iGEM 2019 — Team Lethbridge_HS

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# Team:Lethbridge HS/Notebook

[![](https://static.igem.org/mediawiki/2019/7/75/T--Lethbridge_HS--Mango.PNG)](https://2019.igem.org/Team%3ALethbridge_HS)

* [HOME](https://2019.igem.org/Team%3ALethbridge_HS)
* PROJECT

  [Description](https://2019.igem.org/Team%3ALethbridge_HS/Description)
  [Design](https://2019.igem.org/Team%3ALethbridge_HS/Design)
  [Results](https://2019.igem.org/Team%3ALethbridge_HS/Results)
  [Demonstrate](https://2019.igem.org/Team%3ALethbridge_HS/Demonstrate)
  [Measurement](https://2019.igem.org/Team%3ALethbridge_HS/Measurement)
* PARTS

  [Parts Overview](https://2019.igem.org/Team%3ALethbridge_HS/Parts)
  [Basic Parts](https://2019.igem.org/Team%3ALethbridge_HS/Basic_Part)
  [Composite Parts](https://2019.igem.org/Team%3ALethbridge_HS/Composite_Part)
  [Part Collection](https://2019.igem.org/Team%3ALethbridge_HS/Part_Collection)
  [Improve](https://2019.igem.org/Team%3ALethbridge_HS/Improve)
* LAB

  [Experiments](https://2019.igem.org/Team%3ALethbridge_HS/Experiments)
  [Notebook](https://2019.igem.org/Team%3ALethbridge_HS/Notebook)

  * HUMAN PRACTICES

    [Human Practices](https://2019.igem.org/Team%3ALethbridge_HS/Human_Practices)
    [Engagement](https://2019.igem.org/Team%3ALethbridge_HS/Public_Engagement)
  * TEAM

    [Members](https://2019.igem.org/Team%3ALethbridge_HS/Team)
    [Collaboration](https://2019.igem.org/Team%3ALethbridge_HS/Collaborations)
    [Attributions](https://2019.igem.org/Team%3ALethbridge_HS/Attributions)
    [Sponsors](https://2019.igem.org/Team%3ALethbridge_HS/Sponsors)
  * [HARDWARE](https://2019.igem.org/Team%3ALethbridge_HS/Hardware)
  * [SAFETY](https://2019.igem.org/Team%3ALethbridge_HS/Safety)

NOTEBOOK

---

#


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Lethbridge HS/Experiments

[![](https://static.igem.org/mediawiki/2019/7/75/T--Lethbridge_HS--Mango.PNG)](https://2019.igem.org/Team%3ALethbridge_HS)

* [HOME](https://2019.igem.org/Team%3ALethbridge_HS)
* PROJECT

  [Description](https://2019.igem.org/Team%3ALethbridge_HS/Description)
  [Design](https://2019.igem.org/Team%3ALethbridge_HS/Design)
  [Results](https://2019.igem.org/Team%3ALethbridge_HS/Results)
  [Demonstrate](https://2019.igem.org/Team%3ALethbridge_HS/Demonstrate)
  [Measurement](https://2019.igem.org/Team%3ALethbridge_HS/Measurement)
* PARTS

  [Parts Overview](https://2019.igem.org/Team%3ALethbridge_HS/Parts)
  [Basic Parts](https://2019.igem.org/Team%3ALethbridge_HS/Basic_Part)
  [Composite Parts](https://2019.igem.org/Team%3ALethbridge_HS/Composite_Part)
  [Part Collection](https://2019.igem.org/Team%3ALethbridge_HS/Part_Collection)
  [Improve](https://2019.igem.org/Team%3ALethbridge_HS/Improve)
* LAB

  [Experiments](https://2019.igem.org/Team%3ALethbridge_HS/Experiments)
  [Notebook](https://2019.igem.org/Team%3ALethbridge_HS/Notebook)

  * HUMAN PRACTICES

    [Human Practices](https://2019.igem.org/Team%3ALethbridge_HS/Human_Practices)
    [Engagement](https://2019.igem.org/Team%3ALethbridge_HS/Public_Engagement)
  * TEAM

    [Members](https://2019.igem.org/Team%3ALethbridge_HS/Team)
    [Collaboration](https://2019.igem.org/Team%3ALethbridge_HS/Collaborations)
    [Attributions](https://2019.igem.org/Team%3ALethbridge_HS/Attributions)
    [Sponsors](https://2019.igem.org/Team%3ALethbridge_HS/Sponsors)
  * [HARDWARE](https://2019.igem.org/Team%3ALethbridge_HS/Hardware)
  * [SAFETY](https://2019.igem.org/Team%3ALethbridge_HS/Safety)

EXPERIMENTS

---

# **PROTOCOLS**

|  |  |
| --- | --- |
| [Colony PCR](https://static.igem.org/mediawiki/2019/4/4f/T--Lethbridge_HS--colonypcr.pdf) | [DNA Purification from Enzymatic Reactions](https://static.igem.org/mediawiki/2019/4/44/T--Lethbridge_HS--dnapurification.pdf) |
| [LB Media](https://static.igem.org/mediawiki/2019/9/9f/T--Lethbridge_HS--LB_media.pdf) | [PCR Amplification](https://static.igem.org/mediawiki/2019/7/70/T--Lethbridge_HS--pcramplification.pdf) |
| [PCR Product Purification](https://static.igem.org/mediawiki/2019/a/a2/T--Lethbridge_HS--pcrpurification.pdf) | [Plasmid Miniprep](https://static.igem.org/mediawiki/2019/6/6f/T--Lethbridge_HS--miniprep.pdf) |
| [Chemically Competent Cells](https://static.igem.org/mediawiki/2019/d/d2/T--Lethbridge_HS--chemcompetentcells.pdf) | [Protein Overexpression](https://static.igem.org/mediawiki/2019/7/79/T--Lethbridge_HS--protein_overexpression.pdf) |
| [Restriction Digest](https://static.igem.org/mediawiki/2019/c/c5/T--Lethbridge_HS--restriction_digest.pdf) | [Transformation](https://static.igem.org/mediawiki/2019/3/3e/T--Lethbridge_HS--transformation.pdf) |
| [Ethanol Precipitation](https://static.igem.org/mediawiki/2019/5/5f/T--Lethbridge_HS--ethanolprecipitation.pdf) | [RNase Assay](https://static.igem.org/mediawiki/2019/3/3c/T--Lethbridge_HS--RNase_assay.pdf) |
| [RNase Inhibitor Test](https://static.igem.org/mediawiki/2019/a/ae/T--Lethbridge_HS--RNase_inhibitor.pdf) | [His-tag Purification of Cas13a](https://static.igem.org/mediawiki/2019/b/b4/T--Lethbridge_HS--His-tag_purification.pdf) |
| [Size Exclusion Chromatography Purification of Cas13a](https://static.igem.org/mediawiki/2019/1/17/T--Lethbridge_HS--SEC_purification.pdf) | [Cas13a Activity Assay](https://static.igem.org/mediawiki/2019/d/d4/T--Lethbridge_HS--Cas13a_activity%2Cpdf.pdf) |
| [In vivo Fluorescence Assay](https://static.igem.org/mediawiki/2019/4/44/T--Lethbridge_HS--invivo_fluorescence.pdf) | [RNA Mango Purification](https://static.igem.org/mediawiki/2019/2/22/T--Lethbridge_HS--Mango_purification.pdf) |
| [In vitro transcription](https://static.igem.org/mediawiki/2019/6/6e/T--Lethbridge_HS--invitrotranscription.pdf) | [Flourescent assay](https://static.igem.org/mediawiki/2019/6/61/T--Lethbridge_HS--Flourescent_assay.pdf) |

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Lethbridge HS/Results

[![](https://static.igem.org/mediawiki/2019/7/75/T--Lethbridge_HS--Mango.PNG)](https://2019.igem.org/Team%3ALethbridge_HS)

* [HOME](https://2019.igem.org/Team%3ALethbridge_HS)
* PROJECT

  [Description](https://2019.igem.org/Team%3ALethbridge_HS/Description)
  [Design](https://2019.igem.org/Team%3ALethbridge_HS/Design)
  [Results](https://2019.igem.org/Team%3ALethbridge_HS/Results)
  [Demonstrate](https://2019.igem.org/Team%3ALethbridge_HS/Demonstrate)
  [Measurement](https://2019.igem.org/Team%3ALethbridge_HS/Measurement)
* PARTS

  [Parts Overview](https://2019.igem.org/Team%3ALethbridge_HS/Parts)
  [Basic Parts](https://2019.igem.org/Team%3ALethbridge_HS/Basic_Part)
  [Composite Parts](https://2019.igem.org/Team%3ALethbridge_HS/Composite_Part)
  [Part Collection](https://2019.igem.org/Team%3ALethbridge_HS/Part_Collection)
  [Improve](https://2019.igem.org/Team%3ALethbridge_HS/Improve)
* LAB

  [Experiments](https://2019.igem.org/Team%3ALethbridge_HS/Experiments)
  [Notebook](https://2019.igem.org/Team%3ALethbridge_HS/Notebook)

  * HUMAN PRACTICES

    [Human Practices](https://2019.igem.org/Team%3ALethbridge_HS/Human_Practices)
    [Engagement](https://2019.igem.org/Team%3ALethbridge_HS/Public_Engagement)
  * TEAM

    [Members](https://2019.igem.org/Team%3ALethbridge_HS/Team)
    [Collaboration](https://2019.igem.org/Team%3ALethbridge_HS/Collaborations)
    [Attributions](https://2019.igem.org/Team%3ALethbridge_HS/Attributions)
    [Sponsors](https://2019.igem.org/Team%3ALethbridge_HS/Sponsors)
  * [HARDWARE](https://2019.igem.org/Team%3ALethbridge_HS/Hardware)
  * [SAFETY](https://2019.igem.org/Team%3ALethbridge_HS/Safety)

RESULTS

---

# Results Overview

This year, our team was able to work on both our [diagnostic and therapeutic systems.](https://2019.igem.org/Team%3ALethbridge_HS/Design) We were able to successfully test overexpress all four of our Cas13a proteins, purify two of them, and work experimentally with one. For our crRNAs targeting RNA Mango II, we successfully PCR amplified all four crRNA designs,  *in vitro*  transcribed two, and work experimentally with one. We also purified our snR30-RNA Mango II construct and, to our knowledge, are the first iGEM to work with this RNA aptamer experimentally. We were also able to add characterization data to existing GFP and RFP parts in the registry and successfully performed a dual plasmid transformation with our fluorescent proteins and all of our crRNA designs that target GFP to conduct a control experiment for our therapeutic system. Please see our [Parts pages](https://2019.igem.org/Team%3ALethbridge_HS/Parts) for more information on the design of them.

## **Diagnostic Components**

### CRISPR RNA Production

CRISPR RNA (crRNA) is a crucial aspect of the CRISPR Cas13a system. It seeks out the target RNA sequence so that the Cas13a can cleave the RNA. Our crRNA sequences were synthesized by IDT into the plasmid pUC19. Then we successfully PCR amplified the DNA out of the pUC19 plasmid. The amplified DNA for our Lwa, Lbu, Lsh, and Lba crRNA can be seen in Figures 1-3.

Due to time constraints, our team chose to complete a large scale *in vitro* transcription using only the Lwa and Lbu crRNAs (Figure 4). This allowed our team to use the crRNA with our other project components in later experiments.

![Snow](https://static.igem.org/mediawiki/2019/c/c9/T--Lethbridge_HS--pcr_crrna_lba_lbu.png)

**Figure 1.** 10% DNA PAGE of crRNA products for Lba and Lbu. Left to right: lane 1: empty; lane 2: 100 bp ladder (Thermo Scientific); lane 3: Lbu 57.1; lane 4: Lbu 56; lane 5: Lbu 55; lane 6: Lbu 54; lane 7: Lba 57.1; lane 8: Lba 56; lane 9: Lba 55; lane 10: Lba 54.

![Forest](https://static.igem.org/mediawiki/2019/b/b6/T--Lethbridge_HS--pcr_crrna_lwa_lsh.png)

**Figure 2.** 10% DNA PAGE of crRNA products for Lsh and Lwa. Left to right: lane 1: 100 bp ladder (Thermo Scientific. Lane 2: Lwa 56.5; Lane 3: Lwa 56.8; lane 4: Lsh 56.5; lane 5: Lsh 56.8. Note that all lanes are from the same gel.

![Snow](https://static.igem.org/mediawiki/2019/1/1c/T--Lethbridge_HS--pcr_upscale_lbu_lwa.png)

**Figure 3.** 10% DNA PAGE of Lbu and Lwa PCR products. Left to right: lanes 1-6: Lbu; lanes 7-12: Lwa; lane 13: 100 bp ladder; lanes 14-15: empty.

![Forest](https://static.igem.org/mediawiki/2019/1/16/T--Lethbridge_HS--urea_ivt_lbu_lwa.png)

**Figure 4.** Urea PAGE of *in vitro* transcribed Lbu and Lwa crRNAs. Left to right: lane 1: High Range RiboRuler; lane 2: Lwa after DNase; lane 3: Lwa after *in vitro* transcription; lane 4: Lwa before *in vitro* transcription; lane 5: Lbu after DNase; lane 6: Lbu after *in vitro* transcription; lane 7: Lbu before *in vitro* transcription. Note that all lanes are from the same gel.

### RNA Mango Purification

To characterize the parts for our diagnostic tool, we chose to work with the RNA aptamer, RNA Mango II. For further discussion on our choice of this part, please see our  [here](https://2019.igem.org/Team%3ALethbridge_HS/Design%E2%80%9D%3Edesign%20page%3C/a%3E.%20Our%20team%20received%20an%20RNA%20Mango%20sample%20from%20the%20Kothe%20Lab%20at%20the%20University%20of%20Lethbridge.%20First%2C%20we%20PCR%20amplified%20the%20DNA%20%28Figure%205%29.%20Then%20our%20team%20successfully%20completed%20%3Ci%3Ein%20vitro%3C/i%3E%20transcription%20to%20convert%20the%20DNA%20sample%20into%20RNA%20%28Figure%206%29.%20The%20Kothe%20Lab%20frequently%20works%20with%20run-off%20transcription%20so%20the%20RNA%20Mango%20sequence%20had%20no%20terminator%2C%20which%20increased%20the%20likelihood%20of%20other%20products%20in%20the%20sample.%20Therefore%2C%20our%20team%20chose%20to%20complete%20RNA%20purification%20using%20the%20method%20from%20the%20Panchapakesan%20%3Ci%3Eet%20al.%2C%3C/i%3E%202017%20paper%20%28click%20%3Ca%20href%3D)  ).This method uses streptavidin which binds very strongly to biotinylated thiazole orange. The resulting elutions have RNA that interacts with the biotinylated thiazole orange. Only RNA Mango should interact with the biotinylated thiazole orange, resulting in our sample being pure (Figure 7).

![Snow](https://static.igem.org/mediawiki/2019/2/26/T--Lethbridge_HS--pcr_rna_mango.png)

**Figure 5.** 1% agarose gel of PCR products of amplified RNA Mango. Left to right: lane 1: empty; lane 2: 1 kb ladder; lane 3: RNA Mango PCR product; lane 4: RNA Mango PCR product.

![Forest](https://static.igem.org/mediawiki/2019/f/fb/T--Lethbridge_HS--rna_mango_ivt.png)

**Figure 6.** 8% Urea PAGE of  *in vitro*  transcribed RNA Mango. Left to right: lane 1:; lane 2:; lane 3:; lane 4:; lane 5:; lane 6: *in vitro*  transcribed RNA Mango; lane 7: RNA Mango after DNase; lane 8: size standard from Kothe lab.

 ![](https://static.igem.org/mediawiki/2019/6/68/T--Lethbridge_HS--rna_mango_purif.png)

**Figure 7.** 8% Urea PAGE of streptavidin and biotin purified RNA Mango. Left to right: lane 1:  *in vitro*  transcribed RNA Mango; lane 2: wash 1; lane 3: wash 3; lane 4: elution 1; lane 5: elution 3; lane 6: elution 5; lane 7: elution 8; lane 8: elution 11; lane 9: elution 12; lane 10: elution 13.

.

### RNA Mango RNase Assay

After receiving a question regarding contamination and false positives, our team conducted an RNA Mango RNase Assay. Common RNases in the environment introduce the risk of false positives so we incubated RNase A or saliva with varying concentrations of RNA Mango. Regardless of the concentrations of RNase used, all RNA Mango was cleaved (Figure 8). Therefore, our system has a high likelihood of contamination. In an attempt to combat this issue, our team conducted an RNA Mango inhibitor assay.

 ![](https://static.igem.org/mediawiki/2019/5/55/T--Lethbridge_HS--rnase_assay_combined.png)

**Figure 8.** 10% Urea PAGEs of RNA Mango and varying concentrations of RNase A incubated for increasing lengths of time at 37 ℃. **(A)** Left to right: lane 1: 2.5 μg/mL RNase A, 90 min incubation; lane 2: 2.5 μg/mL RNase A, 60 min incubation; lane 3: 2.5 μg/mL RNase A, 30 min incubation; lane 4: 2.5 μg/mL RNase A, 0 min incubation; lane 5: 1 μg/mL RNase A, 90 min incubation; lane 6: 1 μg/mL RNase A, 60 min incubation; lane 7: 1 μg/mL RNase A, 30 min incubation; lane 8: 1 μg/mL RNase A, min incubation; lane 9: 0 μg/mL RNase A, 90 min incubation; lane 10: 0 μg/mL RNase A, 60 min incubation; lane 11: 0 μg/mL, 30 min incubation; lane 12: 0 μg/mL RNase A, 0 min incubation; lane 13: water control; lane 14: negative control; lane 15: positive control. **(B)**  Left to right: lanes 1-3: empty; lane 4: 10 μg/mL RNase A, 90 min incubation; lane 5: 10 μg/mL RNase A, 60 min incubation; lane 6: 10 μg/mL RNase A, 30 min incubation; lane 7: 10 μg/mL RNase A, 0 min incubation; lane 8: 7.5 μg/mL RNase A, 90 min incubation; lane 9: 7.5 μg/mL RNase A, 60 min incubation; lane 10: 7.5 μg/mL RNase A, 30 min incubation; lane 11: 7.5 μg/mL RNase A, 0 min incubation; lane 12: 5 μg/mL RNase A, 90 min incubation; lane 13: 5 μg/mL RNase A, 60 min incubation; lane 14: 5 μg/mL RNase A, 30 min incubation; lane 15: 5 μg/mL RNase A, 0 min incubation.

### RNA Mango RNase Inhibitor Assay

Our team wanted to see if adding an inhibitor would decrease the likelihood of contamination. To test this we incubated RNA Mango with the RNase inhibitor Murine. We determined that this did not affect the cleaving of RNA Mango. Our team recognizes that this may impact the results of the detection system. One way to combat this would be purifying the RNA from the sample prior to using the detection system. Commercial kits are available to do this from  [ThermoFisher](https://www.thermofisher.com/order/catalog/product/AM1901#/AM1901) and  [Bitesize Bio](https://bitesizebio.com/43279/isolating-bacterial-rna-from-blood/) . In the future, we hope to look into developing a simple RNA purification method that would work with our system. Additionally, we received feedback from [aGEM](https://2019.igem.org/Team%3ALethbridge_HS/Public_Engagement) to incorporate unnatural nucleotides at the ends of the RNA Mango sequence. This may prevent or limit any environmental RNases from cleaving the RNA Mango.

 ![](https://static.igem.org/mediawiki/2019/1/17/T--Lethbridge_HS--rnase_inhib_combined.png)

**Figure 9.** 10% Urea PAGEs of RNA Mango samples with Murine RNase Inhibitor (NEB) and RNase A after incubating for different time points at 37 °C. **(A)** Left to right: lane 1: positive Control; lane 2: negative control; lane 3: water control; lane 4: 0 U Murine, no RNase A; lane 5: 0 U Murine, 0 min incubation; lane 6: 0 U Murine, 30 min incubation; lane 7: 0 U Murine, 60 min incubation; lane 8: 0 U Murine, 90 min incubation; lane 9: 0.5 U Murine, no RNase A; lane 10: 0.5 U Murine, 0 min incubation; lane 11: 0.5 U Murine, 30 min incubation; lane 12: 0.5 U Murine, 90 min incubation; lane 13: 0.5 U Murine, 60 min incubation; lanes 14-15: empty.  **(B)** Left to right: lane 1: 1 U Murine, no RNase A; lane 2: 1 U Murine, 0 min incubation; lane 3: 1 U Murine, 30 min incubation; lane 4: 1 U Murine, 60 min incubation; lane 5: 1 U Murine, 90 min incubation; lane 6: 2.5 U Murine, no RNase A; lane 7: 2.5 U Murine, 0 min incubation; lane 8: 2.5 U Murine, 30 min incubation; lane 9: 2.5 U Murine. 60 min incubation; lane 10: 2.5 U Murine 90 min incubation; lane 11: 5 U Murine, no RNase A; lane 12: 5 U Murine, 0 min incubation; lane 13: 5 U Murine, 30 min incubation; lane 14: 5 U Murine, 60 min incubation; lane 15: 5 U Murine, 90 min incubation.

### Cas13a Purification

Both parts of our system rely on the protein Cas13a, so it was imperative that we purify the protein for future use in our Cas13a activity assay. Our team successfully purified Lbu Cas13a protein while we were unable to purify Lwa Cas13a to a high enough concentration through Nickel Affinity Chromatography (click [here](https://static.igem.org/mediawiki/2019/b/b4/T--Lethbridge_HS--His-tag_purification.pdf))and Size Exclusion Chromatography(click [here](https://static.igem.org/mediawiki/2019/1/17/T--Lethbridge_HS--SEC_purification.pdf)).

Initially, our team tried to express Cas13a in *E. coli* BL21 DE3; however, we had issues with overexpression so we tried again using *E. coli* Rosetta DE3 cells with greater success (Figure 10).

 ![](https://static.igem.org/mediawiki/2019/2/21/T--Lethbridge_HS--OE_lbu_lwa.png)

**Figure 10.**  10% SDS PAGE of overexpression of Lbu and Lwa Cas13a. Run at 180 V for 25 minutes and 200 V for 2.5 hours. Left to right: lane 1: Lbu before induction; lane 2: Lbu after induction; lane 3: Lbu before induction; lane 4: Lbu after induction; lane 5: Lwa before induction; lane 6: Lwa after induction; lane 7: Lwa before induction; lane 8: Lwa after induction; lane 9: 10-250 kDa ladder; lane 10: empty.

Those cells were lysed then centrifuged to separate the supernatant and cell pellet. The lysate was then introduced to a Nickel Sepharose affinity column to isolate the Cas13a protein. It was bound to the Nickel Sepharose due to the histidine tag. Please note that while Lwa Cas13a does not appear well on the scanned gel, a faint band appeared in the wash and elution fractions.

 ![](https://static.igem.org/mediawiki/2019/c/c5/T--Lethbridge_HS--his_purif_combined.png)

**Figure 11.** 10% SDS PAGE of Cas13a proteins after his tag purification.  **(A)**  Left to right: lane 1: Lbu lysate; lane 2: Lbu lysate post-binding; lane 3: Wash (pooled); lane 4: elution 1; lane 5: elution 2; lane 6: elution 3; lane 7: elution 4; lane 8: elution 5; lane 9: elution 6; lane 10: 10-250 kDa ladder.  **(B)**  Left to right: lane 1: Lwa Lysate; lane 2: Lwa lysate post-binding; lane 3: Wash (pooled); lane 4: Elution 1; lane 5: elution 2; lane 6: elution 3; lane 7: elution 4; lane 8: elution 5; lane 9: 10-250 kDa Ladder; lane 10: empty.

To further purify Cas13a, we ran the partially purified protein solution through a column, that contained beads. The beads have small crevices and this causes proteins of different sizes to pass through during different times. Again, the band for Lwa did not show up well after scanning the gel. Since it was likely that a low amount of protein was present in our collected fractions, we chose to move forward only with our purified Lbu. Multiple bands appeared in our purified Lbu Cas13a protein, and further purification steps would aid in ensuring the accuracy of  *in vitro*  data in the future. However, compared to previous purification data from [Munich 2017](https://2017.igem.org/Team%3AMunich/Cas13a), we are working with a much purer sample than has been used in the competition.

 ![](https://static.igem.org/mediawiki/2019/0/0a/T--Lethbridge_HS--sec_lbu_combined.png)

**Figure 12.**  Purification of Lbu Cas13a using size exclusion chromatography.  **(A)**  10% SDS PAGE of Lbu Cas13a after size exclusion chromatography. Left to right: lane 1: Lbu concentrated after affinity chromatography; lane 2: Lbu frac H2; lane 3: Lbu frac H1; lane 4: Lbu frac I1; lane 5: Lbu frac I2; lane 6: Lbu frac I3; lane 7: Lbu frac I4; lane 8: Lbu frac I6; lane 9: Lbu post-SEC and concentrated; lane 10: 10-250 kDa ladder.  **(B)**  Chromatogram generated by AktaPrime of Lbu Cas13a purified using a large Superdex75 column (GE Life Sciences).

 ![](https://static.igem.org/mediawiki/2019/6/67/T--Lethbridge_HS--sec_lwa_combined.png)

**Figure 13.**  Purification of Lwa Cas13a using size exclusion chromatography.  **(A)**  10% SDS PAGE of Lwa after size exclusion purification. Left to right: lane 1: Lwa concentrated post-affinity; lane 2: Lwa frac B7; lane 3: Lwa frac B6; lane 4: Lwa frac H5; lane 5: Lwa frac H3; lane 6: Lwa frac H1; lane 7: Lwa frac I1; lane 8: Lwa I7; lane 9: 10-250 kDa ladder; lane 10: Lwa frac I8.  **(B)**  Chromatogram generated by AktaPrime of Lwa Cas13a purified using a large Superdex75 column (GE Life Sciences).

We noticed a large peak at absorbance 254 nm, which is the level at which nucleic acids absorbs. Since Cas13a binds and interacts with RNA this is likely what is causing the peak. We ran the sample on a urea page to confirm the presence of RNA (Figure 14). Members of the Wieden lab at the University of Lethbridge have worked with proteins that interact with RNA [(Becker  *et al.,* 2012).](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3583859/) To minimize these interactions, they purify this protein using anion exchange chromatography (Q Sepharose). We noticed that previous iGEM teams had not done this purification step in the past. To minimize any issues with other interacting RNAs, we recommend future iGEM teams complete this purification step in between nickel affinity chromatography and size exclusion chromatography. Due to time constraints, we were unable to test this purification methodology to see if it impacted our results.

 ![](https://static.igem.org/mediawiki/2019/7/73/T--Lethbridge_HS--urea_protein_fractions.png)

**Figure 14.**  10% Urea PAGE of Lbu and Lwa protein samples after affinity and size exclusion chromatography purification. Left to right: lane 1: High Range RiboRuler; lane 2: Lwa sample post-concentration after affinity purification; lane 3: Lbu sample post-concentration after size exclusion chromatography; lane 4: Lbu sample post-concentration after affinity chromatography.

### Cas13a Activity Assay

Our team conducted a Cas13a activity assay to test the effectiveness of our enzyme. We incubated 300 nM of Lbu Cas13a complexed with the crRNA with various concentrations of RNA Mango to see the change in fluorescence of the dye thiazole orange. By analyzing our results, we can see that the fluorescence for the RNA Mango at the concentration 25nM sample decreased. This likely indicates that the Cas13a enzyme is cleaving. However, for higher concentrations, there was not a significant change in fluorescence. This may be due to having an insufficient amount of enzyme for proper cleaving to occur or our enzyme not being active enough to cleave larger amounts of RNA Mango in the same amount of time as the 25 nM sample. The sample with concentration 100 nM is excluded from these generalizations. We believe there may have been an error made in the reading. Another reason for the differences seen in the concentrations of RNA Mango could be caused by if our purified RNA is taking on multiple conformations that affect its ability to interact with thiazole orange. If the G Quadruplex is not forming properly, we would not see fluorescence. Graph C shows the results of our control experiment. Similar to the previously mentioned experiment we incubated 300 nM of Lbu Cas13a complexed with the crRNA with various concentrations of RNA, however this time we used RybA. This was a negative control experiment to test the specificity of our CRISPR Cas13a system. It seems that there was no significant change in fluorescence over time thereby indicating that no RybA was cleaved and our CRISPR Cas13a is specific. Since there was no added fluorescence molecule, it is also hard to interpret what any changes would be.

 ![](https://static.igem.org/mediawiki/2019/c/c5/T--Lethbridge_HS--activity_combined.png)

**Figure 15.**  Determining activity of Lbu Cas13a by targeting snR30 containing RNA Mango II by detecting a loss of fluorescence. Excitation occurred at 510 nm and emission at 535 nm and scans were completed for 3 hours. Data was normalized by dividing by the negative control, which contained all components except for RNA.  **(A)**  Raw scans normalized to the negative control of snR30-RNA Mango II at various concentrations in complex with Lbu Cas13a and the crRNA (n=1). Controls are also shown of only snR30-RNA Mango II, the target molecule and Lbu Cas13a, and the target molecule and RNase A (n=2 +/- SD for controls).  **(B)**  Relative fluorescence at the maximum fluorescence of snR30-RNA Mango II at 72 minutes (n=1 for snR30-RNA Mango II at various concentrations, n=2 +/- SD for controls).  **(C)**  Controls for activity of Lbu Cas13a and crRNA. RybA was used as a specificity control for cleavage (n=2 +/- SD for all data shown).

To confirm the effectiveness of our Cas13a enzyme we ran before and after fluorescence scanning samples on a urea page. Additionally, this would allow us to better interpret our experimental controls. This confirmed our speculation that the 25 nM sample was indeed cleaved by Lbu Cas13a. For the other samples, no significant difference is seen between the before and after samples. For the 50 nM before sample, we believe there may have been a loading issue that affected the quantification results seen in table 1. Our controls seen in Figure 16 B and C show that there are no significant changes between before and after scanning the samples. This means that are assay is specific for our targeted molecule needing to be present to have enzyme activation. Cas13a will also not cleave our target molecule without the crRNA in the complex. Please visit our [measurement page](https://2019.igem.org/Team%3ALethbridge_HS/Measurement) to see our further analysis of this assay as being beneficial for future iGEM teams to characterize any RNA cleaving enzymes they are working with!
 ![](https://static.igem.org/mediawiki/2019/2/23/T--Lethbridge_HS--activity_gel_combined.png)

**Figure 16.** 10% Urea PAGEs of Lbu Cas13a activity assay components.  **(A)** Left to right: lanes 1-3: empty; lane 4: RNA Mango 100 nM post-scan; lane 5: RNA Mango 100 nM pre-scan; lane 6: RNA Mango 75 nM post-scan; lane 7: RNA Mango 75 nM pre-scan; lane 8: RNA Mango 50 nM post-scan; lane 9: RNA Mango 50 nM pre-scan; lane 10: RNA Mango 25 nM post-scan; lane 11: RNA Mango 25 nM pre-scan; lane 12: RNA Mango + RNase A post-scan; lane 13: RNA Mango + RNase A pre-scan; lane 14: negative control (no target or control RNA) post-scan; lane 15: negative control (no target or control RNA) pre-scan. **(B)**  Left to right: lane 1: RNase A + RybA pre-scan; lane 2: RNase A + RybA post-scan; lane 3: RybA 25 nM pre-scan; lane 4: RybA 25 nM post-scan; lane 5: RybA 50 nM pre-scan; lane 6: RybA 50 nM post-scan; lane 7: RybA 75 nM pre-scan; lane 8: RybA 75 nM post-scan; lane 9: RybA 100 nM pre-scan; lane 10: RybA 100 nM post-scan. **(C)**  Left to right: lane 1: crRNA pre-scan; lane 2: crRNA post-scan; lane 3: RNA Mango pre-scan; lane 4: RNA Mango post-scan; lane 5: RybA pre-scan; lane 6: RybA post-scan; lane 7: Cas13a Lbu pre-scan; lane 8: Cas13a Lbu post-scan; lane 9: Cas13a Lbu + crRNA pre-scan; lane 10: Cas13a Lbu + crRNA post-scan; lane 11: Cas13a Lbu + RNA Mango pre-scan; lane 12: Cas13a Lbu + RNA Mango post-scan; lane 13: Cas13a Lbu + RybA pre-scan; lane 14: Cas13a Lbu + RybA post-scan. Lane 15: empty.

**Table 1.**  Quantification of figure 16 A lanes 4-11 of decrease in RNA Mango using ImageJ.

| Concentration of RNA Mango (nM) | 25 | 50 | 75 | 100 |
| --- | --- | --- | --- | --- |
| Pre-scan | 49.34% | 20.8% | 45.3% | 50.0% |
| Post-scan | 1.32% | 79.2% | 54.5% | 50.0% |

## **Therapeutic**

### Fluorescent Protein Assays

Our team wanted to create standardized fluorescence curves for GFP and RFP as well as standardized growth curves for the *E. coli* DH5α cells expressing them. Then, when it came time to conduct our *in vivo* fluorescence assay, these curves would prove to be useful references for comparison with our results. We pipetted various samples of GFP and RFP into a 96 well plate, then used a spectrophotometer to observe optical density over time, and also a spectrometer to observe fluorescence over time. Figure 17 showed what we had expected the growth to be with an increase in doubling time likely due to the load placed on the cell based on the plasmid. However, further attempts at replicating this data have been unsuccessful (figure 18). We are uncertain as to the cause of this issue. We suspect that by using overnight cultures, the fluorescent protein may already be matured and thus we did not get reliable fluorescence data or the protein did not fold properly  *in vivo* .

![](https://static.igem.org/mediawiki/2019/c/ce/T--Lethbridge_HS--jul_od_single.png)

**Figure 17.**  Growth of *Escherichia coli* DH5α cells containing GFP or RFP. 3 biological replicates for each fluorescent protein and 7 technical replicates were completed. Shown are all replicates averaged together and the standard deviation. The  *E. coli*  cells expressing GFP average doubling time varied from 38.2 +/- 0.9 minutes to 41.8 +/- 1.6 minutes and for cells expressing RFP the average doubling time varied from 37.8 +/- 0.2 minutes to 41.0 +/- 0.5 minutes.

 ![](https://static.igem.org/mediawiki/2019/8/8f/T--Lethbridge_HS--aug_single_combined.png)

**Figure 18.**  Growth of *Escherichia coli* DH5α cells containing GFP or RFP. 3 biological replicates for each fluorescent protein and 5 technical replicates were completed. Shown are the average technical replicates and the standard deviation.  **(A)**  Optical density of  *E. coli*  cells expressing GFP or RFP with absorbance measured at 600 nm.  **(B)**  Fluorescence of  *E. coli*  cells expressing GFP or RFP. GFP excitation was measured at 475 nm and emission at 508 nm. RFP excitation was measured at 558 nm and emission at 583 nm.

Figure 19 shows a co-culture assay with a 1:1 ratio of GFP and RFP expressed in  *E. coli* . We wanted to see if one culture of fluorescent protein would outcompete the other present. As with the single cultures shown in figure 18, the cells replicated slowly. Our fluorescence data indicated that the GFP we are using is more fluorescent than RFP. The fluorescence of RFP also showed greater variation, indicating that the RFP may not be folding properly to be excited in all replicates.

 ![](https://static.igem.org/mediawiki/2019/3/33/T--Lethbridge_HS--aug_co-culture_combined.png)

**Figure 19.** Co-culture of  *Escherichia coli*  DH5α cells containing GFP or RFP. Equal amounts of each cell type were added into a 96 well plate and grown together to see how they would grow in tandem. 3 biological replicates for each fluorescent protein and 6 technical replicates were completed. Shown are all replicates averaged together and the standard deviation.  **(A)**  Optical density of  *E. coli*  cells expressing GFP or RFP with absorbance measured at 600 nm. **(B)**  Fluorescence of  *E. coli*  cells expressing GFP or RFP. GFP excitation was measured at 475 nm and emission at 508 nm. RFP excitation was measured at 558 nm and emission at 583 nm.

### *In vivo* Dual Plasmid Fluorescence Assay

We completed a dual plasmid transformation. To confirm that we had both plasmids present we completed a digest test because PCR amplification from the colony was not working. This should separate our construct from the plasmid confirming that both parts are present. In figure 20, only linearized cuts are seen. Due to both plasmids being approximately the same size it is difficult to see. However, there are two bands present. In figure 21, only linearized cuts are seen near the 3000 bp mark. Due to both plasmids being approximately the same size it is difficult to see. However, there are two bands present. The second band at approximately 2000 bp is plasmid backbone, while the 1000 bp mark is the GFP insert. The crRNA insert (expected size is ~260 bp) is likely not seen in the gel due to having to run the gel longer to get better resolution for the upper bands.

Initially, our team had planned on doing a triple plasmid transformation of our target fluorescent protein (GFP), crRNA, and Cas13a to test if our system would work  *in vivo* . Additionally, we wanted to have RFP as the fluorescent protein to serve as a specificity control instead of transforming the plasmid containing GFP. We were unsuccessful in getting all three plasmids to transform, but did succeed in getting the fluorescent proteins and crRNA containing plasmids to transform together as seen in figures 20 and 21.

 ![](https://static.igem.org/mediawiki/2019/5/55/T--Lethbridge_HS--rfp.png)

**Figure 20.**  1% agarose of restriction digests using PstI and EcoRI from  *Escherichia coli*  BL21(DE3) cells containing plasmids for RFP and either Lbu crRNA or Lwa crRNA. Left to right: lane 1: 1 kb ladder; lane 2: Lwa crRNA colony 1; lane 3: Lwa crRNA colony 2; lane 4: Lwa crRNA colony 3; lane 5: Lbu crRNA colony 1; lane 6: Lbu crRNA colony 2; lane 7: Lbu crRNA colony 3; lanes 8-13: empty.

 ![](https://static.igem.org/mediawiki/2019/1/19/T--Lethbridge_HS--digest_gfp.png)

**Figure 21.**  1% agarose of restriction digests using PstI and EcoRI from  *Escherichia coli*  BL21(DE3) cells containing plasmids for GFP and Lbu crRNA, Lwa crRNA, Lba crRNA, or Lsh crRNA. Left to right: lane 1: Lwa crRNA colony 1; lane 2: Lwa crRNA colony 2; lane 3: Lwa crRNA colony 3; lane 4: Lwa crRNA colony 4; lane 5: Lsh crRNA colony 2; lane 6: Lba crRNA colony 1; lane 7: Lba crRNA colony 2; lane 8: Lba crRNA colony 3; lane 9: Lbu crRNA colony 1; lane 10: Lbu crRNA colony 2; lane 11: Lbu crRNA colony 3; lane 12: Lbu crRNA colony 4; lane 13: 1 kb ladder.

As an alternative experiment, our team grew cells that expressed the Lbu and Lwa Cas13a protein overnight. We also grew cells that expressed dual plasmids; GFP and crRNA Lwa; GFP and crRNA Lbu; RFP and crRNA Lwa; and RFP and crRNA Lbu. We then lysed the cells that expressed the Cas13a proteins using a French Press and clarified the lysate via centrifugation. Following this, our team pipetted in a 1:1 ratio of clarified cell lysate: fluorescent protein and crRNA into a 96 well plate. This allowed us to observe if there would be an effect from the CRISPR Cas13a system on the fluorescent proteins. We observed that in our optical density data, both dual plasmid systems for GFP and RFP had stunted growth in comparison to only  *E. coli*  cells expressing GFP or RFP or no plasmid (Figure 22C). Adding the lysate may have caused the death of the culture. We neglected to include replicates of the dual plasmid system without adding lysate to observe how that grew. This would be beneficial for any future experiments. Alternatively, there may have been some effect of the protein in the lysate on the GFP fluorescence (Figure 22B). However, we are unsure of the specificity due to the potential of the RFP not folding correctly *in vivo*  as demonstrated by the substantial standard deviation seen in our replicates (Figure 22A).

 ![](https://static.igem.org/mediawiki/2019/9/99/T--Lethbridge_HS--invivo_fluor.png)

**Figure 22.**   *In vivo* fluorescence assay of  *E. coli*  BL21(DE3) cells containing fluorescent protein and crRNA plasmids and  *E. coli*  Rosetta(DE3) cell lysate of overexpressed Cas13a proteins. This assay was conducted with 3 biological replicates and 3 technical replicates.  **(A)**  Fluorescence of  *E. coli*  cells containing only an RFP expressing plasmid, or dual plasmid expression of RFP and crRNAs from Lwa and Lbu that target GFP and the respective cell lysate containing the appropriate Cas13a. RFP excitation was at 558 nm and emission at 583 nm.  **(B)**  Fluorescence of  *E. coli*  cells containing only a GFP expressing plasmid, or dual plasmid expression of GFP and crRNAs from Lwa and Lbu that target GFP and the respective cell lysate containing the appropriate Cas13a. GFP excitation was at 475 nm and emission at 508 nm. **(C)** Optical density of  *E. coli*  cells expressing GFP, RFP, dual plasmid systems mentioned previously, or only  *E. coli*  BL21(DE3) cells with absorbance measured at 600 nm. **(D)** Relative fluorescence at maximum excitation at 81 minutes. GFP excitation was at 475 nm and emission at 508 nm and RFP excitation was at 558 nm and emission at 583 nm.

### References

Becker, M., Gzyl, K.E., Altamirano, A.M., Vuong, A., Urbahn, K., and Wieden, H.-J. (2012). The 70S ribosome modulates the ATPase activity of  *Escherichia coli* YchF.  *RNA Biol 9*(10): 1288-1301.

Panchapakesan, S.S.S., Ferguson, M.L., Hayden, E.J., Chen, X., Hoskins, A.A., and Unrau, P.J. (2017). Ribonucleoprotein purification and characterization using RNA Mango. *RNA 10*: 1592-1599.