 s per step |  | 1 |- *(Note: Shorter annealing/extension times (1 – 10 s) can be used for amplicons <100 bp.
      Longer annealing/extension times (30 – 60 s) can be used for amplicons >250 bp.)*

**5. T7 Endonuclease I**

#### T7 Endonuclease I Assay

The T7 Endonuclease I (T7E1) assay is a widely used method to detect CRISPR-Cas-induced mutations, such as insertions or deletions (indels), at targeted genomic loci. After CRISPR editing, T7E1 recognizes and cleaves mismatched DNA in heteroduplexes formed when edited and unedited DNA strands are hybridised. The resulting DNA fragments can then be visualised using agarose gel electrophoresis. This assay provides a quick and efficient way to assess the efficiency of CRISPR-mediated genome editing by detecting the presence of indels at the target site.

##### Materials

* DirectPCR-Cell lysis reagent
* Proteinase K
* NEBuffer 2
* T7 Endonuclease I

##### Cell Processing

Day 1: Cell Seeding

* Seed 12,500 cells per well into a 96-well plate and incubate overnight at the appropriate conditions for cell growth.

Day 2: Transfection

* Transfect cells by adding 200 ng of plasmid DNA and Lipofectamine 2000 transfection reagent to each well, following the manufacturer’s protocol for optimal transfection efficiency.

Day 5: Cell Lysis and DNA Amplification

* Lysate preparation: Add 70 µL of sterile H₂O, 70 µL DirectPCR Cell Lysis Reagent, and 1.4 µL Proteinase K to each well.
* Incubate cells for 6 hours at 55 °C with gentle shaking (120 rpm).
* Inactivate enzymes by heating at 85 °C for 45 minutes.
* Amplify the targeted gene regions by adding 3 µL of the lysate to a 25 µL PCR reaction using Q5 High-Fidelity DNA Polymerase and specific primers.

##### Post-PCR Processing

* Pour 2% agarose gel in 1xTBE.* Transfer 5 µL of the PCR product into a new tube with 2 µL NEB Buffer 2 and 13 µL nuclease-free water.
  * Subject the sample to a temperature gradient: reduce from 95 °C to 85 °C at a rate of 2 °C/s, then from 85 °C to 25 °C at 0.1 °C/s.
  * **Keep samples on ice from now on!**

##### T7 Endonuclease I Digestion

* Add 0.5 µL of T7 Endonuclease I to the reaction and incubate at 37 °C for 15 minutes to cleave mismatched DNA.

##### Gel Electrophoresis

* Immediately add 5 µL of purple loading dye (no SDS) with 1% GelRed to the digested samples.
* Run the samples on the already prepared gel to visualise the cleaved DNA fragments.
* Analyse the gel images using ImageJ to quantify the extent of cleavage.

**6. Inter-Bacterial Conjugation Assay**

#### Inter-Bacterial Conjugation Assay

##### Solid Media Conjugation Assay

* Transform corresponding plasmids into chemically competent *E. coli* strains.
* Plate the transformed cells on LB agar plates containing the appropriate antibiotics.
* Pick single colonies and set up 5 mL overnight cultures in LB medium with antibiotics.
* Reserve 500 µL of the liquid cultures as glycerol stocks, store at -80°C for future use.
* Centrifuge the remaining cultures at 11,000 rpm for 1 min in separate 1.5 mL tubes.
* Resuspend the pellets in 100 µL of 10 mM magnesium sulfate solution.
* Adjust the OD600 of each suspension to 10 using the magnesium sulfate solution.
* Prepare the experimental groups by mixing 100 µL of recipient and donor cell suspensions.
* Add 1 mL of magnesium sulfate solution to each tube, vortex, and centrifuge at 11,000 rpm for 1 min.
* Remove the supernatant and resuspend the cell pellets in 10 µL of magnesium sulfate solution.
* Pipette 10 µL of the mixture onto an antibiotic-free LB agar plate and let air dry for 10 min.
* Incubate the plates at 37°C and allow conjugation to proceed for 18 hours.
* Collect bacterial patches using sterile inoculation loops and resuspend in 1 mL of magnesium sulfate solution.
* Adjust the OD600 of all suspensions to 2.4.
* Prepare serial dilutions from 10-2 to 10-9 using the OD-adjusted suspensions.
* Plate the dilutions on two types of LB agar plates:
  + One selecting for recipients.
  + One selecting for transconjugants.
* Incubate the plates at 37°C overnight.
* Calculate conjugation efficiency by dividing the number of transconjugant colonies by the number of recipient colonies at a particular dilution.

##### Liquid Media Conjugation Assay

* Use the same *E. coli* clones as in the solid media conjugation assay.
* Follow the first five steps of the solid media conjugation assay.
* Resuspend the pellets in 1 mL of antibiotic-free LB medium.
* Adjust the OD600 of each suspension to 10.
* Mix 120 µL of the OD-adjusted recipient and donor cultures for each experiment group.
* Centrifuge the mixtures for 1 min at 11,000 rpm.
* Resuspend the pellets in 1.2 mL of LB medium.
* Pipette 1 mL of each suspension into a well of a 12-well plate.
* Incubate the plate at 37°C for and allow conjugation to proceed for 18 hours without shaking.
* After incubation, remove the bacterial suspensions from the wells and adjust their OD600 to 2.4.
* Prepare serial dilutions as for solid media conjugation and plate on selective agar plates.
* Incubate the plates at 37°C overnight.
* Calculate conjugation efficiency by dividing the number of transconjugant colonies by the number of recipient colonies at a particular dilution.

**7. Co-Immunoprecipitation (Co-IP) Assay**

#### Co-Immunoprecipitation (Co-IP) Assay

##### Transfection and Lysis of HEK293T Cells

##### Cell Seeding

* Seed HEK293T cells in 6-well plates at a density of 300,000 cells per well.
* Grow them in 2 ml of DMEM supplemented with 10% FCS, 2 mM L-glutamine, and 1% penicillin/streptomycin.
* Incubate for 24 hours at 37°C in a humidified incubator (5% CO₂).

##### Transfection

* After 24 hours, transfect each well with 1 µg of EGFR-GFP plasmid (Addgene #32751) using Lipofectamine 2000 (Thermo Fisher Scientific), as per the transfection protocol.

##### Lysis

* 48 hours post-transfection, lyse cells using an NP 40-based lysis buffer (50 mM HEPES-NaOH (pH 8), 100 mM NaCl, 1 mM EGTA, 0.5% IGEPAL CA-630, 2.5 mM MgCl₂, 1 mM DTT, 10% glycerol) supplemented with protease inhibitors.
* Add 200 µl of ice-cold lysis buffer per well and incubate on ice for 15 minutes.
* Scrape cells, transfer to 1.5 ml tubes, and sonicate (30% power, 15 mins on ice).
* Centrifuge at 20,800g for 30 minutes at 4°C.
* Measure protein concentration (for example using the DC Protein Assay Kit (Bio-RAD)).
* Store the supernatant at -20°C for future use.

##### Induction of Protein Expression and Lysis of *E. coli*

##### Transformation and Culture

* Transform *E. coli* 10-beta cells separately with plasmids: pNeae2\_7D12 and pNeae2.
* Plate cells on LB agar plates with chloramphenicol (25 µg/ml).
* Inoculate 5 ml overnight cultures in LB-chloramphenicol supplemented with 1% glucose for lac repression.

##### Induction

* Dilute overnight cultures 1:100 into 250 ml LB-chloramphenicol.
* Grow until OD600 reaches 0.4-0.6, then induce with 50 µM IPTG.
* Incubate for 6-8 hours at 37°C.

##### Lysis

* Harvest bacteria by centrifugation (5150 rpm, 20 mins, 4°C).
* Resuspend bacterial pellets in NP 40-based lysis buffer with protease inhibitors (2 ml per gram of pellet).
* Incubate with lysozyme (1 mg/ml) for 35 minutes at 4°C.
* Sonicate the lysates (40% power, 15 mins on ice).
* Add DNase I (25 U/ml) and incubate for 30 minutes at 4°C.
* Centrifuge at 5150 rpm for 30 minutes at 4°C.
* Measure protein concentration (for example using the DC Protein Assay Kit (Bio-RAD)).
* Store the supernatant at -20°C for future use.

##### Co-immunoprecipitation (CoIP)

##### Preparation of Anti-myc Beads

* Resuspend agarose anti-myc beads (Proteintech) and transfer 25 µl bead slurry into five 1.5 ml tubes.
* Equilibrate beads with 500 µl of ice-cold dilution buffer (10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, pH adjusted at 4°C).
* Centrifuge at 4500 rpm for 5 minutes at 4°C, discard the supernatant.

##### Binding and Washing

* Dilute HEK293T and *E. coli* lysates (400 µg total protein) in 500 µl of ice-cold dilution buffer.
* Add lysates to equilibrated beads and rotate end-over-end for 1 hour at 4°C.
* Centrifuge at 4500 rpm for 5 minutes at 4°C, discard the supernatant.
* Wash beads with 500 µl of cold wash buffer (10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.05% IGEPAL CA-630, 0.5 mM EDTA) three times.

##### Elution

* Elute bound proteins by resuspending the beads in 80 µl of 2x SDS sample buffer (Laemmli) and boiling for 5 minutes at 95°C.
* Centrifuge at 4500 rpm for 2 minutes, collect the supernatant.

##### Analysis

* Perform SDS-PAGE (using 4-15% precast gradient gel, Bio-RAD).
* Analyze the resolved proteins by performing a Western Blot using primary antibodies against GFP (rabbit anti-GFP, 1:1000) and myc (mouse anti-myc, 1:1000).
* Use fluorophore-coupled secondary antibodies for detection (goat anti-rabbit DyLight 800 4X PEG and goat anti-mouse Alexa Fluor™ 680).

##### Experimental Groups

**Table 21: Samples used to conduct the coIP experiment.**|  | 1 | 2 | 3 | 4 | 5 |
| --- | --- | --- | --- | --- | --- |
| Lysate of HEK293T transfected with EGFR-GFP | + | - | - | + | + |
| Lysate of *E. coli* 10-beta with pNeae2 | - | + | - | + | - |
| Lysate of *E. coli* 10-beta with pNeae2\_7D12 | - | - | + | - | + |
| anti-myc pulldown | + | + | + | + | + |

* Sample 1: Negative control, testing anti-myc bead cross-reaction.
* Samples 2-3: Verify presence of myc epitopes on proteins of interest.
* Samples 4-5: Co-IP experiments for nanobody-antigen interaction (Sample 4 as control, Sample 5 as test group).

**8. Adhesion Assay**

#### Adhesion Assay

##### Preparation of Mammalian Cells

* Coat coverslips in two 12-well plates with poly-D-lysine as follows:

+ Prepare 1:10 poly-D-lysine stock solution.
+ Place one coverslip in each well and add 0.5 ml of poly-D-lysine stock solution, incubate for 15 minutes at RT.
+ Wash twice with 1 ml PBS.
+ Let plates dry by leaving them open in the sterile hood for 2 hours.
+ Close plates and keep them wrapped in parafilm at 4°C for up to two weeks.

* Seed HeLa and HEK293T cells in 12-well plates on coverslips (105 cells/well).
* Let cells grow in the humid CO2 incubator for 24h.

##### Preparation of Fluorescent *E. coli*

* Set up overnight cultures of the various bacterial strains in LB-media with appropriate antibiotics:
  + *E. coli* NEB 10-beta with pHelper\_RP4 (negative control without adhesin).
  + *E. coli* NEB 10-beta with pHelper\_RP4 + pNeae2 (negative control without nanobody);
  + *E. coli* NEB 10-beta with pHelper\_RP4 + pNeae2\_7D12 (test group);* Bring to an OD600 of 0.1 (set up 2 tubes for test group and 2 tubes for each control), let grow until an OD600 of 0.5 and inoculate with 50 µM IPTG to induce adhesin expression.
  * Incubate for 2 hours at 37°C in a shaker.
  * Harvest bacteria by centrifugation (4000xg, 3 min) and bring once again to an OD600 of 0.1, inoculate with 10 µM IPTG and let grow until OD600 of 0.5 (measure tightly to catch the beginning of exponential phase).* Add RADA (stock in DMSO, final concentration in culture = 0.5 mM) in one tube of test group and negative control and incubate at 37°C for 2 hours (culture volume = 3 ml) [keep one culture each without RADA].
    * Harvest bacteria from the tubes by centrifugation (4000xg, 3 min) and wash twice with PBS
    * Check fluorescence with plate reader and compare to cells grown in media without FDAA. Excitation/emission wavelengths of RADA: approx. 554/580 nm.
    * Pellet bacteria (only the samples labelled with RADA) and keep them on ice until resuspension.

##### Infection with *E. coli* Expressing Adhesin

* Remove media from the 12-well flasks containing seeded HeLa and HEK293T cells, add DMEM without FCS and antibiotics (2 ml per well).
* Resuspend bacteria in PBS or DMEM to reach an OD of 0.1 (roughly 3x107 cfu/ml).
* Perform outside sterile hood! Add 1 ml of fluorescently labelled bacteria per well (to reach MOI 300:1), 1 hour infection time at 37°C.

##### Fixing of Cells

* Aspirate media and wash the coverslips five times with 1 ml PBS (RT).
* Fix coverslips with 0.5 ml paraformaldehyde 4% (w/v) + 4% sucrose + 200 mM HEPES solution for 15 min at 4°C.
* Wash with 1 ml PBS containing 0.1 M glycine five times.

##### Staining and Imaging

* Add Hoechst (λEx/λEm (with DNA) = 350/461 nm) at a final concentration of 1 µg/ml and WGA-Alexa Fluor 488 at a final concentration of 5 µg/ml (volume until coverslips are covered).
* Incubate for 5 min at RT in the dark.
* Remove and wash three times with PBS.
* Mount coverslips on glass slides with mounting reagent.
* Perform fluorescence or confocal fluorescence microscopy using appropriate excitation and emission filters.

**9. Inter-Kingdom Conjugation Assay**

#### Inter-Kingdom Conjugation Assay

##### Preparation of mammalian cells

* Seed HEK293T and HeLa cells in T25 flasks (7\*105 cells / flask), in 5 ml DMEM supplemented with 10% FCS, 2 mM L-glutamine and 1% penicillin/streptomycin and incubate in a humid CO2 incubator at 37°C for 24 hours.

##### Preparation of bacterial strains

* Set up 5 ml overnight cultures with LB + appropriate antibiotics:
  + *E.coli*  10 beta with pHelper\_RP4
  + *E.coli*  10 beta with pmob\_m\_CMV
  + *E.coli*  10 beta with pHelper\_RP4 + pNeae2\_7d12
  + *E.coli*  beta with pmob\_m\_CMV + pNeae2\_7d12
  + *E.coli*  10 beta with pHelper\_RP4 + pmob\_m\_CMV (test group without adhesins)
  + *E.coli*  10 beta with pHelper\_RP4 + pmob\_m\_CMV + pNeae2\_7D12 (test group with adhesins)

##### Set up conjugation reaction

* 24 hours after seeding, aspirate media from T25 flasks containing HEK293T cells.
* Wash gently with 1x DPBS twice.
* Harvest bacteria by centrifuging at 13,000 rpm for 1 min.
* Resuspend the bacterial pellets in 5.5 ml DMEM supplemented with 10% FCS, 2 mM L-glutamine, 20 mM HEPES, 5 µg/ml DNaseI-XT and 1 µM cytochalasin D.
* Add 5 ml of the bacterial suspension (OD600 1.0) to the T25 flasks containing HEK293T cells - work outside the sterile hood from here to avoid contamination of the hood.
* Place in a bacteria incubator (static) at 37°C for 12 hours.

##### Elimination of bacteria after conjugation

* Remove media (containing bacteria) from the T25 flasks and wash 2-3 times with PBS supplemented with 150 mM NaCl + 50 µg/ml kanamycin.
* Add 5 ml of fresh DMEM supplemented with 10% FCS, 2 mM L-glutamine, 1% penicillin/streptomycin and 50 µg/ml kanamycin.
* Place in the bacterial incubator again for 2-3 h to neutralize remaining bacteria.
* Check under the microscope to ensure no prevailing signs of live bacteria.
* Thoroughly disinfect the exteriors of the flasks, then transfer to a humid CO2 incubator at 37°C, incubate for 24 h.

##### Read-out

* Perform fluorescence microscopy for qualitative and FACS for quantitative readout of fluorescent reporter expression across the various experimental groups.

## Materials & Methods

### Cas Staples

#### Plasmid Construction

[![Go to results button](https://static.igem.wiki/teams/5237/gotoresults.svg)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D#cas_results_wetlab)

All plasmids created for the Cas staple project were constructed using Golden Gate assembly (GGA), with the exception of the fusion dCas plasmid being created by Gibson assembly . Inserted fragments were either annealed oligonucleotides or PCR amplified DNA fragments. PCRs were performed using Q5 2x Master Mix (New England Biolabs, USA).
The primers carried the corresponding restriction sites of the enzyme used in the GGA. The PCR product was run on a 1% agarose gel in 0.5x TAE buffer, cut out, and extracted using the QIAquick Gel Extraction kit (Qiagen, Netherlands).
Oligonucleotides for the spacers seen in table 1 (Cas staples) were annealed using NEBuffer 2 (New England Biolabs, USA).
For the insertion of PCR products, the backbone to insert ratio was 1:5. For annealed oligonucleotides, 1 µL was used with 100 ng of the backbone plasmid.
The GGA was prepared in a 20 µL reaction volume, using 2 µL T4 ligase buffer (ThermoFisher Scientific, USA), 0.5 µL T4 Ligase, 0.5 µL of the restriction enzyme, and optionally 0.5 µL of DpnI, if all fragments were PCR amplicons.
The tubes were placed in a thermocycler with 15 cycles of 1.5 min at 37°C and 3 min at 16°C, followed by heat inactivation for 10 min at 65°C and 20 min at 80°C.
The Gibson assembly of the fusion dCas was conducted in 20 µL total volume with 10µL of Gibson assembly 2x Master Mix (New England Biolabs, USA). For a efficient assembly the reactions are incubated at 50°C for 60 minutes.
Transformation of the PCR was performed with 7 µL of the reaction mixture to transform 100 µL of chemically competent TOP10 *E. coli* cells.
Cells were kept on ice for 30 min and then heat shocked at 42°C for 30 s.
After another 2 min, cells were plated on Ampicillin-containing agar plates.
Cells were incubated overnight at 37°C. Singular colonies were picked and incubated in LB medium containing Ampicillin overnight at 37°C, shaking at 220 RPM.
DNA was isolated using the QIAprep Spin Miniprep Kit and sequenced by Microsynth using Sanger sequencing.

#### Cell Culture Conditions

HEK293T cells were kept at 37°C and 5% CO2 in 10 mL cell culture medium, containing Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific) with phenol red, supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 10 units penicillin-streptomycin solution (Thermo Fisher Scientific).
Cells were split twice a week using 5 mL Phosphate Buffer Saline (ThermoFisher Scientific, USA) and 1 mL Trypsin-EDTA (Thermo Fisher Scientific).
Cells were then diluted 1:10 into new cell culture medium.

#### T7 Endonuclease I (T7EI) Assay

24h after seeding 12,500 cells per well into a 96-well plate, cells were transfected by adding 200 ng DNA with Lipofectamine 2000 (ThermoFisher Scientific, USA) into each well.
After 72 h, cells were lysed by adding 70 µL H2O, 70 µL DirectPCR-Cell (VWR Chemicals), and 1.4 µL Proteinase K (Sigma-Aldrich, USA) into each well and incubating for 6 h at 55°C and 120 RPM.
Enzymes were then heat inactivated at 85°C for 45 min.
The targeted gene sites were amplified by adding 3 µL of the lysed cells into a 25 µL Q5 reaction mix (New England Biolabs, USA) with the corresponding primers seen in table 2 (Cas staples).
PCR amplicons were then annealed using NEBuffer 2 (New England Biolabs, USA) in a thermocycler by first lowering the temperature from 95°C to 85°C at 2°C per second and then from 85°C to 25°C at 0.1°C per second.
0.5 µL of T7 Endonuclease I (New England Biolabs, USA) was added and samples were incubated at 37°C for 15 min. 5 µL purple loading dye (no SDS, New England Biolabs) was added, and samples were run on a 2% agarose gel in 1x TBE (AppliChem, Germany).
Gel images were analyzed using ImageJ.

#### Dual Luciferase Assay

24h after seeding 12,500 cells per well into a 96-well plate, cells were transfected by adding 200 ng DNA with Lipofectamine 2000 (ThermoFisher Scientific, USA) into each well.
After 48 h, the medium was discarded and cells were lysed for 30 min in Passive Lysis 5X Buffer (Promega, USA) at room temperature, shaking at 500 RPM.
10 µL of each sample was transferred to white 96-well plates.
The plates were analyzed according to the Dual-Luciferase Reporter Assay System (Promega, USA) protocol using the Infinite M200 plate reader (Tecan, Switzerland).

### Staple Functionalization

#### Molecular Cloning of the Cathepsin B-Cleavable Linker GFLG (p10)

[![Go to results button](https://static.igem.wiki/teams/5237/gotoresults.svg)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D#results_cathepsinBliner)

We cloned the SV40-Gal4-GFLG-VP64 construct (p10) to evaluate whether the GFLG linker could be cleaved by cathepsin B *in vivo*. We purchased the GFLG linker as oligonucleotides (fwd\_GFLG\_p10\_CatB, rev\_GFLG\_p10\_CatB) for Golden Gate cloning. To prepare the backbone, we used a plasmid (p3661) already containing the SV40-Gal4-VP64 construct, which had a GS linker between Gal4 and VP64. Using primers fwd\_Esp3I\_CatB\_p10 and rev\_Esp3I\_CatB\_p10, we introduced Esp3I recognition sequences via overhangs and created the amplicon SV40-Gal4-VP64\_backbone. The final plasmid assembly was carried out using Golden Gate cloning with the Type IIS restriction enzyme Esp3I, and the final plasmid was transformed into *E. coli* Top10 cells.

#### Molecular Cloning of the Cathepsin B Expression Cassette (p12)

We amplified the gene block gBlock\_Cathepsin\_B with the primers fwd\_gBlock\_CatB and rev\_gBlock\_CatB. The gene block was composed of an upstream XhoI recognition sequence, followed by a Kozak sequence, the SV40 nuclear localization signal, a GGS linker, the human codon-optimized sequence for cathepsin B, and a downstream BamHI recognition sequence. We then inserted the amplicon into a pcDNA3.1 backbone via restriction cloning and transformed the ligated plasmid into *E. coli* Top10 cells.

#### Molecular Cloning of a Truncated and Mutated Form of Cathepsin B (p12.1)

To generate a truncated and mutated version of cathepsin B, we employed overlap extension PCR. We used the primers CatB\_p12\_frag1\_fwd and CatB\_p12\_frag1\_rev to remove the nucleotide sequence corresponding to the first twenty amino acids of wild-type cathepsin B using the gene block gBlock\_Cathepsin\_B as a template. We introduced three point mutations (D22A, H110A, and R116A) in the nucleotide sequence of cathepsin B using the primers CatB\_p12\_frag2\_fwd, CatB\_p12\_frag2\_rev, CatB\_p12\_frag3\_fwd, CatB\_p12\_frag3\_rev, CatB\_p12\_frag4\_fwd, and CatB\_p12\_frag4\_rev. After generating the amplicon (trunc\_mut\_CatB) with overlap extension PCR, we cloned it into the same pcDNA3.1 backbone used for the wild-type cathepsin B and transformed the ligated plasmid into *E. coli* Top10 cells.

#### Cathepsin B Cleavage Fluorescence Readout Assay

To study cathepsin B-mediated cleavage of different linkers, we conducted a fluorescence readout assay. We seeded 104 HEK293T cells in each well of a 96-well plate with DMEM (10% FCS) and incubated the cells for 24 hours at 37 °C and 5% CO2 before transfection. Plasmid solutions were prepared in OptiMEM, and transfections were performed using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham). For each linker, we included one negative control without the plasmid encoding cathepsin B, and two test samples with 30 ng and 60 ng of the plasmid. After transfection, we incubated the cells at 37 °C and 5% CO2. At 24 hours post-transfection, we added 500 nM doxorubicin to the cell supernatant. We measured eGFP and mCherry fluorescence intensities at 48 hours post-transfection using the Tecan Infinite F200 Pro plate reader (Tecan Group Ltd., Männedorf).

#### Cell Lysis and Western Blotting

To confirm the overexpression of cathepsin B in HEK293T cells, we performed western blotting. We seeded 105 HEK293T cells per well in a 12-well plate with DMEM (10% FCS) and incubated for 24 hours at 37 °C and 5% CO2 before transfecting cells separately with both wild-type and mutated cathepsin B constructs. Transfections were carried out in OptiMEM using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham), followed by incubation at 37 °C and 5% CO2. At 24 hours post-transfection, we supplemented the medium with 500 nM doxorubicin. 48 hours post-transfection, we washed the cells with DPBS. We then lysed the cells in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% Sodium deoxycholate, 0.1% SDS, 1 mM dithiothreitol, 50 mM TRIS-HCl pH 7.6) supplemented with a protease inhibitor cocktail. Protein concentrations were quantified using the DC protein assay (Bio-Rad Laboratories GmbH, Feldkirchen) and measured with the Spark Multimode microplate reader (Tecan Group Ltd., Männedorf). For SDS PAGE, we used a Mini-PROTEAN TGX Stain-Free Precast gel (Bio-Rad Laboratories GmbH, Feldkirchen) and ran the gel in a Mini-PROTEAN Tetra cell system (Bio-Rad Laboratories GmbH, Feldkirchen) at 200 V for 30 minutes. Following SDS PAGE, we transferred proteins to a PVDF membrane via semi-dry western blotting using the Trans-Blot SD system (Bio-Rad Laboratories GmbH, Feldkirchen) at 10 V for 1 hour. The membrane was blocked with I-Block protein-based blocking reagent (Thermo Fisher Scientific, Waltham) and incubated overnight at 4 °C with a polyclonal anti-cathepsin B antibody and a monoclonal anti-beta-tubulin antibody (Proteintech, Rosemont) (1:1000 dilution). After washing thrice with TBS-T buffer, we incubated the membrane with anti-rabbit IgG DyLight 800 (Cell Signaling Technology, Inc., Danvers) and anti-mouse IgG Alexa Fluor 680 (Thermo Fisher Scientific, Waltham) (1:10000 dilution) for 1 hour at 4 °C. Afterwards, we washed the membrane three times with TBS-T buffer and imaged the membrane using the Odyssey XF Imager (LI-COR Biotechnology, Lincoln).

#### Molecular Cloning of an Intein-dCas9 Fusion Protein (p13)

To enable functionalization of Cas staples *in vivo*, we cloned a dCas9-intein fusion protein using HiFi DNA assembly. We PCR-amplified the inteins NpuC and NpuN from the gene block gBlock\_dCas9\_Inteins using the primers SV40\_NpuC\_Cage\_fwd, SV40\_NpuC\_rev, NpuN\_SV40\_fwd, and NpuN\_Cage\_SV40\_rev. We amplified the backbone (p70) and dCas9 (p505) with the primers p70\_BB\_SV40\_fwd, p70\_BB\_SV40\_rev and dCas9\_fwd, dCas9\_rev creating the amplicons p70\_backbone and dCas9, respectively. Finally, we assembled the fragments using the NEBuilder HiFi DNA assembly reaction mix (New England Biolabs, Ipswich).

### Readout System

#### Constructs for FRET Assay

[![Go to results button](https://static.igem.wiki/teams/5237/gotoresults.svg)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D#results_fret_measurment_wetlab)

Plasmid 3 (expression plasmid for scTetR-Oct1, scTetR-mScarlet-I, mNeonGreen-Oct1) was constructed using Gibson
assembly. A plasmid containing tet response elements, a high-copy pUC19
origin, and ampicillin resistance was digested with PstI and XbaI. An operon (SiSt-operon) comprising scTetR-Oct1,
scTetR-mScarlet-I, and mNeonGreen-Oct1 was synthesized by IDT (coralville, IA) as overlapping gBlocks and cloned
into the linearized
vector using NEBuilder HiFi DNA Assembly (NEB, Ipswich, MA).
For the working FRET assay , plasmid 3 was digested with NdeI. The tetR-Oct1 (primer BS-1, BS-2) and
tetR-mScarlet-I (primer BS-3, BS-4) fragments were PCR amplified using specific primer pairs.
Folding plasmid p1.1 was created by PCR amplification of a backbone containing the p15A origin and chloramphenicol
resistance marker (BS-5, BS-6), introducing SalI and XhoI restriction sites. Oct1 target sites were added by
annealing oligos (BS-7, BS-8) and
ligating them into the digested backbone. Additional target sites were added iteratively until 12 sites were
present.

#### Constructs for Protein Expression and Purification

Expression plasmids for simple staple constructs were based on p3. Plasmid p3.6 (Oct1-mNeonGreen) was generated by
inverse PCR with BS-9, BS-10. Plasmid p3.7 (tetR-mScarlet-I) was constructed by digesting p3 with NdeI and XhoI
and assembling the PCR-amplified fragments (p3 template with primer BS-9 + BS-10 and BS-11 + BS-12) using NEBuilder
HiFi DNA Assembly.
Intermediate plasmid p3.8 was created by PCR amplifying p3 with BS-13 and BS-14, and the aforementioned ordered
gBlock (SiSt-operon) with BS-15 + BS-16, and BS-17 + BS-18,
and assembly with NEBuilder HiFi DNA Assembly. p3.9 was cloned by inverse PCR of p3.8 (BS-19 and BS-20) and KLD
treatment.
For GCN4, rGCN4, and bGCN4 expression, intermediate plasmid p4 was constructed by assembling digested p3 (PstI,
XbaI) with a synthesized gene fragment (IDT, MiSt-operon) containing the bGCN4 expression cassette. Plasmid p4.1 and
p4.3 were constructed by inverse PCR
(BS-21 + BS-27 and BS-28 + BS-24 respectively) and KLD treatment. Plasmid p4.2 was constructed by PCR amplifying p4
with BS-23 and BS-24, and NEBuilder HiFi DNA Assembly with the gBlock containing the rGCN4 expression cassette.

#### Protein Expression and *In Vitro* Assays

##### Protein Expression and Purification

Proteins were expressed in *E. coli* BL21 DE3 (NEB, Ipswich, MA) carrying the necessary constructs under a T7
promoter. A 500 mL culture in DYT medium was inoculated 1:100 with an overnight culture, grown to OD600
of 0.4-0.6, and induced with 0.1 mM IPTG. Cells were harvested 16-18 hours post-induction at 32 °C and centrifuged
(30 min, 3270 x g, 4 °C). The general protocol for protein purification were followed. Shortly, the pellet was
resuspended
in NaP10 or lysis buffer supplemented with 1 mg/mL lysozyme and protease inhibitor
cocktail (Sigma-Aldrich, St. Louis, MO). Samples were sonicated (40% power, 30s on/off, 10 min total) and
centrifuged (30 min,
20,000 x g, 4 °C). The supernatant was filtered and proteins were purified using Ni-NTA agarose (Thermo Fisher
Scientific) or Anti-FLAG affinity resin (Thermo Fisher Scientific, Waltham, MA) following manufacturer's
instructions.

##### SDS-PAGE Analysis

The general protocol was followed. Shortly, samples were mixed with 4x Laemmli buffer (Thermo Fisher Scientific) and
heated to 95 °C before loading on 4-15%
precast TGX stain-free gels (Bio-Rad Laboratories). For Coomassie staining, gels were incubated in staining solution
for 30 minutes and destained with a 20% MeOH, 10% AcOH solution until desired contrast was achieved.

##### Electrophoretic Mobility Shift Assay (EMSA)

Purified proteins were incubated with annealed DNA oligos and loaded onto 10% TGX stain-free gels pre-equilibrated
with TBE (Bio-Rad Laboratories). Usually 0.5 µM DNA were loaded with protein ranging from 50 - 0.01 µM in a 10 µL
reaction.
DNA-protein interactions were visualized using SYBR-Safe (Thermo Fisher Scientific, Waltham, MA)
and imaged on a transilluminator.

#### Cell Growth and Fluorescence Assays

Cells were grown overnight to stationary phase and inoculated 1:100 into fresh medium, grown to OD600
0.1. In a 96-well clear flat-bottom plate (200 µL final volume), cells were incubated at 37 °C with shaking at 200
rpm. Measurements were taken every 30 minutes using a Tecan Spark plate reader (Männedorf, Switzerland) for
OD600, mNeonGreen (excitation 506 nm, emission 517 nm), mScarlet-I (excitation 568 nm, emission 591 nm),
and FRET (excitation 506 nm, emission 591 nm).

#### Data Analysis

Data were managed and cleaned using Excel, and final graphs were generated with GraphPad Prism 10.

### Delivery System

#### Cloning of pHelper\_RP4 (Helper Plasmid Encoding the RP4 Conjugation Machinery)

[![Go to results button](https://static.igem.wiki/teams/5237/gotoresults.svg)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D#results_delivery_system)

pHelper\_RP4 was constructed from pTA-Mob 2.0 (addgene #149662) by removing the *oriT* sequence and inserting the gene block (DSg1) using restriction-ligation cloning. The *oriT* was removed by digesting the plasmid with SpeI and SacI, and the linearized plasmid was gel-purified using the Large Fragment DNA recovery kit (Zymo Research). Meanwhile, a gene block encoding the traJ region that was removed and containing the SpeI and SacI restriction sites (DSg1) was PCR amplified, digested with SpeI and SacI and subsequently cleaned up. Then, overnight ligation (at 16°C) of linearized pTA-Mob 2.0 and restricted DSg1 with T4 DNA ligase was set up.

#### Cloning of pmob\_b (Mobilizable Plasmid for Conjugation Between Bacteria)

The pSC101 ori of 2374\_pSC101 was swapped with p15A ori from pRep\_p15A to generate pmob\_b. The backbone for pmob\_b was amplified via PCR from 2374\_pSC101 with the primers DS3 and DS4. The p15A origin of replication (ori) was amplified via PCR from pRep\_p15A with the primers DS5 and DS6. In both cases, the primers were designed to produce the necessary homology regions for subsequent utilization in Gibson Assembly.

#### Cloning of pmob\_m\_CMV (Mobilizable Plasmid for Conjugation between Bacteria and Mammalian Cells)

pmob\_m\_CMV was cloned using four-fragment Golden Gate Assembly. The two parts of the backbone were amplified via PCR from pcDNA3.1 respectively with the primer pairs DS7, DS8 and DS11, DS12. The p15A ori was amplified via PCR from pRep\_p15A with the primers DS9 and DS10. The origin of transfer (*oriT*) sequence was amplified via PCR from 2374\_pSC101 with the primers DS13 and DS14. All resultant PCR amplicons possessed 5’ and 3’ extensions with BsmBI recognition sites and were designed to be assembled directionally after cleavage by BsmBI. Following gel extraction, the four fragments were then assembled via Golden Gate Assembly using BsmBI.

#### Cloning of pmob\_m\_CEA (Mobilizable Plasmid for Conjugation Between Bacteria and Mammalian Cells)

pmob\_m\_CEA was cloned from pmob\_m\_CMV by exchanging the CMV-promoter with the CEA-promoter. The backbone was amplified from pmob\_m\_CMV via PCR with the primers DS15 and DS16. The CEA-promoter was ordered as a gene block (DSg2) and amplified via PCR with the primers DS17 and DS18. Both resultant PCR amplicons carried 5’ and 3’ extensions with BsmBI recognition sites and were designed to be assembled directionally after cleavage by BsmBI. PCR product clean-up was performed for the amplified CEA promoter and the PCR product of the pmob\_m\_CMV backbone was gel purified. The promoter and the backbone were then assembled via Golden Gate Assembly using BsmBI.

#### Cloning of pNeae2\_7D12 (for Surface Expression of Synthetic Adhesin Against EGFR on *E. coli* 10-beta)

The cloning of pNeae2\_7D12 will be done by two-fragment Gibson Assembly using pNeae2 (addgene #168300) as the backbone. The backbone will be amplified using the primers - DS19 and DS20. The gene block encoding wt 7D12 adhesin (DSg3) will be amplified using the primers - DS21 and DS22. The PCR amplicons are designed to possess the necessary homology regions for Gibson Assembly. Following clean-up, the PCR amplicons will then be assembled via Gibson Assembly.

#### Solid Media Bacterial Conjugation Assay

In preparation for the conjugation assay, the corresponding plasmids were transformed into respective chemically competent E. coli strains and plated on appropriate antibiotic-containing LB agar plates to create the necessary donor and recipient cells. Single colonies were picked and 5 ml overnight cultures were set up in LB medium supplemented with the appropriate antibiotics.

The experimental design involved a positive control, two negative controls, and a test group, as outlined in Table 4 below. In all cases, the recipients were made to carry a plasmid conferring resistance against ampicillin (pAmpR) in order to enable selection against donors.

**Table 22: Donors and recipients in the different controls and in the test group used for the bacteria-bacteria conjugation assay.**|  | Donor | Recipient |
| --- | --- | --- |
| Negative control 1 | *E. coli* 10-beta with pHelper\_RP4 | *E. coli* BL21(DE3) |
| Negative control 2 | *E. coli* 10-beta with pmob\_b | *E. coli* BL21(DE3) |
| Positive control | - | *E. coli* BL21(DE3) transformed with pmob\_b |
| Test group | *E. coli* 10-beta with both pHelper\_RP4 and pmob\_b | *E. coli* BL21(DE3) |

The following day, 500 µl of liquid cultures were reserved as glycerol stocks and stored at -80 °C for later use. The remaining liquid bacterial cultures were then centrifuged in separate 1.5 ml tubes at 11,000 rpm for 1 min and the pellets were resuspended in 100 µl of 10 mM magnesium sulfate solution (diluent) as suggested in literature (Silbert *et al.*, 2021). The optical density (OD600) was adjusted to 10 using the diluent. The experimental groups were then prepared for the conjugation assay by mixing together 100 µl of the respective OD-adjusted recipient and donor cell suspensions. To each tube, 1 ml of diluent was then added, the tubes vortexed and centrifuged for 1 min at 11,000 rpm. After removal of the supernatant, the cell pellets were resuspended in 10 µl of the diluent and mixed gently. The 10 µl drop was then pipetted onto an antibiotic-free LB agar plate and let air dry for 10 min. Afterwards, the plates with 10 µl bacterial spots were incubated at 37 °C for 18 hours, after which the bacterial patches were collected using sterile inoculation loops, resuspended in 1 ml of diluent and the OD600 of all suspensions were adjusted to 2.4. Serial dilutions were then prepared in the diluent using the OD-adjusted cell suspensions and the dilutions ranged from 10-2 to 10-9. The dilutions were plated on two types of LB agar plates - one with only ampicillin (selects for recipients) and another with both ampicillin and kanamycin (selects for transconjugants). Three technical replicates were used for the test group. Plates were then incubated overnight at 37 °C.

#### Liquid Media Bacterial Conjugation Assay

Bacteria from the same clones used for conjugation on solid media were used for conjugation on liquid media. The same steps as for solid media conjugation were followed until the first resuspension step. The first resuspension was performed in 1 ml of antibiotic-free LB medium, followed by OD600 adjustment (to 10). For each experiment group, 120 µl of the corresponding OD-adjusted liquid cultures were mixed together and then centrifuged for 1 min at 11,000 rpm. After resuspension of the pellets in 1.2 ml of LB medium each, 1 ml of cell suspension from each experiment group was pipetted into a well of a 12-well plate and incubated at 37 °C for 18 hours without shaking. After incubation, the bacterial suspensions were removed from the wells and their OD600 was adjusted to 2.4 using the diluent. Serial dilutions were prepared and plated on selective agar plates (same as for solid media conjugation). Three technical replicates were used for the test group. The plates were then incubated overnight at 37 °C.

In both solid and liquid media conjugation assays, following the last overnight incubation step, the conjugation efficiency was calculated by counting colonies at a certain dilution on the ampicillin+kanamycin plate (selecting for transconjugants) and dividing them by the number of colonies on the corresponding ampicillin plate (selecting for total number of recipients).

## Sequences

### Cas Staples

**Table 23: List of spacer sequences.**| Plasmid | Spacer sequence | Description |
| --- | --- | --- |
| 8 | TCTCTATCACTGATAGGGAG | gRNA targeting TetO |
| 819 | TGACATCAATTATTATACAT | gRNA targeting CCR5 |
| 4334 | TGCGAATACGCCCACGCGAT ATGCGTCTCTATCACTGATA GGGAG | Fusion guide RNA targeting lacz (Cas12) and TetO (Cas9) with a 5 nucleotide linker |
| 4335 | TGCGAATACGCCCACGCGAT ATGCGAGCTGTCTCTATCAC TGATAGGGAG | Fusion guide RNA targeting lacz (Cas12) and TetO (Cas9) with a 10 nucleotide linker |
| 4336 | TGCGAATACGCCCACGCGAT TCTCTATCACTGATAGGGAG | Fusion guide RNA targeting lacz (Cas12) and TetO (Cas9) |
| 4337 | TGCGAATACGCCCACGCGAT ATGCGTCTCTATCACTGATA GGGAG | Fusion guide RNA targeting lacz (Cas12) and TetO (Cas9) with a 5 nucleotide linker |
| 4338 | TGCGAATACGCCCACGCGAT ATGCGAGCTGTCTCTATCAC TGATAGGGAG | Fusion guide RNA targeting lacz (Cas12) and TetO (Cas9) with a 10 nucleotide linker |
| 4339 | TGCGAATACGCCCACGCGAT TCTCTATCACTGATAGGGAG | Fusion guide RNA targeting lacz (Cas12) and TetO (Cas9) |
| 4364 | TGGCCCCATTCGCACGGCTC | gRNA targeting FANCF |
| 4365 | CTAGGAATATTGAAGGGGGC | gRNA targeting VEGFA |
| 4366 | GTCGGCATGGCCCCATTCGC | gRNA targeting FANCF |
| 4367 | CTAGGAATATTGAAGGGGGC | gRNA targeting VEGFA |
| 4368 | GTCGGCATGGCCCCATTCGC | Fusion guide RNA targeting FANCF (Cas12) and VEGFA (Cas9) |
| 4369 | CTAGGAATATTGAAGGGGGC TGGCCCCATTCGCACGGCTC | Fusion guide RNA targeting VEGFA (Cas12) and FANCF (Cas9) |
| 4370 | GTCGGCATGGCCCCATTCGC | gRNA targeting FANCF |
| 4371 | CTAGGAATATTGAAGGGGGC | gRNA targeting VEGFA |
| 4372 | GTCGGCATGGCCCCATTCGC CTAGGAATATTGAAGGGGGC | Fusion guide RNA targeting FANCF (Cas12) and VEGFA (Cas9) |
| 4373 | CTAGGAATATTGAAGGGGGC TGGCCCCATTCGCACGGCTC | Fusion guide RNA targeting VEGFA (Cas12) and FANCF (Cas9) |
| 4436 | TGCGAATACGCCCACGCGAT TGGCGGCGTGCTGACCGCTA TCTCTATCACTGATAGGGAG | Fusion guide RNA targeting FFluc (Cas9) with an empty spacer for Cas12a and 20 nucleotide stiff linker |
| 4437 | TGCGAATACGCCCACGCGAT CAAAACAACAATCAAAACAA TCTCTATCACTGATAGGGAG | Fusion guide RNA targeting FFluc (Cas9) with an empty spacer for Cas12a and 20 nucleotide poly-A linker |
| 4438 | TCTATCACTGATAGGGAGTG TGGCGGCGTGCTGACCGCTA TGCGAATACGCCCACGCGAT | Fusion guide RNA targeting TetO bottom strand (Cas12a) with an empty spacer for Cas9 and 20 nucleotide stiff linker |
| 4439 | TCTATCACTGATAGGGAGTG CAAAACAACAATCAAAACAA TGCGAATACGCCCACGCGAT | Fusion guide RNA targeting TetO bottom strand (Cas12a) with an empty spacer for Cas9 and 20 nucleotide poly-A linker |
| 4440 | CCACTCCCTATCAGTGATAG TGGCGGCGTGCTGACCGCTA TGCGAATACGCCCACGCGAT | Fusion guide RNA targeting TetO top strand (Cas12a) with an empty spacer for Cas9 and 20 nucleotide stiff linker |
| 4441 | CCACTCCCTATCAGTGATAG CAAAACAACAATCAAAACAA TGCGAATACGCCCACGCGAT | Fusion guide RNA targeting TetO top strand (Cas12a) with an empty spacer for Cas9 and 20 nucleotide poly-A linker |
| 4446 | CTAGGAATATTGAAGGGGGC TGACATCAATTATTATACAT | Fusion guide RNA targeting VEGFA (Cas12) and CCR5 (Cas9) |
| 4447 | CTAGGAATATTGAAGGGGGC TGGCGGCGTGCTGACCGCTA TGACATCAATTATTATACAT | Fusion guide RNA targeting VEGFA (Cas12) and CCR5 (Cas9) with a 20 nucleotide linker |
| p15.1 | TATGCAAATACTGCACTAGTGCAAAACAACAATCAAAACAAATCAAAACAATCTCTATCACTGATAGGGAG | enhancer hijacking 30nt polyA linker |
| p15.2 | TATGCAAATACTGCACTAGTGCAAAACAACAATCAAAACAACAAAACAACAATCAAAACAATCTCTATCACTGATAGGGAG | enhancer hijacking 40nt polyA linker |
| p15.3 | TCTAGGAATATTGAAGGGGGCCAAAACAACAATCAAAACAATGACATCAATTATTATACAT | fluorophore quencher 20nt linker |
| p15.4 | TCTAGGAATATTGAAGGGGGCCAAAACAACAATCAAAACAAATCAAAACAATGACATCAATTATTATACAT | fluorophore quencher 30nt linker |
| p15.5 | TCTAGGAATATTGAAGGGGGCCAAAACAACAATCAAAACAACAAAACAACAATCAAAACAATGACATCAATTATTATACAT | fluorophore quencher 40nt linker |
| p15.6 | CGGCGCTCTTCGAGATCTAGGAATATTGAAGGGGGCTTGGGCTCGAGAAGGATGTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGTCTAAGAACTTTAAATAATTTCTACTCTTGTAGATTATGGGAGCCCTCACTTCACTGACATCAATTATTATACATGTTCGAAGAGCGCGG | fusion guide RNA processing |
| p15.7 | ATGCAAATACTGCACTAGTG | crRNA MbCas12a targeting Oct1 |
| p15.14 | TATGCAAATACTGCACTAGTGCAAAACAACAATCAAAACAATCTCTATCACTGATAGGGAG | enhancer hijacking 20nt polyA linker |

**Table 24: List of primers for T7EI assay.**| No. | Sequence | Target gene |
| --- | --- | --- |
| 477 | TCCAGATGGCACATTGTCAG | VEGFA fwd |
| 478 | AGGGAGCAGGAAAGTGAGGT | VEGFA rev |
| 1016 | GAGCCAAGCTCTCCATCTAGT | CCR5 fwd |
| 1017 | GCCCTGTCAAGAGTTGACAC | CCR5 rev |
| 6265 | CACCTCATGGAATCCCTTCT | FANCF fwd |
| 6266 | CCACAGGCTGCTGAGAAAAC | FANCF rev |
| 6267 | GAAGCAACTCCAGTCCCAAA | VEGFA fwd |
| 6268 | CCAAGGTTCACAGCCTGAAA | VEGFA rev |
| 6379 | ATCATCTCGCACGTGGTTCC | FANCF fwd |
| 6380 | TTCCAAAGCGAAAGGAAGCG | FANCF fwd |
| 6381 | AAGGCAGCAAAGACTTCCGA | FANCF rev |
| 6382 | AGAAGCCAGTGGACTAGCAC | FANCF rev |

**Table 25: List of primers for cloning fusion Cas.**| Primer name | Sequence | Plasmid name |
| --- | --- | --- |
| N-termSpCas9\_bb+Linker\_fwd | AAAACGTCTCCGAGGGTCAGGGGGAGGAGGAAGTGACAAGAAGTACAGCATCGGCC | fusion Cas |
| --- | --- | --- |
| N-termSpCas9\_bb\_rev | TCACGTCTCAGGCTGCTGGGACTCCG | fusion Cas |
| MbCas12a\_fwd | TCACGTCTCAAGCCATGCTGTTCCAGGACTTCACC | fusion Cas |
| MbCas12a\_Linker\_rev | AAAACGTCTCACCTCCACCAGATCCGCCACCTCCTCTGTTCTGGGCGAAATTCAGCC | fusion Cas |
| AsCas12a\_Esp3I\_fwd | TCACGTCTCGAGCCATGACACAGTTCGAGGGCTT | fusion Cas |
| AsCas12a\_Linker\_rev | AAAACGTCTCCCCTCCACCAGATCCGCCACCTCCGTTGCGCAGCTCCTGGA | fusion Cas |
| \_D914A\_\_D1324A1\_fwd | GATCGGCATCGCCCGGGGCGAGC | fusion dCas |
| \_D914A\_\_D1324A1\_rev | TGGTGCCGATGGCCAGGCCGATG | fusion dCas |
| \_D1324A\_\_H2154A2\_fwd | CATCGGCCTGGCCATCGGCACCA | fusion dCas |
| \_D1324A\_\_H2154A2\_rev | GCTCTGAGGCACGATAGCGTCCACATCG | fusion dCas |
| \_H2154A\_\_D914A3\_fwd | CGATGTGGACGCTATCGTGCCTCAGAGC | fusion dCas |
| \_H2154A\_\_D914A3\_rev | GCTCGCCCCGGGCGATGCCGATC | fusion dCas |
| SV40NLS-MbCas12a-GSG-SpCas9-NLS\_Esp3I\_fwd | AAAACGTCTCACACCATGGCCCCAAAGAAGAAGCG | fusion Cas P2A Puro |
| SV40NLS-MbCas12a-GSG-SpCas9-NLS\_RE\_rev | AAAACGTCTCATGCCGAATTCCTTTTTCTTTTTTGCCTGG | fusion Cas P2A Puro |
| GSG-P2A-Puro\_Esp3I\_fwd | AAAACGTCTCAGGCAGTGGAGCAACGAATTTTTC | fusion Cas P2A Puro |
| GSG-P2A-Puro\_Esp3I\_rev | AAAACGTCTCAAGCCCCAGGCTTGCGGGTCATAC | fusion Cas P2A Puro |
| pSplit-4280\_RE\_fwd | AAAACGTCTCAGGCTTAGCTGAAGTTCATCTGCACCAAAG | fusion Cas P2A Puro |
| pSplit2-4280\_Esp3I\_rev | AAAACGTCTCAGGTGGCACCGCTCGAGTCTAGAG | fusion Cas P2A Puro |
| RLuc\_CCR5\_KLD\_fwd | TACATCGGGCTCCGGCACTTCGAAAGTTTATGATCCAGAACAAAGGA | double cut ren luc reporter |
| RLuc\_CCR5\_KLD\_rev | TAATAATTGATGTCAGACATGGTGGCTAGCCTATAGTGAG | double cut ren luc reporter |
| fwd\_11RE1.1 | GATCCAAGCTTGCATGCCTGCAGGTCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACT | Oct1 5xUAS |
| fwd\_11RE1.2 | GTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGACTCTAGAGGGTATA | Oct1 5xUAS |
| fwd\_11RE2 | TAATGGATCCTTTGATGCAAATACTGCACTAGTGCTATGCGTTTGATGCAAATACTGCACTAGTGCTATGCGTTTGATGCAAATACTGCACTAGTGCC | Oct1 5xUAS |
| rev\_11RE1.1 | GAGGACAGTACTCCGCTCGGAGGACAGTACTCCGCTCGGAGGACAGTACTCCGACCTGCAGGCATGCAAGCTTG | Oct1 5xUAS |
| rev\_11RE1.2 | CCATTATATACCCTCTAGAGTCTCCGCTCGGAGGACAGTACTCCGCTCGGAGGACAGTACTCCGCTCG | Oct1 5xUAS |
| rev\_11RE2 | TCGAGGCACTAGTGCAGTATTTGCATCAAACGCATAGCACTAGTGCAGTATTTGCATCAAACGCATAGCACTAGTGCAGTATTTGCATCAAAGGAT | Oct1 5xUAS |

### Staple Functionalization

**Table 26: List of oligos for staple functionalization experiments.**| Description | Sequence |
| --- | --- |
| fwd\_GFLG\_p10\_CatB | CAGCGGTTCAGGCAGCGGCAGCGGTTTTTTAGGTGGCAGCGGTTCGGGCTCAGGCAGC |
| rev\_GFLG\_p10\_CatB | CGTCGCTGCCTGAGCCCGAACCGCTGCCACCTAAAAAACCGCTGCCGCTGCCTGAACC |
| fwd\_Esp3I\_CatB\_p10 | ACACGTCTCAGACGCATTGGACGATTTTGATC |
| rev\_Esp3I\_CatB\_p10 | TCACGTCTCCGCTGACCGTCAATTGC |
| fwd\_gBlock\_CatB | AAGCCTCTCGAGCAC |
| rev\_gBlock\_CatB | CCTAGTCGGATCCCTAG |
| CatB\_p12\_frag1\_fwd | CATGGTGATGCGGTTTTGGCAG |
| CatB\_p12\_frag1\_rev | TCACGTCTCCGCTGACCGTCAATTGC |
| CatB\_p12\_frag2\_fwd | AAGTAGGCGGCAGTCCAAGCTTTCATCCCCTGTCC |
| CatB\_p12\_frag2\_rev | CCGCAAGAGCCCTGAGCCCGAATTTCC |
| CatB\_p12\_frag3\_fwd | ATTCGGGCTCAGGGCTCTTGCGGCAG |
| CatB\_p12\_frag3\_rev | AAGGGGGTGCGCTGCCGTTCACGTGGGCCTCGCATGGTG |
| CatB\_p12\_frag4\_fwd | CGTGAACGGCAGCGCACCCCCTTGCACAG |
| CatB\_p12\_frag4\_rev | CTTGGTACCGAGCTCGGATCC |
| SV40\_NpuC\_Cage\_fwd | GCGGTGCCACCATGATGCCTAAAAAGAAACGCAAAGTTGG |
| SV40\_NpuC\_rev | CTGTACTTCTTGTCGCAGTTGGAAGCGATGAAACC |
| NpuN\_SV40\_fwd | AGCTGGGAGGCGACTGCCTTTCTTATGAAACCGAAATCC |
| NpuN\_Cage\_SV40\_rev | CAGATGAACTTCAGTCAAACCTTCCGCTTCTTCTTAGG |
| p70\_BB\_SV40\_fwd | AGCGGAAGGTTTGACTGAAGTTCATCTGCACCAAAGCTTAAG |
| p70\_BB\_SV40\_rev | TTCTTTTTAGGCATCATGGTGGCACCGCTCGAGTCTAG |
| dCas9\_fwd | TCGCTTCCAACTGCGACAAGAAGTACAGCATCGGCCTGG |
| dCas9\_rev | TCATAAGAAAGGCAGTCGCCTCCCAGCTGAGACAG |

**Table 27: List of gene fragments for staple functionalization experiments.**| Description | Sequence |
| --- | --- |
| SV40-Gal4-VP64\_backbone | ACACGTCTCAGACGCATTGGACGATTTTGATCTGGATATGCTGGGAAGTGACGCCCTCGATGATTTTGACCTTGACATGCTTGGTTCGGATGCCCTTGATGACTTTGACCTCGACATGCTCGGCAGTGACGCCCTTGATGATTTCGACCTGGACATGCTGTGAGCTTGAATTCCACCACACTGGACTAGTGGATCCGAGCTCGGTACCAAGCTTAAGTTTAAACCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACGGGCCCTCTAGACTCGAGGGAGATGCCCAAAAAGAAGCGAAAAGTAGGCGGCAGTGGCGGGAGTAAGCTCCTCAGTTCTATAGAACAGGCGTGCGATATCTGTCGACTGAAAAAACTGAAGTGTAGTAAAGAAAAGCCAAAGTGTGCAAAGTGTTTGAAGAACAATTGGGAATGCAGATATTCACCGAAGACCAAACGGAGCCCTCTTACTCGCGCCCATTTGACGGAAGTTGAATCCAGGCTCGAAAGGTTGGAGCAACTCTTTCTTTTGATTTTCCCGAGGGAAGATCTTGACATGATTTTGAAAATGGATTCATTGCAAGATATTAAAGCACTGCTTACGGGGTTGTTTGTCCAAGACAACGTGAACAAGGACGCTGTCACAGATCGCCTGGCTAGTGTTGAAACAGATATGCCGTTGACTTTGAGGCAACACAGGATTAGCGCAACCTCCTCTTCTGAAGAATCCTCTAATAAAGGGCAGCGGCAATTGACGGTCAGCGGAGACGTGA |
| gBlock\_Cathepsin\_B | GAAGCCTCTCGAGCACCATGCCCAAAAAGAAGCGAAAAGTAGGCGGCAGTATGTGGCAGCTGTGGGCCAGCCTGTGCTGCCTGCTGGTGCTGGCCAATGCCCGCAGCCGGCCAAGCTTTCATCCCCTGTCCGACGAACTGGTGAACTATGTGAACAAGAGAAACACAACATGGCAGGCCGGCCACAATTTCTACAATGTGGACATGAGCTACCTGAAGCGGCTGTGCGGCACCTTTCTGGGAGGCCCTAAGCCCCCTCAGAGGGTGATGTTCACCGAGGATCTGAAACTGCCTGCCAGCTTCGACGCCAGGGAACAGTGGCCTCAGTGTCCCACCATCAAGGAAATTCGGGACCAGGGCTCTTGCGGCAGCTGCTGGGCCTTCGGAGCAGTGGAAGCAATCAGCGACAGAATTTGCATTCATACAAACGCCCACGTGTCCGTCGAGGTGTCCGCTGAGGATCTGCTGACCTGCTGCGGCAGCATGTGCGGGGATGGCTGCAACGGCGGCTATCCCGCTGAAGCCTGGAATTTCTGGACCAGGAAAGGCCTGGTCAGCGGAGGCCTCTACGAGTCCCACGTGGGATGTAGACCATACTCCATTCCACCATGCGAGCACCACGTGAACGGCAGCAGACCCCCTTGCACAGGCGAGGGGGATACTCCTAAGTGCTCCAAGATCTGCGAGCCCGGATACAGCCCCACATATAAACAGGATAAACACTATGGATACAACAGCTACTCTGTGTCTAATAGCGAGAAGGATATTATGGCTGAGATCTACAAGAATGGGCCCGTGGAGGGCGCCTTCTCCGTGTATAGCGATTTCCTGCTGTACAAATCCGGGGTGTATCAGCACGTGACCGGTGAGATGATGGGCGGCCATGCCATCAGAATCCTGGGCTGGGGAGTCGAGAACGGCACCCCTTACTGGCTGGTGGCCAACAGCTGGAATACCGACTGGGGCGACAACGGGTTCTTCAAAATCCTGCGGGGCCAGGATCACTGCGGCATTGAGTCTGAGGTGGTGGCCGGCATTCCCCGCACAGATCAGTACTGGGAGAAGATCTAGGGATCCGACTAGGC |
| trunc\_mut\_CatB | CATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACGGGCCCTCTAGACTCGAGCACCATGCCCAAAAAGAAGCGAAAAGTAGGCGGCAGTCCAAGCTTTCATCCCCTGTCCGACGAACTGGTGAACTATGTGAACAAGAGAAACACAACATGGCAGGCCGGCCACAATTTCTACAATGTGGACATGAGCTACCTGAAGCGGCTGTGCGGCACCTTTCTGGGAGGCCCTAAGCCCCCTCAGAGGGTGATGTTCACCGAGGATCTGAAACTGCCTGCCAGCTTCGACGCCAGGGAACAGTGGCCTCAGTGTCCCACCATCAAGGAAATTCGGGCTCAGGGCTCTTGCGGCAGCTGCTGGGCCTTCGGAGCAGTGGAAGCAATCAGCGACAGAATTTGCATTCATACAAACGCCCACGTGTCCGTCGAGGTGTCCGCTGAGGATCTGCTGACCTGCTGCGGCAGCATGTGCGGGGATGGCTGCAACGGCGGCTATCCCGCTGAAGCCTGGAATTTCTGGACCAGGAAAGGCCTGGTCAGCGGAGGCCTCTACGAGTCCCACGTGGGATGTAGACCATACTCCATTCCACCATGCGAGGCCCACGTGAACGGCAGCGCACCCCCTTGCACAGGCGAGGGGGATACTCCTAAGTGCTCCAAGATCTGCGAGCCCGGATACAGCCCCACATATAAACAGGATAAACACTATGGATACAACAGCTACTCTGTGTCTAATAGCGAGAAGGATATTATGGCTGAGATCTACAAGAATGGGCCCGTGGAGGGCGCCTTCTCCGTGTATAGCGATTTCCTGCTGTACAAATCCGGGGTGTATCAGCACGTGACCGGTGAGATGATGGGCGGCCATGCCATCAGAATCCTGGGCTGGGGAGTCGAGAACGGCACCCCTTACTGGCTGGTGGCCAACAGCTGGAATACCGACTGGGGCGACAACGGGTTCTTCAAAATCCTGCGGGGCCAGGATCACTGCGGCATTGAGTCTGAGGTGGTGGCCGGCATTCCCCGCACAGATCAGTACTGGGAGAAGATCTAGGGATCCGAGCTCGGTACCAAG |
| gBlock\_dCas9\_Inteins | CGTTCCTAGCATGCCTAAAAAGAAACGCAAAGTTGGGGAGCAGGAAGTCTTTGAATATTGTCTGGAGGATGGTTCTTTGATTAGGGCTACAAAAGACCACAAGTTTATGACGGTAGATGGTCAGATGCTCCCGATTGACGAAATTTTCGAAAGGGAATTGGACTTGATGCGGGTAGACAACTTGCCAAATTCTGGCAGCGGAAGCGGGTCTGGATTTCTTGGGGGTTCAGGCTCAGGGAGTGGGTCCATCAAGATCGCTACTCGAAAATACTTGGGCAAACAAAACGTCTACGATATAGGCGTGGAACGCGATCACAACTTTGCGCTCAAGAACGGTTTCATCGCTTCCAACTGCCCTAAAAAAAAACGGAAAGTGCACCTACCAAAAAAGAAGAGGAAGGTTTGCCTTTCTTATGAAACCGAAATCCTTACCGTAGAGTACGGTCTGCTTCCGATAGGTAAAATCGTTGAAAAGCGGATCGAGTGTACTGTATATAGCGTGGACAACAACGGGAATATATATACTCAGCCGGTGGCACAGTGGCATGATCGCGGGAAGCAGAAGGTCTTTGAGTACTGCTTGGAAGACGGAAGCCTCATAAGAGCCACGAAAGACCACAAATTCATGACCGTAGATGGACAGATGTTGCCGATAAAGGAGATATTCCGCCGGAAGCTCGACCTGATGAGAGTTGATAATCTTCCCAATAGCGGTTCTGGGTCCGGTTCAGGATTTCTGGGCGGTTCCGGCTCTGGAAGTGGTAGCATCGAGATCGCTACGGAAAAATATCTCGGAGAACAGAACGTCTATGACATCGGGGTTGAGCGAGACCACAATTTCGCCTTGAAAAATGGGGGCTATTTTCAAGGTATCGAAATTGCTACTGAAAAGTACCTGGGTGAGCAAAATGTTTATGACATAGGAGTAGAGCGCGACCACAACTTTGCCCTTAAAAATGGCCCTAAGAAGAAGCGGAAGGTTTGACGTGTAATGA |
| NpuC | GCGGTGCCACCATGATGCCTAAAAAGAAACGCAAAGTTGGGGAGCAGGAAGTCTTTGAATATTGTCTGGAGGATGGTTCTTTGATTAGGGCTACAAAAGACCACAAGTTTATGACGGTAGATGGTCAGATGCTCCCGATTGACGAAATTTTCGAAAGGGAATTGGACTTGATGCGGGTAGACAACTTGCCAAATTCTGGCAGCGGAAGCGGGTCTGGATTTCTTGGGGGTTCAGGCTCAGGGAGTGGGTCCATCAAGATCGCTACTCGAAAATACTTGGGCAAACAAAACGTCTACGATATAGGCGTGGAACGCGATCACAACTTTGCGCTCAAGAACGGTTTCATCGCTTCCAACTGCGACAAGAAGTACAG |
| NpuN | AGCTGGGAGGCGACTGCCTTTCTTATGAAACCGAAATCCTTACCGTAGAGTACGGTCTGCTTCCGATAGGTAAAATCGTTGAAAAGCGGATCGAGTGTACTGTATATAGCGTGGACAACAACGGGAATATATATACTCAGCCGGTGGCACAGTGGCATGATCGCGGGAAGCAGAAGGTCTTTGAGTACTGCTTGGAAGACGGAAGCCTCATAAGAGCCACGAAAGACCACAAATTCATGACCGTAGATGGACAGATGTTGCCGATAAAGGAGATATTCCGCCGGAAGCTCGACCTGATGAGAGTTGATAATCTTCCCAATAGCGGTTCTGGGTCCGGTTCAGGATTTCTGGGCGGTTCCGGCTCTGGAAGTGGTAGCATCGAGATCGCTACGGAAAAATATCTCGGAGAACAGAACGTCTATGACATCGGGGTTGAGCGAGATCACAATTTCGCCTTGAAAAATGGGGGCTATTTTCAAGGTATCGAAATTGCTACTGAAAAGTACCTGGGTGAGCAAAATGTTTATGACATAGGAGTAGAGCGCGACCACAACTTTGCCCTTAAAAATGGCCCTAAGAAGAAGCGGAAGGTTTGACTGAAGTTCATCTG |
| p70\_backbone | AGCGGAAGGTTTGACTGAAGTTCATCTGCACCAAAGCTTAAGTTTAAACCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACGGGCCCTCTAGACTCGA |
| dCas9 | TCGCTTCCAACTGCGACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACGCTATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACTGCCTTTCTTATGA |

### Readout System

**Table 28: List of oligos for basic staple cloning and assays.**| Number | Sequence |
| --- | --- |
| BS-1 | actcatatgtcccgccttgacaaaag |
| BS-2 | tcaggtctcattggtatttctcctctttagatttattac |
| BS-3 | actggtctctccaaatgtcccgccttgacaaaag |
| BS-4 | acacatatggtatttctcctctttaatctcg |
| BS-5 | actaaagtggatccaaactcgag |
| BS-6 | gtctgtcgactctggcgaaaatgagacg |
| BS-7 | tcgactttgatgcaaatactgcactagtgctatgcgtttgatgcaaatactgcactagtgctatgcgtttgatgcaaatactgcactagtgcgc |
| BS-8 | cgagcgcactagtgcagtatttgcatcaaacgcatagcactagtgcagtatttgcatcaaacgcatagcactagtgcagtatttgcatcaaag |
| BS-9 | catcaccacgtatctaaaggtgaggaagataatatggc |
| BS-10 | gtgatgatgcatatggtatttctcctctttagatggtac |
| BS-11 | actcatatgtcccgccttgacaaaag |
| BS-12 | actctcgagttagtggtgatggtgatgatgtttatacagttcatccattcctcc |
| BS-13 | gacacatgcatgccatagc |
| BS-14 | ccgcatcatcaccatcaccactaatgaggtaccaaactcgagtaag |
| BS-15 | agtgaaagtcgagctcg |
| BS-16 | aattgttcaagctcctctaagtc |
| BS-17 | gggtctggtggatcc |
| BS-18 | acctcattagtggtgatggtgatgatgcgggttgatacgcttttcc |
| BS-19 | tcccgccttgacaaaagtaag |
| BS-20 | catatggtatttctcctctttagatg |
| BS-21 | gagaaataccatatgaagcagttagaggacaag |
| BS-22 | ctctttagatttaacgctctcctactaactttttaag |
| BS-23 | ttagtaggagagcgttaattaggtaccaactcgagtaag |
| BS-24 | cttatcgtcgtcatccttgtaatccatatggtatttctcctctttagatgg |
| BS-25 | gattacaagggatgacgacgataagaaagacccggcggccctg |
| BS-26 | tttggtacctcattaacgctctcctactaactttttaag |
| BS-27 | aagcagttagaggacaagg |
| BS-28 | cttatcgtcgtcatccttgtaatccatatggtatttctcctctttagatgg |

**Table 29: List of gene fragments for basic staple cloning and assays.**| Gene fragment | Sequence |
| --- | --- |
| SiSt operon | agtgaaagtcgagctcggtacgctatggcatgcatgtgtcgcgacctgcataatacgactcactatagggtaccatctaaagaggagaaataccatat gtctcgcttagataaatccaaggttattaatagcgcattggaattgctgaatgaagtcggtattgagggattgactacgcgcaaacttgcacaaaaat taggggtagagcaacccacgctgtattggcacgttaaaaacaagcgtgccttattagacgcacttgcaatcgagatgttagaccgccatcatactcac ttctgcccattagagggcgagtcttggcaggacttcctgcgcaacaatgctaagtcttttcgttgtgccctgttaagccatcgcgacggagctaaagt acacttgggcacacgcccaacggagaaacagtacgagacattggagaaccaattggccttcttatgtcagcaaggatttagtcttgaaaacgcattat acgctttaagtgcggtaggacattttactcttggttgtgtgttagaggatcaggagcaccaagtcgcaaaggaagaacgcgaaacccctaccaccgac tcgatgcccccgctgcttcgtcaggcgattgagctgttcgatcatcaaggggcagagcctgcattcctgtttgggctggagttgattatttgcggtct tgagaaacaattgaaatgcgaatctgggtcaggcgggggaggaagcgggggaggcggatcgggagggggtggttcaggtgggggcggttccggcggcg gtggttccggaggaggcggatcatcccgccttgacaaaagtaaggtaatcaattcagcgctggaacttttgaacgaagtgggtattgagggtctgact acccgtaaactggcacaaaaattgggcgtagaacagcccaccttatattggcatgttaagaacaaacgtgctctgcttgatgcgctggcgatcgagat gctggatcgtcatcacacacatttttgccccctggagggcgaaagctggcaggatttcctgcgcaacaatgctaagagcttccgctgcgcacttcttt cgcaccgtgacggagcgaaggtccatttaggaacgcgtccgacagagaaacagtatgaaacgcttgaaaatcagcttgcattcctttgtcagcaaggg tttagtttagaaaacgcgctgtatgctctttccgctgtcggtcactttaccctgggatgcgtattggaagatcaggagcaccaggtagcaaaagaaga gcgcgaaactcctaccacagatagcatgcctcccttacttcgtcaagctatcgagttgtttgatcatcaaggcgcggaacctgccttcttgtttggat tggagctgattatttgtggcctggaaaaacagctgaagtgcgaatcgggctctgggtctggtggatccgaggagccaagtgacttagaggagcttgaa caattcgccaaaaccttcaaacagcgccgtattaagttgggcttcacacagggcgatgtcggtctggcgatgggcaagctgtatggtaacgacttttc acagactacgatttcccgttttgaagccttgaacctgtcttttaagaacatgtgcaagttaaaaccccttttggaaaaatggttgaacgacgctgaga acttaagttcagattcttcgttgtcatcaccgtcggcacttaactctcctgggatcgaagggttgtcacgccgtcgtaaaaagcgcacgtccattgag actaacattcgtgtggctttggagaaaagcttcttggagaatcagaagccaacctcagaagagatcacgatgattgcagatcaattgaatatggagaa ggaggtgatccgtgtgtggttttgcaatcgtcgtcaaaaggaaaagcgtatcaacccgtaatgaatctaaagaggagaaataccatatgtctcgctta gataaatccaaggttattaatagcgcattggaattgctgaatgaagtcggtattgagggattgactacgcgcaaacttgcacaaaaattaggggtaga gcaacccacgctgtattggcacgttaaaaacaagcgtgccttattagacgcacttgcaatcgagatgttagaccgccatcatactcacttctgcccat tagagggcgagtcttggcaggacttcctgcgcaacaatgctaagtcttttcgttgtgccctgttaagccatcgcgacggagctaaagtacacttgggc acacgcccaacggagaaacagtacgagacattggagaaccaattggccttcttatgtcagcaaggatttagtcttgaaaacgcattatacgctttaag tgcggtaggacattttactcttggttgtgtgttagaggatcaggagcaccaagtcgcaaaggaagaacgcgaaacccctaccaccgactcgatgcccc cgctgcttcgtcaggcgattgagctgttcgatcatcaaggggcagagcctgcattcctgtttgggctggagttgattatttgcggtcttgagaaacaa ttgaaatgcgaatctgggtcaggcgggggaggaagcgggggaggcggatcgggagggggtggttcaggtgggggcggttccggcggcggtggttccgg aggaggcggatcatcccgccttgacaaaagtaaggtaatcaattcagcgctggaacttttgaacgaagtgggtattgagggtctgactacccgtaaac tggcacaaaaattgggcgtagaacagcccaccttatattggcatgttaagaacaaacgtgctctgcttgatgcgctggcgatcgagatgctggatcgt catcacacacatttttgccccctggagggcgaaagctggcaggatttcctgcgcaacaatgctaagagcttccgctgcgcacttctttcgcaccgtga cggagcgaaggtccatttaggaacgcgtccgacagagaaacagtatgaaacgcttgaaaatcagcttgcattcctttgtcagcaagggtttagtttag aaaaacgcgctgtatgctctttccgctgtcggtcactttaccctgggatgcgtattggaagatcaggagcaccaggtagcaaaagaagagcgcgaaact cctaccacagatagcatgcctcccttacttcgtcaagctatcgagttgtttgatcatcaaggcgcggaacctgccttcttgtttggattggagctgat tatttgtggcctggaaaaacagctgaagtgcgaatcgggctctggatccgggtctggtgtaagcaaaggagaagcggttatcaaagagttcatgcgct ttaaagtgcacatggaaggaagtatgaatggacacgaatttgaaattgagggagagggggaagggcgcccttacgaaggcacccagaccgcgaaatta aaggttacgaagggaggtcccttgccgtttagttgggacattttgtcaccccaatttatgtatgggtcgcgcgcatttattaaacaccctgctgatat tccagactattacaagcagagcttcccggaagggttcaagtgggaacgcgtaatgaacttcgaagatggaggggcggtaactgtgacacaagacacca gcctggaagacggaactctgatctataaagtaaagttacgtggaaccaacttcccccccgat |
| MiSt operon | agtgaaagtcgagctcggtacgctatggcatgcatgtgtcgcgacctgcataatacgactcactatagggtaccatctaaagaggagaaataccatat gaaagacccggcggccctgaaacgcgcccgcaatactgaagctgcacgtcgcagccgtgcccgcaagctgcaacgtgttaaacaacttgaggataaagt agaggaattgttgtcgaaaaattatcacttagagaacgaggttgcgcgccttaaaaagttagtaggagagcgtggatccgggatgaagcagttagagga caaggttgaggagctgctttccaagaactatcatttggaaaacgaagtagcacgtttgaagaagcttgttggggagcgccttcagaagttgcagcgtgt aaagcgtgctcgtaacacagaagcagcacgtcgctcgcgcgctcgcaaggcggcgttgaagggctaatgaggtaccaaactcgagtaagga tctccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctagaatcttccgct tcctcgctcactgactcgctgcgctcggtcgttcg |

### Delivery System

**Table 30: List of oligos for conjugation experiments.**| Type | ID | Sequence |
| --- | --- | --- |
| Primer | DS1 | caagcgatccactagtatcttcc |
| Primer | DS2 | cctagagctcgatcgtcttgc |
| Primer | DS3 | ccccatacgatataagttgtactactttagtcagttccgcagtattacaaaag |
| Primer | DS4 | ctttcgactgagcctttcgtggccggcctacggc |
| Primer | DS5 | aggctggccgtaggccggccacgaaaggctcagtcgaaagac |
| Primer | DS6 | gcggaactgactaaagtagtacaacttatatcgtatggggctgact |
| Primer | DS7 | CTATCGTCTCGcggaagatcctttgatcttttctacg |
| Primer | DS8 | CTATCGTCTCCgttggggataccccctagag |
| Primer | DS9 | CTATCGTCTCCcaacgaaaggctcagtcgaa |
| Primer | DS10 | CTATCGTCTCGttacaacttatatcgtatggggc |
| Primer | DS11 | CTATCGTCTCTgtaattctgtggaatgtgtgtcagttagggtg |
| Primer | DS12 | CTATCGTCTCAcccaacgccagcaacgcgg |
| Primer | DS13 | CTATCGTCTCGtgggcaggataggtgaagtaggc |
| Primer | DS14 | CTATCGTCTCCtccggccagcctcgcagag |
| Primer | DS15 | TACGCGTCTCCCActctggctaactagagaacc |
| Primer | DS16 | TACGCGTCTCGggaacgcgtatatctggccc |
| Primer | DS17 | TACGCGTCTCAttccatccaccttgccgaaa |
| Primer | DS18 | TACGCGTCTCTagTGCTGTCTGCtctgtcct |
| Primer | DS19 | ATCACCATCATCATCATgccgccgaacaaaaactcatct |
| Primer | DS20 | CTCTTCCAATTTCACCTGctgggccggttccag |
| Primer | DS21 | cgctggaaccggcccagCAGGTGAAATTGGAAGAGTCAG |
| Primer | DS22 | gagtttttgttcggcggcATGATGATGATGGTGATGC |
| gBlock | DSg1 | caagcgatccactagtatcttccgctgcataaccctgcttcggggtcattatagcgattttttcggtatatccatcctttttcgcacgatatacaggattttgccaaagggttcgtgtagactttccttggtgtatccaacggcgtcagccgtccgcccgagggcagagccatgacttttttagccgctaaaacggccggggggtgcgcgtgattgccaagcacgtccccatgcgctccatcaagaagaggcacttcgagctgtaagtacatcaccgacgagcaaggcaagacgatcgagctctagg |
| gBlock | DSg2 | ccatccaccttgccgaaaagatttgtctgaggaactgaaaatagaagggaaaaaagaggagggacaaaagaggcagaaatgagaggggaggggacagaggacacctgaataaagaccacacccatgacccacgtgatgctgagaagtactcctgccctaggaagagactcagggcagagggaggaaggacagcagaccagacagtcacagcagccttgacaaaacgttcctggaactcaagctcttctccacagaggaggacagaGCAGACAGCA |
| gBlock | DSg3 | taatacgactcactatagggggcccagccggccCAGGTGAAATTGGAAGAGTCAGGCGGGGGGTCGGTTCAAACAGGGGGATCTCTGCGTTTAACGTGTGCTGCTAGTGGTCGTACATCACGTAGTTACGGCATGGGATGGTTTCGCCAGGCCCCCGGAAAGGAACGTGAGTTCGTGTCGGGAATTTCATGGCGCGGTGACTCGACGGGCTACGCGGATTCTGTGAAAGGACGCTTCACCATCTCACGCGACAACGCAAAGAATACGGTGGACTTGCAGATGAATTCCCTGAAGCCAGAGGACACTGCGATTTATTATTGCGCTGCTGCTGCGGGTTCGGCTTGGTATGGGACTCTTTACGAATACGACTATTGGGGTCAAGGGACACAGGTTACCGTTTCTTCGGCCTTAGAGCATCACCATCATCATCATgcggccgcccgctgagcaataactagc |

### **References**

Silbert, J., Lorenzo, V. De, & Aparicio, T. (2021). Refactoring the conjugation machinery of promiscuous plasmid RP4 into a device for conversion of Gram-negative isolates to Hfr strains. *ACS Synthetic Biology*, 10(4). <https://doi.org/10.1021/acssynbio.0c00611>

{% 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 %}

# Results

### **PICasSO Enables 3D Genome Engineering**

Our **PICasSO** toolbox (**P**lasmid-**I**ntegraded **Cas** **S**tapled **O**rigami), can be used to **selectively induce proximity** between two custom DNA loci or strands, **enabling future scientists** to investigate the effects of spatial genome perturbations on **cell development and pathogenesis**. We established a range of **novel DNA linking proteins** that we call **protein staples**. These range from minimal and easy to express, to larger Cas-based staples that allow for **flexible target design**. Furthermore, we established different **functionalization modules**, allowing for **cell-specific staple creation, depletion and control**. We successfully validated our Cas staples in mammalian cells and programmed functional DNA interactions in trans. To facilitate efficient delivery of large constructs we engineered bacteria capable of **inter-kingdom conjugation**.

[Scroll down](#Abstract)

[### CRISPR/Cas Staples

* Highly **specific**, easily **programmable** tool, based on the CRISPR/Cas system, that allows targeting of **any location** within the genome
* Multiple Cas staples with **fusion guide RNAs and fusion Cas proteins** tested for their ability to induce **proximity of genetic elements**
* We show **successful engineering** of the **3D genome structure** with Cas staples

![Results Icon](https://static.igem.wiki/teams/5237/wetlab-results/results-overview-cas-staple-correct.svg)](#CasStaples)
[### Staple Functionalization

* **Tumor-specific activation** of Cas stapling
* **Reliable** readout system for the identification of **functional linkers**
* We show that the **peptide linker GFLG** can be **selectively cleaved** by the protease cathepsin B *in vivo*

![Results Icon](https://static.igem.wiki/teams/5237/wetlab-results/resluts-overview-functionalization.svg)](#StapleFunctionalization)
[### Readout Systems

* FRET based system to **measure *in vivo* DNA stapling**
* *In vitro* EMSA assay to **calculate DNA binding affintiy**
* Successfully developed **Simple staple** to establish **new measurement systems for PICasSO**

![Results Icon](https://static.igem.wiki/teams/5237/wetlab-results/results-overview-readout-system.svg)](#ReadoutSystems)
[### Delivery System

* Re-purposing the bacterial conjugation machinery to **deliver Cas staples to mammalian cells**
* Utilizing the **RP4 system** to catalyze the transfer of an *oriT* carrying mobilizable plasmid
* Designed procedures to characterize **adhesion-enhancing nanobodies** and to test **inter-kingdom conjugation**

![Results Icon](https://static.igem.wiki/teams/5237/wetlab-results/results-overview-delivery-system.svg)](#DeliverySystem)

* [CRISPR/Cas Staples](#CasStaples)
* [Staple Functionalization](#StapleFunctionalization)
* [Readout Systems](#ReadoutSystems)
* [Delivery System](#DeliverySystem)
* [References](#References)
* [Back to the top](#top)

## CRISPR/Cas Staples

Actively utilizing the **3D DNA conformation** to engineer gene expression requires a tool
that is **highly specific** while still allowing for targeting **any location** within the genome. Recently,
CRISPR/Cas-based systems that manipulate three-dimensional DNA conformation to regulate cellular processes
have been developed. However, these systems are often complex and have a limited range of applications.
Here we present the design and application of **fusion guide RNAs (fgRNAs)** and fused Cas-nucleases to create
a **simple, adjustable system** for the precise programming of DNA-DNA contacts. We **successfully** demonstrated
the use of these Cas staples and their application in **gene expression control** by **artificially inducing spatial proximity of otherwise separate regulatory elements**.
Taken together, our results establish Cas staples as an **innovative and powerful** tool for remodeling the
three-dimensional landscape of the genome.

### Introduction

### Background Information

#### The CRISPR/Cas System as a Gene Editing Tool

In 2012, Jinek *et al.* described the use of the Clustered Regularly
Interspaced Short Palindromic Repeats (CRISPR)/Cas system, to induce DNA double-strand breaks.
Since their seminal work, gene editing with CRISPR/Cas has come a long way as exemplified by a
multitude of applications in all areas of the life sciences and even first CRISPR-based gene
therapies (Sheridan, 2023). At their core the CRISPR/Cas gene editors
are constituted by a ribonucleoprotein complex consisting of a Cas nuclease and one or two RNA
molecules responsible for guiding the nuclease to a specific genomic site. The most widely applied
CRISPR nuclease is Cas9 from *Streptococcus pyogenes*. It binds a CRISPR RNA
(crRNA) and a transactivating CRISPR RNA (tracrRNA) that can be combined into a single guide RNA
(sgRNA) (see **Fig. 1A**) (Pacesa *et al.*, 2024).
The sgRNA includes a scaffold required for its binding to the Cas nuclease and an interchangeable
20 nucleotide (nt) spacer sequence that defines the DNA target via complementary base pairing.
Once Cas9 binds to a matching DNA strand, it efficiently cleaves the target DNA (Cong *et al.*, 2013). Furthermore, a specific three nucleotide sequence (NGG) on the
3’ end in the targeted DNA is required for binding and cleavage. This is referred to as the
protospacer adjacent motif (PAM)
(Sternberg *et al.*, 2014).

![](https://static.igem.wiki/teams/5237/wetlab-results/cas-staple-svg/background-cas9-cas12a-principle.svg)

**Figure 1: The CRISPR/Cas system.** *A* and *B*, schematic structure
of Cas9 and Cas12a with their sgRNA/crRNA, sitting on a DNA strand with the PAM. The spacer
sequence forms base pairings with the dsDNA. In case of Cas9 the spacer is located at the 5’ prime
end, for Cas12a at the 3’ end of the gRNA. The scaffold of the gRNA forms a specific secondary
structure enabling it to bind to the Cas protein. The cut sites by the cleaving domains, RuvC and
HNH, are symbolized by the scissors.

Over the following years additional Cas proteins with different functional
properties have been discovered, including Cas12a from Acidaminococcus sp. (AsCas12a) and
Moraxella bovoculi (MbCas12a)(Zetsche *et al.*, 2015). In contrast to Cas9,
the gRNAs of Cas12a carry a 5’-scaffold (see **Fig. 1B**). Similarly, the PAM (TTTN) is also on
the 5’ side of the spacer
(Pacesa *et al.*, 2024).
Important to our work, Cas12a is capable of processing arrays of multiple consecutive crRNA
repeats into individual crRNAs/gRNAs enabling the expression of multiple gRNAs from a single
expression cassette (Paul and Montoya, 2020).
The introduction of specific mutations allows the generation of catalytically dead Cas variants
(Koonin *et al.*, 2023) (Kleinstiver *et al.*, 2019). By
fusing dCas proteins to transactivator domains and targeting them to an endogenous promoter region
via a specific gRNA, they can be harnessed for programmable transcriptional activation or “CRISPR
activation” (CRISPRa) (Kampmann, 2017).

#### Strategies to Program DNA Interactions

The three-dimensional conformation of genomes is crucial for the correct
interpretation of the genetic code and the phenotypic plasticity of cells. Interacting genetic
elements often occur in close proximity in cis but it was shown that long-range interactions of
genomic loci separated by thousands of bases or even located on different chromosomes are
similarly important (Cramer, 2019). Here, the three-dimensional (3D) conformation of the genome
comes into play and directly influences biological functions (Lieberman-Aiden *et
al.*, 2009)(Dixon et al., 2012). Being able to artificially influence DNA interactions would
allow us to study and program the 3D genome organization and to study and potentially cure
pathophysiological rearrangements. One complex example to achieve this, makes use of the
CRISPR/Cas system in the form of light-activated dynamic looping for endogenous gene expression
control (LADL) by Kim *et al.* (2019). In this system, two dCas9 proteins bind
to specific endogenous loci through their respective sgRNAs. The dCas9 are fused to photoreceptors
that cluster upon activation by blue light resulting in the inducible bridging of two genomic
loci. (see **Fig. 2**).

![](https://static.igem.wiki/teams/5237/wetlab-results/cas-staple-svg/dcas9-dcas9-cry2-cibn-loop.svg)

**Figure 2: Light-activated dynamic looping for endogenous gene expression
control.** The Cas proteins bind to the
DNA via the sgRNA. They are fused to CIBN, which, upon exposure to blue light, forms a heterodimer
with CRY2. CRY2 also forms oligomers with other CRY2 proteins, resulting in the bridging between
two different Cas9 protein complexes. This induces the looping of DNA segments (adapted from Kim *et al.* (2019)).

#### Aim of This Subproject

Although systems for the manipulation of 3D DNA conformations principally exist, they
require several components and multiple gRNAs, resulting in very complex experimental setups. The arguably most prominent tool, called LADL, relies on the unspecific clustering of dCas9 rendering multiplexing impossible. Related less complex approaches that are
based on smaller proteins like zinc finger domains, remain difficult to adapt to any genomic locus of
choice (Kim and Kini, 2017).
Here we present the usage of CRISPR/Cas proteins to reliably connect or "staple" two DNA strands.
We created fusion guide RNAs (fgRNAs) that are composed of Cas12a and Cas9 gRNAs, connected via their
spacers (Kweon *et al.*, 2017). These Cas staples solely
require the two Cas proteins dCas9 and dCas12a and an fgRNA to bridge two separate DNA strands. This allows for precise and adjustable genomic targeting while keeping the systems complexity to minimum.
Cas12a's ability to process concatenated crRNAs will allow for simple multiplexing by expressing several fgRNAs in repeat from a single promoter (Gonatopoulos-Pournatzis *et al.*, 2020).
In the first part of this project we established functional fgRNAs. We then continued to show that
these fgRNAs can be used in combination with Cas12a and Cas9 to form complete Cas staples that bring two separate
DNA loci into proximity.

### Results

### **Fusion Guide RNA Entry Vectors Allow for Efficient Creation of fgRNAs**

To successfully induce proximity of two DNA strands we had to connect two different DNA binding
elements. We selected SpyCas9 and MbCas12a as DNA-binders due to their programmability. To
physically connect both proteins, we decided to link their gRNAs. In contrast to fusing the Cas
proteins, this comes with the advantage that one could easily multiplex the system, while still
guaranteeing that specific pairs of genomic loci are connected. Specifically, the 3'-end of the
Cas12a gRNA was fused to the 5'-end of the Cas9 gRNA. Via this approach the two spacer sequences
are linked directly, ensuring a minimal distance between the two DNA strands. The design also
facilitates interchangeability, as the central portion of the fgRNA construct, which includes both
spacers (for Cas9 and Cas12a) together with an optional linker, can be easily replaced in one
cloning step. The scaffold components remain integrated within the backbone of the construct. To
implement this compact composition and ensure an efficient cloning procedure, we [engineered](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%7D%7D) an entry
vector for the fgRNAs . We inserted a gene block encoding ccdbB between the scaffolds of Cas12a and Cas9 guide RNA (gRNA). SapI cut sites were incorporated between the scaffolds and the ccdB gene.
Therefore, all fgRNAs can be created by a simple
Golden Gate Assembly when adding the spacer sequence as annealed oligonucleotides to the entry
vector (see **Fig. 3**). CcdB ensures bacteria transformed with the unchanged plasmid to die due to
the toxicity of ccdB. This concept was proven by our own clonings throughout the project resulting
in a very high success rate for the transformations. Picking one colony for sequencing was
sufficient in all cases but one.

![](https://static.igem.wiki/teams/5237/wetlab-results/entry-vector.svg)

**Figure 3: Construction process of fgRNAs using the entry vector.** The
ccdB gene can be excised using SapI in a Golden Gate assembly. By inserting oligonucleotides
with the desired spacer sequences and matching overhangs, the complete fgRNA can be assembled into the entry vector. Due
to the cytotoxic nature of ccdB, only cells with the oligonucleotides as inserts survive.

### **Fusion Guide RNAs Can Be Used For Multiplex Genome Editing**

#### Editing Endogenous Loci With fgRNAs

To develop and test functional fgRNA Cas staples we determined two essential applications of the
fgRNAs to establish first. These are the utilization for multiplex genome editing and the
implementation in CRISPRa (see **Fig. 4**) (Kweon *et al.*, 2017).

![](https://static.igem.wiki/teams/5237/wetlab-results/cas-staple-svg/background-crispr-cas-system-fgrna-past.svg)

**Figure 4: Applications of the Fusion Guide RNA.**
Fusion Guide RNAs can be used for multiplex genome
editing by guiding active Cas12a and Cas9 to two distinct loci. Similarly, fgRNAs allow for
CRISPRa, by guiding the Cas9-VP64 transcriptional activator towards a target locus.

To prove that our fusion gRNAs can still form functional ribonucleocomplexes together with Cas9 and Cas12a, a series of different
fgRNAs was created, each carrying spacers specific to the VEGFA and FANCF gene. HEK293-T cells
were transfected with constructs encoding the catalytically active Cas proteins and gRNAs.
The gene editing rates were evaluated 72 h post transfection by measuring indel frequencies with a T7 endonuclease I assay.
Initially we used AsCas12a and SpCas9 targeting the endogenous VEGFA (AsCas12a) and FANCF (SpCas9) loci. As controls, we included samples containing separate single gRNAs
co-transfected with a plasmid encoding the corresponding Cas protein, and compared them to samples including fgRNAs in combination with either one of the Cas proteins or both Cas combined in a single sample (see **Fig. 5**).
We detected high editing rates (45% for VEGFA and 15% for FANCF),
when using individual sgRNAs. Of note, the editing efficiencies for FANCF with fgRNAs resulted in noticeable indel frequencies of about 10%, with either SpCas9 and even when both Cas orthologs were co-transfected. Similar results were observed for VEGFA. In this case indel frequencies of even 40 % were measured for the sample in which both Cas variants were combined with the fgRNA These initial results confirmed our [engineering approach](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D) and prove that fgRNA show editing efficiencies comparable to commonly used gRNAs.

![](https://static.igem.wiki/teams/5237/wetlab-results/results-ascas-2.svg)

**Figure 5: FgRNAs Enable Efficient Editing of Endogenous Loci.**Editing efficiencies were measured by assessing indel rates 72h post transfection via T7EI assay. Editing % was calculated through band intensities. The schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts are included in each sample.

#### Functional Fusion Guide RNAs Can Be Designed for Different Cas Orthologs

To further evaluate the capabilities of the fgRNAs, we tested them in combination with
different Cas12a orthologs. [After some initial testing,](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D) we decided on using MbCas12a together with
SpCas9.
Additionally, to test if the differences in editing rates from the preliminary assay resulted from
the targeted loci or the different Cas orthologs, the spacers were tested in both arrangements.
Once with Cas12a targeting FANCF and SpCas9 targeting VEGFA and once vice versa. To better assess
the impact that the utilization of a fgRNA has on the editing rates, the sgRNAs were tested
separately and in one sample.
Having the sgRNA with single Cas proteins in the same sample resulted in no clear
difference in the indel frequencies (see **Fig. 6A and Fig. 6B**). The fusion of the gRNAs resulted in
a lower editing rate overall. While the editing for VEGFA stayed at about 20% in all
cases, the editing for FANCF dropped significantly. When targeting the same gene under the same
conditions, the editing rates for MbCas12a were overall lower than the ones from SpCas9.

![](https://static.igem.wiki/teams/5237/wetlab-results/results-mbcas-2.svg)

**Figure 6: Fusion gRNA Editing Rates In Combination with MbCas12a.**
*A* and *B*, Editing efficiencies were measured by assessing indel rates 72h post transfection via T7EI assay. Editing % was calculated through band intensities. The schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts are included in each sample. *A* and *B* display both orientations of the two spacers for VEGFA and FANCF.

#### fgRNAs Are Compatible With Linkers of Various Lengths

To further assess the effect of the genomic locus on the editing rate, we included CCR5 as an
additional gene target.
For this assay, a fgRNA with a 20 nt long linker was included between the two spacers.
The editing rate for VEGFA was again relatively consistent throughout the samples (see **Fig. 7**).
For CCR5, the editing rate with sgRNAs was approximately the same at about 30%.
However, it dropped below 10% for the fgRNA. The addition of the 20 nt
linker had no effect on the editing rates compared to no linker.

![](https://static.igem.wiki/teams/5237/wetlab-results/results-ccr5-2.svg)

**Figure 7: Fusion gRNA Editing Rates for Multiplexing CCR5 and VEGFA** Editing efficiencies were measured by assessing indel rates 72h post transfection via T7EI assay. Editing % was calculated through band intensities. The schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts are included in each sample. Cas12a targets VEGFA and Cas9 targets CCR5.

### **Using Fusion Guide RNAs to Induce Gene Expression**

#### fgRNAs Enable Efficient Activation of Gene Expression via CRISPRa

To establish the foundation for their use as protein scaffolds, we identified the next step as
demonstrating the use of fgRNAs for CRISPR activation. For this, we intend to recruit the
transcriptional activator VP64 to a firefly luciferase gene
to induce expression. The VP64 protein is attached to the catalytically inactive
Cas9 protein, which is then guided by gRNAs to the luciferase gene. The gRNAs target
a TetO sequence, which is positioned in front of the luciferase gene in multiple repeats.
The firefly luciferase activity was then quantified as photon counts and normalized against
Renilla luciferase, which is expressed on a separate plasmid under an ubiquitous promoter.
In two biological replicates we saw similar Relative luciferase activity with fgRNA as a guide
compared to a sgRNA (see **Fig. 8**). For further insight into the engineering behind these
findings, we recommend to take a look at the [engineering cycle fgRNA iteration 4 and 5.](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)

![](https://static.igem.wiki/teams/5237/wetlab-results/results-crispra-2.svg)

**Figure 8: CRISPRa Induced Luciferase Expression for sgRNAs and fgRNAs.**
Firefly luciferase activity was measured 48h post transfection and normalized against ubiquitously expressed Renilla luciferase. The tetO repeats were targeted by Cas9-VP64, once with a sgRNA and once with a fgRNA that had a non-targeting sequence for the Cas12a spacer. The schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts are included in each sample.

#### "Stapling" Two DNA Strands Together Using fgRNAs

After showing the general capability of the fgRNA to work for editing and for CRISPR activation,
the next step was to use it to staple two DNA loci together, and thereby induce proximity between
two separate functional elements. For this, an enhancer plasmid and a reporter plasmid was used.
The reporter plasmid has firefly luciferase behind several repeats of a Cas9 targeted sequence.
The enhancer plasmid has a Gal4 binding site behind several repeats of a Cas12a targeted sequence.
By introducing a fgRNA staple and a Gal4-VP64, expression of the luciferase is induced (see **Fig. 9A**).
Different linker lengths were tested. Cells were again normalized against ubiquitous renilla
expression. Further information on our learnings from this assay can be found in the Cas staple
engineering cycle iteration 2.
Using no linker between the two spacers showed similar relative luciferase activity to the
baseline control (see **Fig. 9B**). An extension of the linker from 20 nt up to 40 nt resulted in an
increasingly higher expression of the reporter gene.

![](https://static.igem.wiki/teams/5237/wetlab-results/results-eh-2.svg)

**Figure 9: Applying Fusion Guide RNAs for Cas staples.**
*A*, schematic overview of the assay. An enhancer plasmid and a reporter plasmid are brought into proximity by a fgRNA Cas staple complex binding both plasmids. Target sequences were included in multiple repeats prior to the functional elements. Firefly luciferase serves as the reporter gene, the enhancer is constituted by multiple Gal4 repeats that are bound by a Gal4-VP64 fusion. *B*, results of using a fgRNA Cas staple for trans activation of firefly luciferase. Firefly luciferase activity was measured 48h post transfection and normalized against ubiquitously expressed Renilla luciferase. Statistical significance was calculated with ordinary One-way ANOVA with Dunn's method for multiple comparisons (\*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; mean +/- SD). The assay included sgRNAs and fgRNAs with linker lengths from 0 nt to 40 nt.

### **Using Fusion Cas Proteins in Cas Staples to Induce Proximity**

To further improve the efficiency, we introduced a GSG-linker between the Cas proteins. Similar to
the initial tests for the fgRNAs, the capability of our fusion Cas constructs was tested by
assessing the indel frequencies via a T7EI assay. For this, the same target sequences as before were
used, namely FANCF and VEGFA in both configurations. We included biological duplicates in this
assay.
The fusion Cas proteins allowed for editing in general, with single gRNAs and fgRNAs (see **Fig.
10A and 10B**). The editing rate with Cas9 was higher overall. Especially for the fgRNA fusion Cas
combinations, the Cas12a editing rates were significantly lower, dropping to about 1%. At the same
time, targeting VEGFA resulted in a higher editing efficiency than FANCF, as one can see in our
fgRNA engineering cycle iteration 3.

![](https://static.igem.wiki/teams/5237/wetlab-results/results-fcas-2.svg)

**Figure 10: Editing rates for fusion guide RNAs with fusion Cas
proteins.** *A* and *B*, editing efficiencies were measured by assessing indel rates 72h post transfection via T7EI assay. Editing % was calculated through band intensities. The schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts are included in each sample. Cas proteins linked by a dash ("–") were fused to each other. Biological replicates are marked as individual dots.

To further investigate the characteristics of the fusion Cas system, CCR5 was again included as a
different target site, as well as a 20 nt linker between the two spacers.
In this case, while the combination of fusion Cas proteins with sgRNAs allowed for a high editing
rate of 15% to 25% at both target sites, VEGFA in combination with Cas12a was much more consistent
at around 20% than CCR5 with Cas9 at about 2% (see **Fig. 11**). The inclusion of a linker had no
significant impact on the editing rates with a fgRNA.

![](https://static.igem.wiki/teams/5237/wetlab-results/results-fcas-ccr5-2.svg)

**Figure 11: Targeting VEGFA and CCR5 with fusion Cas proteins and fgRNAs.** Editing efficiencies were measured by assessing indel rates 72h post transfection via T7EI assay. Editing % was calculated through band intensities. The schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts are included in each sample. Cas proteins linked by a dash ("–") were fused to each other.

Continuing the procedure in a similar manner as for the fgRNAs, we focused on inducing proximity
between genetic loci next. The same assay was used, with one enhancer plasmid and one reporter
plasmid. Though less distinct than the results for using just fgRNAs, the fusion Cas proteins can
be used to increase expression levels of the reporter firefly luciferase (see **Fig. 13**). While
using sgRNAs results in similar relative luciferase activity as for the negative control between
0.1 and 0.2., using a fgRNA with a 20 to 30 nt linker consistently resulted in activities at 0.25.
Fusion guide RNAs without a linker and with a 40 nt linker had on average about the same activity,
but with a higher spread over the biological replicates. Further learnings from this assay and how
we want to continue in the future is layed out in our [Cas staples engineering cycle iteration 2.](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)

![](https://static.igem.wiki/teams/5237/wetlab-results/results-fcas-eh-2.svg)

**Figure 12: Results of Implementing Fusion Cas Proteins in Trans Activation
of a Reporter**  Firefly luciferase activity was measured 48h post transfection and normalized against ubiquitously expressed Renilla luciferase. Statistical significance was calculated with ordinary One-way ANOVA with Dunn's method for multiple comparisons (\*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; mean +/- SD). Fusion Cas proteins were paired with sgRNAs and fgRNAs with linker lengths from 0 nt to 40 nt.

### Discussion

### **We Successfully Engineered Functional Fusion Guide RNAs**

The use of fgRNAs for multiplex gene editing with Cas9 and Cas12a was shown to be effective. Though the
fgRNAs have proven to allow for similar editing rates compared to sgRNAs in some cases, a number of
factors have been identified that have a higher impact on the efficiency of fgRNAs, including the targeted
genomic locus and the Cas ortholog. The editing rate varies considerably for different genes. In the
various assays conducted in this subproject, VEGFA showed a relatively consistent and high editing
rate. The editing rate of FANCF was observed to be fluctuating and lower in most cases.

This is likely due to differences in chromatin accessibility, which allows the Cas proteins
to reach some parts of the DNA more effectively than others (Klemm *et al.*, 2019).
Cas9 and Cas12a appear to not only have a varying editing rate overall, but also show
different responses to the fusion of the gRNAs or the Cas proteins themselves. Comparing
editing rates on individual genes indicates an overall increased performance of Cas9 with
fgRNAs.

One potential explanation for this observation might be a higher tolerance of Cas9 to
modifications made to the gRNA. In contrast, the addition of a linker appears to have
no impact on the editing rates. A reason for that might be that either both Cas proteins
are not able to bind together in general, or that 20 nt are not enough space between the
spacers to fit both Cas proteins.

While the fusion Cas protein constructs also worked in combination with fgRNAs, their overall perfomance
was better in presence of sgRNAs.
Having two individual connections in the Cas staple complex, might result in a more rigid assembly that
would require precise coordination of the discrete linkers. To achieve this, a vast range of protein and
gRNA linkers would need to be tested in different combinations, allowing for a better assessment on the
exact effect they have on the system and how these linkers would need to be combined to allow for an
effective forming of the Cas staple complex.

In comparison to the results presented by Kweon *et al.*, Cas9 was also observed to
have
a higher editing rate in general. However, the difference in editing rates for different
genes was not significant and the results show the editing rates for FANCF were in fact
higher. To further assess the actual difference the targeted gene makes, a large scale screen
of multiple different genes that are targeted under the same conditions would be necessary.

### **Fusion Guide RNA based Cas Staples Facilitate a Promising New Approach of 3D Genome Engineering**

The main objective of this project was to establish fgRNA based Cas staples as an effective tool to bring
genetic loci of choice into proximity. We first showed the fgRNAs ability to bind DNA in conjunction with
a dead Cas protein, by applying CRISPRa.
In the subsequent assays, we were able to show that our fusion Cas construct is able to bring and hold two
genomic loci in proximity in a way that allows for gene activation. By this we not only confirm that
changes in the 3D genome structure can determine whether a gene is activated, but also show that our Cas
staple system is capable of exactly that.

Though, compared to simple CRISPRa, the fold changes of
expression are way lower, this may allow for way more precise manipulation of gene expression. By
expanding the system to endogenous loci and enhancers, it could easily be linked to complex regulatory
systems within the cell, permitting a precisely engineered gene expression for the locus of choice.

Introducing fusion Cas proteins into the Cas staple system, showed first promising results, though not
significant enough to consider it as a clear increase in gene expression. This again shows the requirement
of further assays to understand the characteristics of fusion Cas proteins, especially in the context of
binding DNA.

The use of CRISPRa with fgRNA aligns with the results previously published in this regard, also showing an
increase in gene expression through the recruitment of an activator (Kweon *et al.*,
2017).
Changing the 3D structure to induce expression with fgRNA staples is to our knowledge a completely new
approach. In a similar way, this has been done with the LADL system (Kim *et al.*,
2019). Other approaches that use a more complex, modified version of Cas9 also showed that gene expression
can be altered by chemically inducing rearrangements on a 3D-level (Morgan *et al.*,
2017). These publications also show, that hijacking genomic loci for this procedure is feasible.

#### Outlook

To further prove our Cas staples to be applicable in shaping the human genome, a few new assays were
designed to be conducted in the future.
We are already in the process of designing constructs for the next experiments, which would focus on using
human genome enhancers to increase expression of target genes.
This extension of the system would further show how the introduction of Cas staples changing the
arrangement of DNA is enough to influence expression levels at will.

In a very recent publication the usage of so-called double guide RNAs (dgRNAs) for a similar application
was proposed (Yang *et al.*, 2024). These dgRNA consist of two subsequent Cas9 gRNAs
rather than a Cas12a and a Cas9 gRNA.
While this publication only showed application in bacterial context, we want to properly assess and
compare both systems in the eukaryotic environment. We are currently in the cloning phase of dgRNAs that
are usable in HEK293-T cells and plan to perform the same assays as we did with our fgRNA system to
thoroughly compare these to ways of using gRNA fusions as the basis of DNA stapling.

On the one hand, the fgRNA system would be able to solve complex systems via multiplexing different target
sites, for which we already have an experiment running, confirming this. On the other hand, the dgRNA
needs only one Cas ortholog to form the staple complex, lowering the amount of protein coding DNA required
to be introduced into the host cell. In this may lie the possibility of making it easier to package in
delivery
systems like adeno-associated viruses and thereby enabling the use in gene therapy.
Having both of these systems available in our toolbox, further increases the range of applications that we
can provide to researchers.

Furthermore, we plan on improving the fusion Cas staple, to get similar expression levels as seen for the
fgRNAs. First thoughts on new approaches can be read about in our Cas staples engineering cycle iteration
2. This would again include the screening of different protein linkers combined with different fgRNAs. The
establishment of a stable protein linker is the basis of making it responsive to outside stimuli like
certain chemicals or proteases. In the context of staple extension we show the successful cleavage of a
peptide linker by cathepsin B, allowing for the functionalization of a Cas staple construct. We would thereby enable our system to be adaptable to different conditions, that can
be specific to certain types of cancers through the likes of tumor micro environments or overexpression of
distinct proteins (Anderson & Simon, 2020).

## Staple Functionalization

To enable the **functionalization** of our PICasSO toolbox for a wide range of therapeutic and synthetic biology applications, we designed cathepsin B-responsive peptide linkers to selectively control the connection of our protein staples. Cathepsin B is a **lysosomal protease** enriched in various cancer types. We overexpressed cathepsin B in HEK293T cells to investigate its ability to cleave **different peptide linkers** using a fluorescence readout assay. We successfully demonstrated that the **GFLG linker, which can be incorporated into our staple constructs, was efficiently cleaved** by cathepsin B *in vivo* when cells were treated with doxorubicin. Furthermore, we showed that wild-type cathepsin B matured into its **active forms** under these conditions.

### Introduction

### Background Information

Cathepsin B is a cysteine protease typically located in lysosomes or secreted outside the cell, where it degrades proteins of the extracellular matrix. It plays a critical role in apoptosis and is significantly overexpressed in various cancer types, including breast and colorectal cancer (Ruan *et al.*, 2015). The overexpression of cathepsin B is associated with tumor invasion and metastasis. Various stimuli, such as ischemia, bile acids and TNFα, can induce cathepsin B-mediated apoptosis. In this process, lysosomes release large amounts of cathepsin B into the cytosol, where it cleaves anti-apoptotic factors like Bcl-2 and XIAP, leading to an increase in apoptotic proteases, such as caspase-3 (Bien *et al.*, 2010).
The significance of cathepsin B in cancer progression is well-documented, with studies showing elevated cathepsin B levels in cancerous tissues compared with non-cancerous tissues (Ruan *et al.*, 2015). Research has shown a connection between elevated levels of cathepsin B and enhanced angiogenesis, invasion and metastasis (Ruan *et al.*, 2015). Given its important role in tumor progression, cathepsin B is considered a potential therapeutic target (Ruan *et al.*, 2015) or prodrug-activating enzyme (Zhong *et al.*, 2013). Proteolytic cleavage of pro-biologics allows for the precise temporal and spatial regulation of biopharmaceutical activity in therapeutic strategies (Bleuez *et al.*, 2022).

#### Aim of This Subproject

We wanted to explore the potential of our PICasSO platform approach for therapeutic applications by designing protein-based DNA staples that are cleaved in response to the overexpression of cathepsin B in cancerous tissues. Furthermore, we demonstrated doxorubicin-dependent cathepsin B cleavage of one out of five documented linkers (Jin *et al.*, 2022; Shim *et al.*, 2022; Wang *et al.*, 2024) in HEK293T cells.
Additionally, we developed a construct consisting of a dead Cas9 (dCas9) connected to two SV40 nuclear localization sequences and two caged intein fragments (NpuN, NpuC) (Gramespacher *et al.*, 2017). The cages were connected to the split intein via cathepsin B-responsive linkers, preventing fragment association and consequent protein *trans*-splicing. In cancer cells overexpressing cathepsin B, the linkers would be cleaved, thus uncaging the inteins and resulting in association of NpuN and NpuC. In the subsequent protein *trans*-splicing reaction NpuN and NpuC cleave themselves out of the construct, linking dCas9 proteins to each other.
With these approaches, we aimed to induce structural and functional changes in protein-stapled DNA selectively in cancerous tissue.

### Results

### **Mature Cathepsin B is Expressed in HEK293T Cells**

To achieve cathepsin B cleavage-induced Cas stapling, catalytically active cathepsin B needs to be expressed in the cytosol. Therefore, we investigated the expression of different cathepsin B constructs under different conditions in HEK293T cells. In addition to wild-type (wt) cathepsin B, we also cloned a truncated and mutated version of cathepsin B (Δ1-20, D22A, H110A, R116A) and compared protein expression of both constructs in doxorubicin-treated and untreated conditions.
**Figure 13** shows a Western blot of the wild-type (wt) version of cathepsin B as well as the [truncated and mutated version of cathepsin B](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D) (Δ1-20, D22A, H110A, R116A). The truncated and mutated version of cathepsin B lacked an N-terminal signal peptide responsible for co-translational targeting to the rough endoplasmic reticulum. This would lead to the cytoplasmic expression of cathepsin B (Müntener *et al.*, 2005). Additionally, three point mutations would disrupt the conformation of an occluding loop increasing cathepsin B activity in the cytoplasm (Nägler *et al.*, 1997). Cells of both cathepsin B versions were treated with 500 nM doxorubicin (dox) 24 hours post-transfection and incubated for additional 24 hours. For each condition, three replicates were blotted. We observed no differences in protein expression levels between the dox-treated and untreated wt versions of cathepsin B. For the truncated and mutated version of cathepsin B, however, only the untreated samples showed the corresponding band at approximately 36 kDa expected for this version of cathepsin B. Additionally, the bands of the truncated and mutated version appeared much weaker than the ones of the wt, indicating poorer protein expression. The household protein β-tubulin is visible in all samples at approximately 55 kDa. The wt cathepsin B additionally showed bands for pro-cathepsin B at approximately 42 kDa, a mature single-chain version of cathepsin B at approximately 33 kDa and a mature double-chain version at approximately 26 kDa.

![]( https://static.igem.wiki/teams/5237/wetlab-results/catb-wb-w.svg)

**Figure 13: Western Blot of Two Versions of Cathepsin B With and Without Doxorubicin.** From left to right: protein ladder, wild-type (wt) cathepsin B with (+) and without (-) doxorubicin, truncated and mutated version of cathepsin B with (+) and without (-) doxorubicin. The household protein, β-tubulin, is visible in all samples at 55 kDa. The wt cathepsin B also shows bands for pro-cathepsin B at 42 kDa, mature single-chain cathepsin B at 33 kDa and mature double-chain cathepsin B at 26 kDa. The band for the truncated and mutated version of cathepsin B can be seen in the samples without doxorubicin at 36 kDa.

### **Implementation of a Cleavage-Responsive Fluorescence Readout Assay**

Based on the results of the Western blot, we decided to use wt cathepsin B to investigate the proteolytic cleavage of different peptide linkers. To this end, we used a fluorescence readout assay based on VP64-induced mCherry expression. We successfully identified one linker that could be cleaved efficiently by cathepsin B *in vivo*.
To investigate cathepsin B cleavage of different linkers, we incorporated five peptide linkers from literature (GFLG, FFRG, FRRL, VA, FK) (Jin *et al.*, 2022; Shim *et al.*, 2022; Wang *et al.*, 2024) in between the DNA binding domain (DBD) of Gal4 and the transactivator domain VP64 (as previously described in Muench *et al.* (2023)). Binding of Gal4-DBD upstream of a gene encoding the fluorescence protein mCherry induces overexpression of mCherry by VP64. Consequently, separation of Gal4-DBD and VP64 by cathepsin B cleavage of the peptide linker reduces mCherry expression (see **Fig. 14**).

![](https://static.igem.wiki/teams/5237/wetlab-results/catb-gal4-vp64-mechanism.svg)

**Figure 14: Schematic Illustration of the Cathepsin B Fluorescence Readout Assay.**The DNA binding domain (DBD) of Gal4 is conjugated to the transactivator domain VP64 via a cathepsin B-cleavable peptide linker. Binding of the Gal4-DBD to the upstream activating sequence (UAS) in proximity to the mCherry gene induces mCherry overexpression via VP64. Cathepsin B cleavage of the linker separates Gal4-DBD and VP64 and consequently reduces mCherry expression.

We conducted fluorescence readout assays in HEK293T cells. The transfected plasmids encoded mCherry, the Gal4-VP64 constructs with different linkers, and cathepsin B (see **Fig. 15**). Additionally, a stuffer plasmid and a plasmid encoding eGFP were transfected for normalization. By [conducting preliminary tests](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D), we determined adding doxorubicin 24 hours post transfection in a final concentration of 500 nM to the cell supernatant as the optimal procedure. This induces the lysosomal escape of mature cathepsin B (Bien *et al.*, 2004). 48 hours post transfection, we measured the fluorescence intensities of mCherry and eGFP and took micrographs of the transfected cells with a fluorescence microscope.

![]( https://static.igem.wiki/teams/5237/wetlab-results/catb-transfection-plasmids1.svg)

**Figure 15: Transfection Plan of HEK293T Cells for Fluorescence Readout Experiments.** HEK293T cells in a 96-well plate were transfected with plasmids encoding mCherry, the Gal4-VP64 constructs with different linkers, and cathepsin B (CatB). Additionally, a stuffer plasmid and a plasmid encoding eGFP were transfected for normalization.

### **mCherry and eGFP Can be Used as a Reporter System to Measure Cleavage Efficiency in HEK293T Cells**

In this experiment, mCherry and eGFP were evaluated as reporters to quantify the efficiency of cathepsin B-mediated cleavage of Gal4-Linker-VP64 constructs in HEK293T cells.
**Figure 16** shows micrographs taken with a fluorescence microscope of three different conditions: the null control, the negative control and the test sample. **Figure 17** shows the corresponding graphs. All samples were transfected with plasmids encoding eGFP and mCherry. The null control and the negative control were not transfected with the plasmid encoding cathepsin B. The null control was also not transfected with any of the plasmids encoding Gal4-Linker-VP64 constructs. The test sample was transfected with 30 ng of the plasmid encoding cathepsin B and with the plasmid encoding Gal4-GFLG-VP64. As expected, the null control exhibited no detectable mCherry signal, with corresponding fluorescence intensity measurements at baseline levels. Since no plasmid encoding a Gal4-V64 construct was transfected, mCherry overexpression via VP64 could not be induced. However, we observed a high fluorescence intensity for eGFP, indicating that the transfection was successful. The negative control showed strong signals of both mCherry and eGFP. Therefore, it can be assumed that the transfection was successful and that our mCherry readout system is functional. Interestingly, there are some cells which either seem to only express mCherry or eGFP and some cells that show no fluorescence signal. The test sample showed less eGFP and mCherry fluorescence compared to the negative control. We expected to observe reduced fluorescence intensity of mCherry, as the transfected cells would express cathepsin B, which cleaves the linker, thereby decreasing mCherry expression.

![](https://static.igem.wiki/teams/5237/wetlab-results/catb-fluorescence-microscope-w1.png)

**Figure 16: Micrographs of HEK293T Cells in Two Control Conditions and One Test Condition.** Micrographs were taken with a fluorescence microscope 48 hours post transfection. An overlay of brightfield, eGFP and mCherry is shown. All samples were transfected with plasmids encoding eGFP. The null control and the negative control were not transfected with the plasmid encoding cathepsin B. The null control was also not transfected with any of the plasmids encoding Gal4-Linker-VP64 constructs. The test sample was transfected with 30 ng of the plasmid encoding cathepsin B and with the plasmid encoding Gal4-GFLG-VP64. The micrograph of the test sample is not from the same biological replicate as the micrographs of the two controls.

![](https://static.igem.wiki/teams/5237/wetlab-results/catb-results/catb-fluorescence-readout-null-negative-test-w.svg)

**Figure 17: Fluorescence Readout After 48 Hours for Two Control Conditions and One Test Condition.** The fluorescence intensity for mCherry was measured for the GFLG linker and normalized against a baseline eGFP fluorescence intensity. The null control and the negative control were not transfected with the plasmid encoding cathepsin B. The null control was also not transfected with any of the plasmids encoding Gal4-Linker-VP64 constructs. The test sample was transfected with 30 ng of the plasmid encoding cathepsin B and with the plasmid encoding Gal4-GFLG-VP64.

### **The Peptide Linker GFLG Is Cleaved by Cathepsin B *in Vivo***

This experiment investigated the cleavage of five peptide linkers by cathepsin B in vivo, analyzing mCherry fluorescence intensity across the different linkers and transfection conditions.
**Figure 18** shows the fluorescence intensity of mCherry for five different peptide linkers (GFLG, FFRG, FRRL, VA, FK). The negative control was not transfected with the plasmid encoding cathepsin B. We investigated two different test conditions, in which we either transfected 30 ng or 60 ng of the plasmid encoding cathepsin B. The fluorescence intensity of mCherry was normalized by the measured fluorescence intensity of eGFP in each condition. Additionally, the values for 30 ng and 60 ng cathepsin B were normalized against the corresponding negative controls. One data point for the VA linker, transfected with 60 ng of the plasmid encoding cathepsin B, was excluded due to severe deviation from the other values. We conducted a two-way analysis of variance (ANOVA) to assess the significance of the observed differences between the negative control and the test conditions for each linker. As the negative control did not contain the plasmid encoding cathepsin B, we expected the measured fluorescence intensity of mCherry to be the highest in these conditions. However, this was only observed for the GFLG and FK linkers. Contrary to our expectations, the fluorescence intensity of the negative control was the lowest out of the three conditions tested for the remaining linkers. It appears that the addition of the plasmid encoding cathepsin B increases mCherry fluorescence intensity when the linker is not cleaved. However, this increase is only significant for the FFRG linker in the 60 ng condition. For the GFLG linker, we observed significant decreases in fluorescence intensity between the negative control and both test conditions, with no difference between the 30 ng and 60 ng conditions. For the FK linker, no significant decreases in fluorescence intensity between the negative control and the test conditions were observed.

![](https://static.igem.wiki/teams/5237/wetlab-results/catb-results/catb-fluorescent-readout-final-results-w.svg)

**Figure 18: Fluorescence Readout After 48 Hours for Five Different Peptide Linkers and Three Different Conditions.** The fluorescence intensity for mCherry was measured for five different linkers and normalized against a baseline eGFP fluorescence intensity. The negative control was not transfected with the plasmid encoding cathepsin B. The fluorescence intensity of the negative control was set to one. Two different test conditions were investigated, in which either 30 ng or 60 ng of the plasmid encoding cathepsin B were transfected. The fluorescence readout was analyzed using a two-way ANOVA. Medium: DMEM (10% FCS). P values: ns, P > 0.05; \*, P ≤ 0.05; \*\*, P ≤ 0.01; \*\*\*, P ≤ 0.001; \*\*\*\*, P ≤ 0.0001.

### Discussion

### **GFLG Is a Promising Candidate for Targeted Applications in Environments With Upregulated Cathepsin B Activity**

Initially, Western blot analysis confirmed that overexpressed wild-type cathepsin B was processed into its mature single-chain and double-chain forms as previously reported in the literature (Mentlein, Hattermann, Held-Feindt, 2012). This confirms that active cathepsin B is present inside of the cells. However, since lysis of the cells also disrupts the lysosomes, we can not conclude whether this active cathepsin B is also present in the cytosol *in vivo*. For the truncated and mutated version of cathepsin B, we only observed protein bands in samples that were not treated with doxorubicin. The three samples incubated with doxorubicin for 24 hours showed only faint bands for the housekeeping protein β-tubulin, indicating generally low protein levels. This suggests that the cells were subjected to stress, possibly through doxorubicin or inadequate handling. Along with low transfection efficiency, these factors may have contributed to the low protein levels observed.
Using fluorescence microscopy, we demonstrated cathepsin B-mediated linker cleavage in our reporter assay setup. This assay employed a Gal4-VP64 system, in which cleavage of the peptide linker by cathepsin B reduced mCherry expression, providing a reliable measure of cleavage efficiency. The successful cleavage of linkers was indicated by reduced mCherry fluorescence intensity in cells overexpressing cathepsin B. It was also observed that not all cells were fluorescent in accordance with an expected transfection efficiency. We also noticed that some cells seemed to only express eGFP or mCherry. However, most cells seemed to express both eGFP and mCherry as indicated by their yellow fluorescence.
Finally, we investigated the cleavage of different peptide linker variants by cathepsin B with help of our fluorescence readout assay. Five different peptide linkers (GFLG, FFRG, FRRL, VA, FK) were tested. A two-way ANOVA revealed a significant reduction in fluorescence between the negative control and the two test conditions for the GFLG linker in the presence of cathepsin B, demonstrating efficient cleavage. Surprisingly, a significant increase in fluorescence was observed between the negative control and 60 ng test condition for the FFRG linker. However, since the increase between the negative control and the 30 ng test condition of the same linker was not significant, this difference is likely due to biological variability between the samples.
Additionally, our cathepsin B-cleavable linker can be combined with caged inteins (Gramespacher *et al.*, 2017) conjugated to a dead Cas9 to selectively induce Cas-stapling in the presence of cathepsin B. Corresponding parts can be found in our part collection and in the iGEM registry, but still require characterization.
In conclusion, these findings demonstrate that our fluorescence-based readout assay can reliably detect cathepsin B-mediated cleavage of peptide linkers, with the GFLG linker showing particular susceptibility to cleavage. This makes GFLG a promising candidate for targeted applications in environments with upregulated cathepsin B activity, such as cancer tissues.

#### Outlook

Future experiments will investigate the influence of different doxorubicin concentrations on the activity of cathepsin B in the cytosol. Different linker lengths or a repeat of the GFLG linker could also be tested. Additionally, this system could be used for other proteases that are involved in certain diseases, such as different caspases in neurodegenerative conditions (Espinosa-Oliva *et al.*, 2019).

## Readout Systems

By developing custom **EMSA and FRET** assays, we established **key tools** for the **validation
and characterization of various protein staples**. Beginning with the successful construction of **basic
staples**, these assays provided **key insights** into the characteristics of DNA binding proteins and stapling **mechanisms**. By rigorously **testing our workflow,** we developed **foundational techniques** that future **iGEMers and researchers**
can leverage to engineer and optimize **protein-based DNA-folding** systems.

### Introduction

### Background Information

#### Selection of DNA binding Proteins tetR, Oct1 and GCN4

To establish easy-to-use assays for the characterization of protein staples, we started building on basic protein parts. Instead of Cas proteins, we used the Tetracycline Repressor (TetR) and the human transcription factor Oct1 as DNA-binding domains as they are well-characterized proteins with known binding properties. TetR is a bacterial transcriptional repressor that binds specifically to the tetO operator sequence and dissociates in the presence of tetracycline. It is widely adopted as a synthetic gene regulation tool, both in prokaryotic and eukaryotic systems (Berens & Hillen, 2004). Similarly, Oct1, a POU domain transcription factor involved in immune cell regulation and stress response, has been shown to bind tightly to its octamer DNA motif (Lundbäck *et al.*, 2000; Stepchenko *et al.*, 2021). It was shown that the DNA-binding domain of Oct1 can be readily fused to other proteins, for increased protein solubility and strong DNA-binding capabilities, even during protein purification (J. H. Park *et al.*, 2013; Y. Park *et al.*, 2020). Fusing TetR to Oct1 we created "simple staple proteins" able to bind both operator sequences simultaneously.
In addition to TetR and Oct1, we additionally used small basic-region leucine zipper (bZip) proteins with DNA-binding capabilities as they are among the most compact DNA-binding domains known to date. The motif consists of a coiled-coil leucine zipper dimerization domain, and a highly charged basic region that binds to DNA (Hollenbeck & Oakley, 2000). One well characterized example is the General Control Protein 4 (GCN4), a well-characterized transcriptional activator from yeast (Arndt & Fink, 1986). At its N-terminus, GCN4 contains basic residues, the so-called bZip domain, through which it binds specifically to the CRE (cyclic AMP response element) DNA sequence (Hollenbeck *et al.*, 2002). A variant of GCN4 with the DNA binding bZip-domain at the C-terminus (rGCN4) has been engineered to bind to the inverted CRE sequence, INV2 with similar affinity (Hollenbeck *et al.*, 2001). By genetically fusing GCN4 to rGCN4, we created a small bivalent DNA binding staple with less than 150 amino acids.

#### Förster Resonance Energy Transfer (FRET)

Förster Resonance Energy Transfer (FRET) is a distance-dependent physical process: in which energy is transferred non-radiatively from an excited donor fluorophore to an acceptor fluorophore via dipole-dipole coupling. The efficiency of energy transfer is highly sensitive to the distance between the donor and acceptor, typically in the range of 1-10 nm, making FRET ideal for studying molecular proximity (Hochreiter *et al.*, 2019). This proximity sensitivity is governed by the Förster radius (R₀), which is the distance at which 50% energy transfer occurs. Factors affecting FRET efficiency include the overlap of the donor's emission spectrum with the acceptor’s absorption spectrum, the quantum yield of the donor, and the relative orientation of the fluorophores (Wu & Brand, 1994). These characteristics allow FRET to detect interactions such as protein-DNA binding or DNA-DNA contacts in real time.
For our assay, we selected mNeonGreen and mScarlet-I as donor and acceptor fluorophores, respectively, due to their strong fluorescence, spectral overlap, and minimal photobleaching, ensuring high FRET efficiency in our system (Bindels *et al.*, 2017; Shaner *et al.*, 2013). FRET's sensitivity to small changes in distance makes it especially powerful for analyzing molecular interactions in living cells (Okamoto & Sako, 2017).

#### Electrophoretic Mobility Shift Assay (EMSA)

The Electrophoretic mobility shift assay (EMSA) is a widely adopted method used to study DNA-protein interactions. EMS exploits the fact that nucleic acids bound to proteins have reduced electrophoretic mobility, compared to their unbound counterpart. (Hellman & Fried, 2007). Mobility-shift assays can be used to qualitatively assess DNA binding capabilities as well as for the quantitative determination of binding stoichiometries and kinetics such as the apparent dissociation constant (Kd (Fried, 1989).

#### Aim of This Subproject

Engineering a solid and versatile toolbox is a huge challenge, especially when working with complex systems such as DNA-bound protein staples. To systematically characterize these components, we first set out to develop a well-tested collection of assays that lay the foundation for the investigation of the more complex aspects of our PICasSO toolbox. These assays provide essential tools for studying protein-DNA interactions and DNA-DNA proximity. We used electrophoretic mobility shift assays (EMSA) to analyze protein-DNA binding kinetics and sequence specificity *in vitro*  and a Förster resonance energy transfer (FRET) assay to detect interactions *in vivo*. Together, these assays form the backbone of our experimental approach and were utilized to systematically analyze and create new staples.

### Results

### **FRET Measurement Can Be Used to Detect DNA-DNA Proximity *in vivo***

The FRET assay was developed using a two-plasmid system in bacterial cells. [After testing different constructs,](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D) our final expression plasmid
contains a tetR binding site and expresses three key proteins under the control of a single T7
promoter in a polycistronic operon: (1) tetR-Oct1, our simple staple fusion protein that acts as a
bivalent DNA binding protein, tethering two plasmids via tetR and Oct1 binding sites; (2)
Oct1-mNeonGreen, serving as the FRET donor; and (3) tetR-mScarlet-I, the FRET acceptor. This
ensures all three proteins are co-expressed in similar stoichiometry. The folding plasmid contains
an Oct1 binding site for the staple and FRET donor binding.

When tetR-Oct1 binds the respective sites on both plasmids, mNeonGreen and mScarlet-I are brought into
into proximity, facilitating FRET between the two fluorophores (see **Fig. 19**). Successful stapling of the
plasmids results in increased energy transfer from mNeonGreen to mScarlet-I, which can be detected
by exciting mNeonGreen and measuring enhanced emission from mScarlet-I. A positive control,
consisting of a direct fusion of mNeonGreen and mScarlet-I, ensures maximal FRET efficiency and
serves as a benchmark for the assay.

![basic-staple-fret](https://static.igem.wiki/teams/5237/figures-corrected/basic-staple-fret.svg)

**Figure 19: Overview of DNAs tapling and FRET measurement**

Fluorescence intensity, normalized to OD600, of mNeonGreen and mScarlet-I was measured 18 h after inducing staple protein expression with varying concentrations of IPTG (see **Fig. 20A and 20B**). Counterintuitively, an increasing fluorescence was detected for decreasing IPTG concentrations, likely due to slower culture growth due to the high burden of strong induction. Fluorescence intensity of the positive control was significantly stronger compared to the negative control and staple. The negative control and staple, both of which carried the same expression plasmid construct, had similar fluorescence intensity for mNeonGreen and mScarlet-I down to approximately 0.05 mM. Lower concentrations resulted in strong discrepancies. To ensure comparability between the negative control and staple, further fluorescence intensity measurements were conducted after induction with 0.05 mM IPTG. In this experiment, only minor differences were detected for the individual fluorescent proteins between the non-connected negative control and the stapled sample When we measured FRET efficiency instead, we detected a strong increase in FRET efficiency for the staple as compared to the negative control, indicating successful induction of spatial proximity between both DNA strands (see **Fig. 20C**).

![fluo-tit](https://static.igem.wiki/teams/5237/wetlab-results/sist-results-panel-fret.svg)

**Figure 20: Fluorescence measurement of mNeonGreen, mScarlet-I and FRET.**
Fluorescent measurement, normalized to cell count, of mNeonGreen (ex. 490 nm, em. 530 nm),
mScarlet-I (ex. 560 nm, em. 600 nm), and FRET (ex. 490 nm, em. 600 nm) in *E.
coli*, 18 h
after induction with 0.025 mM IPTG. Data is presented as mean +/- SD.
**A**, **B** Fluorescence intensity of mNeonGreen and mScarlet-I with different IPTG
concentrations.
**C** Fluorescence intensity of FRET pair after induction with 0.05 mM IPTG. (n = 3) Statistical
significance was
determined with Ordinary two-way ANOVA with Šidák's multiple comparison test, with a single
pooled
variance. \*p < 0.05, \*\*\*\*p < 0.001. Only significant results are shown.

### **DNA Binding Proteins Can Be Readily Expressed And Purified**

To better characterize them and perform EMSA experiments, we aimed to purify all mini staples described above. The DNA binding proteins TetR and Oct1-DBD were fused to mScarlet-I and mNeonGreen, respectively, and to each other, each harboring a His6-tag. The bZip proteins GCN4, rGCN4 and their fusion bGCN4 were fused N-terminally to a FLAG-tag (DYKDDDDK). All proteins could be readily expressed under control of a T7 promoter in *E. coli BL21 DE3* and purified with Ni-NTA, or Anti-FLAG affinity columns for His-tagged and FLAG-tagged proteins, respectively
(see **figure 21**).

![](https://static.igem.wiki/teams/5237/wetlab-results/bs-sds-expression-panel-horizontal.svg)

**Figure 21: SDS-PAGE analysis of purified DNA binding
proteins.**
A) Analysis of fractions eluate of purified protein taken during Ni-NTA affinity chromatography.
b) Analysis of fractions eluate of purified protein taken during Anti-FLAG affinity chromatography
1 µL of each sample was prepared with Leammli buffer and loaded on 4-15% TGX-Gel. Correct bands of interest are highlighted by red boxes.

### **Optimized EMSA Conditions Enable the Successful Demonstration of DNA-staple Interactions**

To assess possible DNA binding, a qualitative EMSA was performed with the purified Oct1-DBD, bGCN4 and TetR staple proteins and additionally three different buffer systems TetR. DNA binding could be detected for the single purified proteins including Oct1-DBD, TetR, bGCN4, but not for the bGCN4 fusion (see **Fig. 22**).
Binding buffer 1 (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2HPO4, 1.8 mM KH2HPO4, 0.1 % (v/v) IGEPAL® CA-360, 1 mM EDTA), also described by Hollenbeck (2001)) was the best performing buffer and used for subsequent experiments.

![](https://static.igem.wiki/teams/5237/wetlab-results/sist-emsa-quali-buffer-opt.svg)
![](https://static.igem.wiki/teams/5237/wetlab-results/sist-emsa-staple.svg)
![](https://static.igem.wiki/teams/5237/wetlab-results/mist-emsa-quali.svg)

**Figure 22: Qualitative EMSA results.** Gelelectrophoresis was performed in TBE buffer with 10 % TGX-Gel pre-equilibrated with TBE. Bands are visualised by post-staining with SYBR-Safe. A, 8 µM purified mNeonGreen-Oct1 fusion-protein were equilibrated with different DNA concentrations (1000 nM, 100 nM, 10 nM) containing three Oct1 binding sites. in different buffer compositions (Binding buffer 1: 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2HPO4, 1.8 mM KH2HPO4, 0.1 % (v/v) IGEPAL® CA-360, 1 mM EDTA; Binding buffer 2: 10 mM Tris, 50 mM KCl; NaP250: 50 mM NaH2PO4, 150 mM NaCl, 250 mM Imidazol). B, purified TetR-Oct-1 fusion protein was incubated with 0.5 µM DNA containing either a TetR or Oct-1 binding site. C, 200 µM purified protein were equilibrated with 0.5 µM DNA containing one target site.

### **Successful Quantification of DNA Binding Calculation of Dissociation Constants**

To further analyze DNA binding, quantitative shift assays were performed for GCN4 and rGCN4. Here 0.5 µM DNA were incubated with varying concentrations of protein until equilibration. After electrophoresis, bands were stained with SYBR-Safe and quantified based on pixel intensity. The obtained values were fitted to equation 1, describing formation of a 2:1 protein-DNA complex.

Θapp = Θmin + (Θmax - Θmin)
Ka2 [L]tot2
1 + Ka2 [L]tot2

*Equation 1*

Here [L]tot describes the total protein monomer concentration, Ka corresponds to the apparent monomeric equilibration constant. The min/max values arSe the experimentally determined site saturation values (For this experiment 0 and 1 were chosen for min and max respectively). GCN4 binds to its optimal DNA binding motif with an apparent dissociation constant Kk of (0.2930.033)×10-6 M, which is almost identical to the rGCN4 binding affinity to INVii a d of (0.2980.030)×10-6 M (see **Fig. 23**).

![](https://static.igem.wiki/teams/5237/wetlab-results/mist-leucine-zipper-kd-plot.svg)

**Figure 23: Kd Calculation of GCN4 and rGCN4 Quantitative assessment of binding affinity for GCN4 and rGCN4.** Proteins of varying concentrations were incubated with 0.5 µM DNA in Binding buffer 1, and the bound fraction analyzed by dividing pixel intensity of bound fraction with pixel intensity of bound and unbound fraction using ImageJ. At least three separate measurements were conducted for each data point. Values are presented as mean +/- SD.

### Discussion

### **We Detected In Vivo Stapling and Measured Protein-DNA Binding Constants**

The results from the fluorescence intensity measurements showed stronger expression of the fluorescent
proteins for the positive control. Based on our preliminary testing, explained in [our engineering
cycles](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D), we suspect a strong metabolic burden to influence expression levels, especially for the
staple construct. This could be both due to the polycistronic expression of multiple protein, as well as
the strong T7 promoter resulting in too high mRNA levels. The negative control and staple showed strong discrepancies in mNeonGreen and mScarlet-I fluorescence for IPTG concentrations below 0.05 mM. This is surprising as both samples have the same expression plasmid and only the folding plasmid of the negative control is missing the binding site for Oct1. One possible explanation could be off-target binding in the *E. coli* genome, resulting in changes in the expression pattern or similar. With 84 specific Oct1 binding sequences previously reported in the genome this could be a major factor. (Y. Park *et al.*, 2020). Studies also showed binding affinity of Oct1 to different target sites, albeit with lower affinity, possibly resulting in even more potential genomic binding sites(Verrijzer *et al.*, 1992). The folding plasmid, harboring a p15A origin of replication, is expected to have around 11 copies per cell (Shao *et al.*, 2021), resulting in approximately 130 Oct1 binding sites, which stand in competition to genomic binding sites. Further experiments are needed to better understand this observation.
All proteins for in vitro characterization could be expressed and purified. Some nonspecific proteins still remained in the eluate as detected by SDS-PAGE, but this is to be expected given well known unspecific binding of cellular proteins to the Ni-NTA affinity matrix. Initial qualitative tests showed successful binding of the single DNA binding proteins to the DNA. Since TetR and Oct1 were fused to mScarlet-I and mNeongreen, respectively, we could show that these proteins accept fusions, while still being able to bind DNA, which also matches previous observations (Gossen & Bujard, 1992; J. H. Park *et al.*, 2013b). Bound and unbound fractions were visualized on the gel by SYBR-Safe staining and illumination with a trans-illuminator, resulting. To enable exact quantification, and to further characterize additional DNA binding proteins the bZip proteins were chosen for quantitative gel shift assays. For the purified proteins GCN4, rGCN4 and the fusion bGCN4 a qualitative gel was run with high protein concentration. Bands for GCN4 and rGCN4 were visible but no band for bGCN4 could be detected, indicating a lack of DNA binding. This suggests that the dimerization, necessary for simultaneous binding of two DNA strands is disrupted by the GSG-linker (Ellenberger *et al.*, 1992; Liu *et al.*, 2006; Lupas *et al.*, 2017; Woolfson, 2023). To better understand possible problems in dimerization circular dichroism could be used to analyze secondary structure and proper coiled coil formation (Greenfield, 2006). Further engineering will be required including the investigation of various linkers with specific properties to ensure correct folding and dimerization (Chen *et al.*, 2013). The apparent binding kinetics calculated for GCN4 ((0.2930.033) × 10-6 M) and rGCN4
((0.2980.030) × 10-6 M) are
approximately a factor 10 higher then those described in literature ((9 ± 6) × 10-8 M for
GCN4 and (2.9 ± 0.8) × 10-8 M for rGCN4) (Hollenbeck *et al.*, 2001). The differences could be explained by the lower sensitivity of SYBR-Safe staining compared to radio-labeled oligos. Most likely, the protein concentration was miscalculated due to the presence of additional (lower intensity) bands in the SDS-PAGE analysis, indicating the co-purification of small amounts of unspecific proteins.

The FLAG-tag fusion to the N-terminus of proteins could potentially decrease binding affinity, likely due to steric hindrance affecting the interaction with DNA. Interestingly, the differences in binding affinity between GCN4 and rGCN4 appear negligible. Since GCN4 binds to DNA via its N-terminus and rGCN4 binds C-terminally, the FLAG-tag likely does not directly influence DNA binding. However, it may influence the dimerization of the proteins, which is necessary for DNA binding. To further investigate this, the FLAG-tag can be cleaved using an enterokinase and potential changes in binding affinity could be analyzed.

#### Outlook

Future work will focus on quantifying FRET interaction *in vivo*  to assess stapling efficiency and DNA-DNA proximity more accurately. Additionally, the development of mini staples can be further improved by testing alternative linkers predicted by [our dry lab simulations.](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) By systematically assessing different linker sequences, we aim to create functional staples and furthermore lay the foundation for future engineering of small bZip DNA binding domains.
Finally, we are also currently testing out our measurement system with the more complex Cas staples.

## Delivery System

As part of the PICasSO toolbox, we propose **bacterial conjugation** as a **cheap, simple and scalable alternative** to conventional **DNA delivery methods**, particularly suited for large plasmid constructs **encoding protein-based staples for controlled modulation of mammalian 3D genome organization**.
Bacterial conjugation is one of the key mechanisms for **horizontal gene transfer** between bacteria on solid media. We validated the proficiency of the **RP4 conjugative machinery** in mediating conjugation between bacteria on solid media. Future experiments will expand the **DNA delivery capabilites** of the **Type IV secretion system** to mammalian cells by employing **synthetic adhesins** to enhance **cell-cell contact.**

### Introduction

### Background Information

The bacterial T4SS is implicated in conjugation, a conserved mechanism of horizontal gene transfer among gram-negative (De La Cruz *et al.*, 2010) and gram-positive (Grohmann *et al.*, 2003) bacteria. In nature, conjugation is a major driver of bacterial genome evolution in several niches ranging from soil and water to biofilms in animal hosts, enabling the utilization of new metabolites, conferring pathogenic properties, and even resistance to heavy metals and antibiotics, under selection pressure (Virolle *et al.*, 2020).

The mechanism of conjugation and the myriad of factors required are well-characterized, although several questions remain open and the function of some of the structures still remains elusive. For instance, the fundamental question of what triggers conjugation still remains poorly understood as we learnt from [Dr. Christian Lesterlin](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D), an expert in the field.

Moreover, different conjugative systems have been described in gram-negative bacteria (IncF, IncW, IncP, IncN, etc.) all of which are functionally similar. In all these systems, following the establishment of pilus-mediated contact between bacteria (mating pair formation), a mobilizable plasmid is processed in the donor bacterium and a single strand of the plasmid is covalently attached to a protein (relaxase). This DNA-protein complex is then transferred through T4SS into the recipient bacterium, where it is re-circularized and maintained. One of the key elements in this process is the origin of transfer (*oriT*) sequence in the mobilizable plasmid, which is recognized and nicked by the relaxase to initiate the transfer.

Dr. Christian Lesterlin recommended us to work with the RP4 system (IncP) encoding short and rigid pili as it is a well-characterized and established broad host-range conjugative system. As a consequence of the short and rigid nature of pili encoded by the RP4 system, conjugation rates tend to be two to four orders of magnitude higher in solid media than in liquid media (Robledo *et al.*, 2022), where shear forces have a destabilizing effect on the mating pair.

Notably, all the events leading to DNA transfer by conjugation are driven by the donor bacterium and its protein components are typically plasmid encoded. Thus, it is technically possible for any type of cell to serve as the recipient (Waters, 2001). In line with this claim, there have been some studies that report on conjugational DNA transfer not only between different bacterial species (Hamilton *et al.*, 2019) but also from bacteria to members of other kingdoms – *Agrobacterium tumefaciens* that is able to deliver DNA to plants (Gelvin, 2003) being a well-known example. Besides DNA transfer to plants by *A. tumefaciens*, other bacteria like *Bartonella henselae* have been shown to deliver DNA to human cells (Schröder *et al.*, 2011) via their T4SS during infection, albeit with very low efficiency.

Fascinated by this phenomenon, we wondered whether conjugation can be engineered as a generalized DNA delivery tool. Indeed, a few studies have been successful in generating bacteria capable of delivering DNA to yeast using their T4SS (Soltysiak *et al.*, 2019) but there is a lack of research done on engineering bacteria to conjugate with mammalian cells. Taking inspiration from the work of VL Waters (2001), who showed that it is possible to deliver DNA to mammalian cells by conjugation using the RP4 machinery, we contemplated ways to rationally engineer this system to enable higher efficiency and selectivity.

Since the primary trigger for conjugation remains obscure, we hypothesized that cell-cell contact might be one of the main determinants for conjugation to occur efficiently. In this line, Robledo *et al.*(2022) showed that enhanced cell-cell contact mediated by synthetic adhesins led to an increase in conjugation efficiency between bacteria. Specifically, a 100-fold increase in conjugation efficiency was observed in liquid matings between bacteria carrying the RP4 conjugation system.

This combination of knowledge motivated us to test inter-kingdom conjugation between bacteria and mammalian cells using synthetic adhesins against EGFR (a common mammalian surface receptor) to potentially increase the probability of DNA transfer. We received valuable feedback from Dr. Robledo herself, who said that if cell-cell contact were to be the limiting factor, we are likely to succeed with our approach.

### **What are the benefits of our system?**

By establishing our conjugation-based DNA delivery system, we seek to contribute to the available array of DNA delivery methods with an innovative alternative. The advantages of using conjugation over commonly used gene delivery methods include:

* The ability to deliver large plasmids (~ 100 kb in size): It is known that lipofection efficiency decreases with increasing plasmid size (Kreiss *et al.*, 1999), so conjugation can be employed as an alternative tool to deliver large plasmids to mammalian cells *in vitro*. Furthermore, our system circumvents the limited coding capacity of AAVs which can only be used to deliver DNA up to ~ 5 kb (Wang *et al.*, 2019).
* Low cost and easy scalability: propagating bacteria transformed with the conjugative helper plasmid and the mobilizable plasmid is easy to scale up and is considerably cheaper than transfection reagents available on the market.
* Tunable specificity: The synthetic adhesin module can be modified to target bacteria to specific cell types and therefore allows targeted DNA delivery in a heterogeneous cellular environment, such as in 3D *in vitro* cancer models.

#### Aim of This Subproject

We sought to explore the potential of using the bacterial Type IV secretion system (T4SS) as a novel platform to deliver genetic payloads to mammalian cells. Intrigued by the work of VL Waters (2001), who showed conjugation-mediated DNA transfer between bacteria and mammalian cells, we brainstormed ways to engineer conjugation as a generalized DNA delivery tool. Recognizing the importance of cell-cell contact as an important parameter for efficienct conjugation between bacteria (Robledo et al., 2022), we were curious to test the effects of the same in promoting conjugation between bacteria and mammalian cells. To this end, we planned to incorporate adhesins into our conjugation system that increase conjugation efficiency and direct plasmid transfer to specific cell types.

### Results

### **RP4 Machinery Is Effective in Mediating Conjugation on Solid Media**

Bacterial conjugation can be mediated by plasmids in either a *cis* or *trans* configuration. The *cis* configuration involves a single plasmid that encodes for the conjugation machinery as well as carries the *oriT* sequence. Very high conjugation efficiencies can be achieved with this system (frequencies in the order of 10-2 in 24 hours (Hamilton *et al.*, 2019)) as every successfully conjugated recipient automatically becomes a donor. The *trans* configuration, on the other hand, is a dual-plasmid-system where the conjugation machinery and the *oriT* sequence are present on separate plasmids and thus, only the plasmid carrying the *oriT* is mobilized between bacteria. As can be imagined, the conjugation efficiencies are modest while using the *trans* configuration (frequencies in the order of 10-5 in 24 hours (Hamilton *et al.*, 2019)). Nevertheless, we decided to use the *trans* set up since we were not interested in delivering the entire T4SS arsenal to our future recipients - mammalian cells. As a second benefit of this strategy, recipient cells do not become conjugation competent themselves, increasing the safety of our system. Therefore, we aimed to assess the functionality of the RP4 system in the *trans* configuration.

Our dual-plasmid system consists of pHelper\_RP4 (RP4 conjugation helper plasmid) and pmob\_b (mobilizable plasmid carrying the *oriT* sequence). For information on how we cloned these plasmids, please refer to the [experiments page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D). We chose to use pTA-Mob 2.0 as the backbone for pHelper\_RP4 not only because it encodes the RP4 conjugation machinery, but also since it has been shown to be successful in mediating inter-kingdom conjugation between bacterial and yeast cells (Soltysiak *et al.*, 2019). Finally, as the RP4 system encodes short and rigid pili, the conjugation efficiency is considerably lower in liquid media than in solid media (Robledo *et al.*, 2022) and hence our decision to test the proficiency of our system in both solid and liquid media.

The donors in our test group carried two plasmids (pHelper\_RP4 and pmob\_b) and all recipients carried a plasmid conferring resistance to ampicillin (to allow for selection against donor bacteria). Origin of replication (ori) compatibilities between the different plasmids used was ensured.

In addition, prior to mixing the experimental donor and recipient groups (presented in Table 1), the bacterial suspensions were either brought to an OD600 of 1.2 or 10 to test the effect of cell density on conjugation efficiency.

**Table 1: Donors and recipients in the different controls and in the test group used for the bacteria-bacteria conjugation assay.**

|  |  | Donor |  | Recipient |
| Negative control 1 |  | *E.coli* 10-beta carrying the RP4 helper plasmid |  | *E.coli* BL21(DE3) |
| Negative control 2 |  | *E.coli* 10-beta carrying the mobilizable plasmid |  | *E.coli* BL21(DE3) |
| Positive control |  | - |  | *E.coli* BL21(DE3) carrying the mobilizable plasmid |
| Test group |  | *E.coli* 10-beta carrying both the RP4 helper and the mobilizable plasmid |  | *E.coli* BL21(DE3) |

We observed efficient conjugation in all test groups (calculated as transconjugants per recipient), with conjugation efficiencies differing depending on the OD600 of the donors and recipients used at the start of conjugation. Notably, a 1000-fold increase in conjugation efficiency was observed between the test groups upon increasing the OD600 from 1.2 to 10 (see **Fig. 24**). This provides a clear indication that cell density and therefore, enhanced cell-cell contact is an important parameter for conjugation on solid media. Interestingly, at an OD600 of 10, we also observe some natural transformation happening in case of the negative control 2 samples, which could be a consequence of high cell density resulting in increased chances of natural transformation. In liquid media however, we could not show any conjugation happening, which confirms the inefficiency of the wild-type RP4 system to mediate conjugation in liquid media. Moreover, the positive controls that were chemically transformed grew on selective agar plates, suggesting that the apparent lack of conjugation in liquid media is not due to the experimental conditions but rather the inefficiency of the conjugative system itself, under liquid conditions.

![](https://static.igem.wiki/teams/5237/wetlab-results/conjugation-plot2.svg)

**Figure 24: Efficiency of conjugation on solid media.** Bar charts depicting the conjugation efficiency (reported as transconjugants/recipient) of the various experimental groups. Three technical replicates were used for the OD600 10 test group.

### **Characterization of Conjugation Enhancing Nanobodies**

The general workflow for a coIP experiment involves using antibodies to pull down the target protein (bait) from a crude or pre-cleared cell lysate. The target protein tends to form complexes with its interaction partners (prey) which now gets pulled down along with the bait protein. Following immunoprecipitation, the protein complexes bound to the beads can be analyzed by resolving them using SDS-PAGE and subsequently detecting them via Western Blot.

In our case, we plan to use coIP to prove interaction between the [anti-EGFR adhesin](https://parts.igem.org/Part%3ABBa_K5237015) and EGFR *in vitro*. This will be achieved by checking for co-immunoprecipitation of GFP-tagged EGFR along with myc-tagged anti-EGFR adhesins following a myc pulldown. **Figure 25** provides a graphic illustration of the concept of this assay.

![](https://static.igem.wiki/teams/5237/figures-corrected/conjugation-coip.svg)

**Figure 25. Co-immunoprecipitation assay.** Anti-myc beads bind to the myc-tagged adhesin, which carries a nanobody against EGFR (wild-type 7D12). The nanobody mediates high affinity binding of the adhesin to EGFR which is fused to GFP. After the myc pull down, the beads are washed and the proteins are eluted from the beads. The eluted proteins are then analysed by Fluorescent Western Blot analysis against both myc and GFP.

The coIP itself is yet to be executed but preliminary experiments have already been performed to validate proper expression of EGFR-GFP by HEK293T cells and the adhesins (without nanobodies) by *E.coli* 10-beta. In addition, since the proteins of interest are rather large (~180 kDa for EGFR-GFP and ~76 kDa for adhesin without nanobody), we also tested two different sample preparation conditions (boiling for 5 mins at 95 ℃ and heating for 30 mins at 37 ℃) and blotting conditions (15V, 30 mins at 4 ℃ and 15V, 1 hour at 4 ℃). The results revealed successful EGFR-GFP expression after transfection in HEK293T cells and adhesin (without nanobody) expression after induction in *E.coli* 10-beta (see **Fig. 26**). Moving forward, these results affirm expression of full-length intimin by *E.coli* 10-beta, allowing for their confident utilization in upcoming conjugation assays.

![](https://static.igem.wiki/teams/5237/wetlab-results/pre-test-for-coip.png)

**Figure 26: Fluorescent Western Blot scans after staining against GFP (green) and myc (red) and subject to different sample preparation and blotting conditions.** Contents of samples 1,2,3 and 4 are presented in the text under the figure. The proteins of interest are indicated using arrows: white arrows point at the location of EGFR-GFP (~180 kDa) and blue arrows point at the location of myc-tagged adhesin without nanobody (~76 kDa).

For the planned coIP assay, HEK293T cells will be transfected with a plasmid encoding EGFR-GFP and lysed. *E.coli* lysates will be generated after transformation and induction of protein expression. Please refer to the [experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3Dexperiments%29%20%7D%7D) page for a more detailed description of the procedures. The experimental design is planned to include pulldowns of various lysate combinations using anti-myc beads:

* Pulldown of HEK293T cell lysate that would reveal any cross-reaction of the anti-myc beads with proteins in the HEK293T cell lysate;
* Pulldown of *E.coli* lysates (both adhesins without and with nanobody) to validate the beads and also to test proper expression of full-length adhesins by *E.coli* 10-beta;
* coIP of HEK293T and *E.coli* lysate expressing adhesins without nanobody to test for any interaction between the bait and prey proteins that is not mediated by the nanobody;
* coIP of HEK293T and *E.coli* lysate expressing adhesins with the nanobody to prove specific interaction between the bait and prey proteins.

### **Adhesion Assay to Visualize Bacterial Accumulation on the Mammalian Cell Surface**

To showcase the improved adhesion of conjugative bacteria expressing anti-EGFR adhesins to the surface of mammalian cells, we aimed to compare the accumulation of anti-EGFR adhesin-expressing bacteria on the surface of HEK293T and HeLa cells against bacteria expressing adhesins without nanobodies.

We chose to conduct a fluorescence microscopy-based assay to test whether adhesins expressed on bacteria enable better binding to the surface of mammalian cells. Following IPTG induction of adhesin expression, *E.coli* 10-beta were labeled by growing them in LB-media supplemented with fluorescently labeled D-amino acids (RADA). In preliminary tests we confirmed this to result in the incorporation of fluorescent D-amino acids into the bacterial cell wall during synthesis, thereby labeling the peptidoglycan layer. However, the adhesion assay itself could not be performed due to time constraints.

In the future, we aim to confirm adhesin-dependent binding of fluorescently labeled bacteria by adding them to HEK293T or HeLa cells grown on coverslips, followed by incubation, washing and fixation for microscopy. Following fixation and subsequent staining with Hoechst and WGA-Alexa Fluor 488, the coverslips will be mounted on glass slides to be visualized under a fluorescence microscope. A more thorough description of the assay can be found in the [experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3Dexperiments%29%20%7D%7D) page. The experimental design is planned to include the following bacteria:

* *E.coli* 10-beta transformed with pHelper\_RP4: to assess natural bacterial adhesion to mammalian cell surfaces;
* *E.coli* 10-beta transformed with pHelper\_RP4 and pNeae2: no nanobody control;
* *E.coli* 10-beta transformed with pHelper\_RP4 and pNeae2\_7D12: test group.

Through this assay, we hope to prove correct extracellular display of nanobodies by conjugative bacteria and also gain qualitative insights into the enhanced affinity of nanobody-expressing bacteria to the mammalian cell surface.

### **Bacteria-Mammalian Conjugation to Probe Inter-Kindgdom Conjugational DNA Transfer**

The goal of the bacteria-mammalian conjugation assay is to explore the avenue of inter-kingdom conjugation and potential improvements in conjugation efficiency due to enhanced cell-cell contact. This concept can be seen on **figure 27**.

![](https://static.igem.wiki/teams/5237/figures-corrected/conjugation-mammalian-bacteria.svg)

**Figure 27: Bacteria-mammalian conjugation mediated by synthetic adhesins.** DNA delivery to mammalian cells via the T4SS by adhesin-expressing bacteria docked on the mammalian cell surface.

We aim to perform the bacteria-mammalian conjugation assay using a custom protocol that we designed. It involves seeding HEK293T cells in T25 flasks and growing them for 24 hours prior to the experiment. Then, conjugation-competent bacteria carrying a mobilizable plasmid encoding EGFP under a SV40 promoter (for constitutive expression in mammalian cells) will be added to the flasks containing adherent HEK293T cells. Conjugation will be allowed to proceed for 12 hours in media supplemented with DNaseI (prevents natural transformation) and cytochalasin D (prevents endocytosis-mediated bacterial uptake) as done by Waters, V (2001). 12 hours later, the media will be removed and the mammalian cells will be washed to remove most of the attached bacteria. The remaining bacteria shall be neutralized by adding fresh media supplemented with appropriate antibiotics to the flasks. After checking successful elimination of live bacteria, the HEK293T cells will be placed back into the humid CO2 incubator. 24 hours later, the HEK293T cells will be imaged under a fluorescence microscope to visualize reporter protein expression. FACS can be performed for a more quantitative read-out. For a more detailed description of the assay, please refer to the [experiments page](%7B%7B%20url_for%28%27pages%27%2C%20page%3Dexperiments%29%20%7D%7D).

Several controls will be included in the experimental design to allow proper interpretation of results. In all cases, bacteria transformed with different plasmid combinations shall be added to the HEK293T cells. Following are the bacteria that have been planned to be utilized in the bacteria-mammalian conjugation assay:

* No adhesin negative controls:  *E.coli* 10-beta expressing either:
  + pHelper\_RP4 or
  + pmob\_m\_CMV
* Adhesin expressing negative controls:  *E.coli* 10-beta expressing either:
  + pHelper\_RP4 + pNeae\_7D12 or
  + pmob\_m\_CMV + pNeae\_7D12
* Conjugation test groups:  *E.coli* 10-beta expressing:
  + pHelper\_RP4 and pmob\_m\_CMV (no adhesin test group)
  + pHelper\_RP4 and pmob\_m\_CMV + pNeae\_7D12 (test group with adhesins)
* Positive control for comparison against other groups:
  + Transfection with pmob\_m\_CMV.

We are yet to conduct the experiment and obtain data. Nevertheless all the necessary materials are in place and the assay shall be duly executed.

### Discussion

We started by validating the functionality of the RP4 helper plasmid in our dual-plasmid system as well as of the *oriT* sequence in the mobilizable plasmid by performing conjugation assays between bacteria on both solid and liquid media. We observed that the conjugation efficiency achieved using our experimental design was comparable to published literature (Hamilton *et al.*, 2019), thereby validating our RP4 helper plasmid and its ability to catalyze transfer of the *oriT* carrying mobilizable plasmid. Moreover, we noted a 1000-fold increase in conjugation efficiency between the test groups upon increasing the OD600 from 1.2 to 10. This provided a clear indication that cell density and therefore, enhanced cell-cell contact is an important parameter for conjugation on solid media. Also, almost no conjugation could be observed when perfomed in liquid media, pointing at the inefficiency of the RP4 counjugative machinery to mediate conjugation in destabilizing conditions. Thus, we laid the foundation for our future conjugation experiments between bacteria and mammalian cells.

Western Blot analysis confirmed expression of myc-tagged adhesins (without nanobodies) by *E.coli* 10-beta. This knowledge allows for their utilization as the donor strain in upcoming conjugation assays in spite of not being an optimal strain for protein expression. We also established a coIP workflow to prove specific interaction between the nanobody and its target protein *in vitro*, which can be easily adapted to other nanobodies and their respective target proteins. Validation of nanobody-target protein interaction is vital to interpret results from the adhesion assay designed to examine the enrichment of nanobody-expressing bacteria on the surface of mammalian cells. The results from the adhesion assay will be used to verify adhesin display in the extracellular space and also give us a qualitative understanding of whether the presence of adhesins result in an increased affinity between the conjugative bacteria and mammalian cells. This knowledge will also help us to complement and support the results from the coIP experiment.

Finally, through the bacteria-mammalian conjugation assay, we hope to delineate the role of cell-cell contact in mediating inter-kingdom conjugation between bacteria and mammalian cells. In the future, we also aim to test our system on different cell lines to examine their susceptibilities to receiving DNA from bacteria via the T4SS. We also seek to further tune the specificity of our delivery system using cell type-specific promoters, the constructs for which have already been cloned.

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