iGEM 2024 — Team exeter

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## June

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**Week 1 [17/6/24 - 21/6/24]**

##### --- **Monday 17th of June**

* Lab induction [everyone]
* Writing project description [Ruby, Ellin]
* Allocating team roles [everyone]
* Planned Rosetta experiment [George, Oli, Becky, Yara]
* Began developing the wiki [Anouska, Nigella, Ben] using HTML - pages and links were created, and images were added.

##### --- **Tuesday 18th of June**

* Rosetta experiment planning continued [George, Oli, Becky, Yara]
* Continued writing project description [Ruby, Ellin]
* Protein purification practice experiments took place [Ruby, Yara]
* Culturing and incubation for Rosetta experiment [George, Oli, Becky]
* Initial draft of ethics form produced [Ellin]
* Began work on project branding - developing a colour scheme [Anouska, Nigella], thinking up some names [Anouska, Nigella] and creating some logo ideas [Ben].
* Edited the CSS style sheets for the wiki, allowing colours and fonts to be changed.[Anouska, Nigella, Ben]
* Created a 'wiki cheat sheet' PowerPoint containing the basics of HTML [Ben] and CSS [Anouska] to help the rest of the team.

##### --- **Wednesday 19th of June**

* Rosetta cultures failed to grow! Too many heat shocks were performed... protocol from yesterday was repeated [George, Oli, Becky].
* Project description was further developed, following a meeting with John [Ruby].
* Researches mycobacterium tuberculosis bovis quorum sensing, via academic papers [Ellin].
* Added the notebook page to the wiki, with attributions [Ben].
* BL21 transformation was attempted, with Cas 12 and Cas 13a plasmids [Louise, Tom]

##### --- **Thursday 20th of June**

* Western blot practice experiments were performed [Ruby, Yara]
* BL21 experiment for Cas 13a was performed successfully, but not for the Cas 12. The Cas 13a is now being grown in a liquid culture for a gel, ready for an experiment of Friday [Louise], while the Cas 12 is being transformed again [Tom].
* Project description was finalised [Ruby]
* Western blot lab protocol was refined [Yara]
* mCherry was inoculated into liquid culture [George, Becky]
* Agar plates were produced, with varying concentrations of ampicillin and chloramphenicol [Oli, George, Becky]
* More HTML tweaks were performed to better organise certain pages [Ellin, Ben]
* The notebook page was reworked to contain a photo diary and collapsible sections, and updated with additional attributions [Ben]
* A new template for wiki text boxes was developed, with shadow effects and curved edges [Anouska]
* Comprehensive aesthetic changes (colours, container shapes, fonts, and menu bar appearance) were effected [Anouska, Nigella]
* More gRNA literature was researched [Ellin]

##### --- **Friday 21th of June**

* Group meeting to discuss the week and plans for week 2 [everyone]
* A tweak to the CSS left all the pictures on the notebook wiki page random sizes - this was fixed, and dividers were created [Ben]
* General wiki edits, tidying up some of our previous HTML experiments [Ben, Nigella, Talia, Anouska]
* mCherry Rosetta culture was plated onto agar-antibiotic plates [Oli, Becky, George]
* mCherry Rosetta culture was placed into a checkerboard 64 well plate. Fluorescent intensity was then measured for photon wavelengths 587-610 nm. The OD (optical density) at 600 nm is being measured as well [George, Becky, Oli].
* Rosetta experiment was written up, as part of our contribution section of the wiki [Becky]
* The Western blot experiment didn’t work [Ruby, Yara]
* Ran a practice Qubit protein concentration experiment [Ruby, Yara]
* Some of the non-wiki team began learning HTML [Ruby, Yara]

![Two laptops and an ipad, displaying the 'team' page of our wiki, which contains an enormous picture of Ben's face. Ben stands next to them, pulling a similar expression to that in the picture.](https://static.igem.wiki/teams/5124/notebook/4bens.webp)

Wiki development is underway!

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![Two petri dishes on a worktop - the leftmost dish shows red fluorescence in small spots across the surface.](https://static.igem.wiki/teams/5124/notebook/results-2.webp)

Growing our first Rosetta culture - the mCherry batch (left) has come out a lot better.

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![Ruby uses a pipette in an experiment, showing enormous concentration.](https://static.igem.wiki/teams/5124/notebook/ruby-2.webp)

Ruby hard at work.

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![Yara is ecstatic about a miraculous discovery in the lab.](https://static.igem.wiki/teams/5124/notebook/yara-1.webp)

Very exciting when the results come back!

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**Week 2 [24/6/24 - 28/6/24]**

##### --- **Monday 24th of June**

* Integrated human practices were researched [Ruby, Talia]
* Contact was made with farmers, via John [Ruby]
* Past wikis were scoured in search of inspiration [Ruby]
* The team page was reformatted and filled out [Ben], using a new layout
* The logo was given tweaks, The new name was added (BoviTect) and the menu bar was tweaked [Nigella, Anouska]* Notebook sections were expanded to include week 2, and the images were properly resized (trust me it's harder than it sounds) [Ben]
  * Rosetta has a lower chloramphenicol resistance than expected as the cultures didn't grow on the agar, either with cam present or in the wells. Agar plates with just amp or lower concentrations of cam have now been produced, and the mCherry rosetta cultures will plated on them tomorrow. [Oli, Becky, Ellin]
  * Learning HTML and editing the wiki [Yara, Ruby]
  * Research into presentation and promotional video requirements [Yara]

##### --- **Tuesday 25th of June**

* Ethical approval form & rewrite info sheet and consent form for 2024. [Ellin, Anouska]
* Produced overnight mCherry Rosetta culture, which will then be plated on the individual antibiotic plates tomorrow, and again left to grow overnight. We should have results from this experiment on Thursday. [Oli, Becky]
* IHP Research and learning to code the wiki. [Ruby, Yara]
* Re-nickling columns ready for BL21 protein purification tomorrow. [Ruby, Yara]
* General wiki tweaks, mostly editing the teams page. [Ben]

##### --- **Wednesday 26th of June**

* Plated up cultures on the antibiotic agar plates… and now we wait. [Becky, Oli]
* Meeting with Dr John Love. [et al]
* Final ethics form completed [Anouska]
* Installed flutter, to begin app development. Researched modelling possibilities. Drafted hardware concepts. [Oli, Ben, Talia]
* Writing the lab protocol for transformation for publication on the Wiki [Becky]
* Wiki tweaking [Nigella]
* Making up buffers for protein purification [Yara, Ruby]
* Lysed cells for protein purification [Louise, Tom]
* Made liquid culture for mCherry and blank plasmid [Louise]

##### --- **Thursday 27th of June**

* Rosetta grew on some of the agar plates. Our previous question of Rosetta not being very chloramphenicol resistant, is confirmed to be true, as no cultures grew on any of the plates with just chloramphenicol, besides the 1.1μg/mL conc plate; This yielded a similar result to the control, implying that it had no effect on growth. [Oli, Becky, George]
* Inoculated mCherry and blank plasmid into autoinduction media [Louise, Anouska]
* Miniprep of the mCherry and blank [Tom]
* COSHH form done for autoinduction media [Louise]
* Attempting to put in animations using JSON and Lottie [Anouska]
* Redesigning logo for letter headings, etc. [Anouska, Nigella]
* crRNA attempting to attach the cr and tracrRNA [Tom]
* The checkerboard test will need to be repeated 2 more times. [Oli, George, Becky]
* Protein purification of Cas 12a and Cas 13, renickeling column and running 2x Qubits [Yara, Ruby]

##### --- **Friday 28th of June**

* Optimized the process for producing graphs for our checkerboard test, by using matplotlib in conjunction with for loops in python. Produced dose response curves for ampicillin. [Oli]
* Lysed cells for protein purification mCherry and pX1900 [Louise]
* Working on the Wiki: Back to top button, designing animations [Anouska]
* Drawing graphics for wiki using Procreate + wiki tweaks [Nigella]
* Lab photography for wiki [Yara]
* Purification of the blank and mCherry controls [Yara, Anouska, Nigella]
* Writing questions for interviews (Integrated HP) [Becky, Talia]
* Learning how to use dartpad for app development[Talia]
* Completing attaching tracrRNA and spacer sequence together (with added buffer region) and calculating crRNA shapes [Tom]

![The wiki team sat at a table, with the profile photos of the team members visible on a laptop screen. Mysteriously, everyone's faces have been replaced by a still of Barry Bee from Bee Movie (2007).](https://static.igem.wiki/teams/5124/notebook/team2.webp)

Wiki development continues!

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![three students in the lab running protien purification through nickel columns ](https://static.igem.wiki/teams/5124/notebook/screenshot-2024-07-01-at-10-26-12.webp)

Anouska, Nigella and Yara carrying out the purification of the blank and mCherry controls through the nickel columns!.

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![Columns of lines at various intervals, shown in white on a dark grey background. Someone more closely involved with the lab work should probably write this alt text to be honest...](https://static.igem.wiki/teams/5124/notebook/linediagram.webp)

Actual (left) and simulated (right) results of a test to check for the presence of our plasmids. Left-to-right, these are Cas13a, Cas12, mCherry and a blank control plasmid.

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![The wiki team, standing up in the corner of a room, appearing to be taking part in some kind of bizarre iGEM yoga. Ben looks particularly distressed.](https://static.igem.wiki/teams/5124/notebook/imagetwo.webp)

To keep morale high, the occasional team-building activity has taken place as a brief respite from iGEMing - such as this impromptu exercise session...

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## July

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**Week 3 [1/07/24 - 5/07/24]**

##### --- **Monday 1st of July**

* Re-designed non-biological hardware concept (using suggestions from the Dundee 2013 team), so that the challenge of implementing a phone camera into the design can be neglected all together; instead focusing on light-sensitive photodiodes connected to a raspberry pi, which can send signals to a mobile device through an app. This should be much easier to implement from both a hardware and software perspective [Oli]
* Transformed Ampicillin-resistant, mCherry Rosetta again for our 8x8 well plate. Also plated Rosetta onto 2 separate agar plates containing Chloramphenicol from different stocks, to determine if there is a contamination issue. [Oli, George]
* After facing issues with the desktop Android Studio, app development was resumed on the online version [Ben]
* Writing more interview questions and short explanation of our project for each person we interview [Becky]
* Qubit concentrations for Cas12a, Cas13, mCherry and blank plasmid [Yara, Nigella]
* Wiki tweaking [Nigella]
* Stash design, setting up team page [Anouska]
* Redid a final agarose gel for all the plasmids, looks pretty [Louise]
* Writing up the protocols for plasmid to protein pipeline [Louise]

##### --- **Tuesday 2nd of July**

* Starting on CAD design for hardware [Anouska]
* Meeting with Becky for help with coding to fix parts of Wiki (sticky contents bar, header background) [Nigella, Anouska]
* Drew final version of wiki banner [Nigella]
* Fixed code for DNA sequences. Sequences can now be printed with their complementary strain and their inverse. [Oli]
* Produced overnight liquid cultures for 8x8 well plate tomorrow. Also verifying that ampicillin-resistant mCherry plasmid cannot be expressed in chloramphenicol environment. [Oli, George]
* Ran a western blot on our lovely 34 samples [Ruby, Yara]
* Had a meeting with Mark and Chloe on how to optimise our protein expression, cell lysis and western blot [Ruby, Yara]
* Became a lab assistant for the morning and was Very Helpful ⭐ (except when I forgot to change the pipette end) [Ben]
* Continued Flutter development - now working on accessing the camera through the app [Ben]
* BLAST sequencing of genes [Becky]
* Set up the team email, added a signature and started drafting emails to people we would like to interview [Becky, Louise]
* Lab photography [Yara, Ruby]

##### --- **Wednesday 3rd of July**

* Update from Chloe on the lab work required over the next few weeks and allocating dates for everyone to be in the lab [everyone]
* Working on new version of hardware on CAD [Anouska]
* Stash orders and design [Anouska]
* Image hover on the Wiki - modifying teams page [Nigella]
* Western blot results - WE GOT CAS!!! [Yara, Ruby]
* Restriction digest with linear DNA that is maltose binding protein [Ruby]
* Running agarose gel [Ruby, Becky]
* Running an SDS-PAGE on the pellet to see if more Cas proteins were there [Louise, Ben]
* Uploaded some photos [Louise]
* Lab protocol write ups from last week's lab work [Yara]
* Showed Louise and Ben how to do SDS-Page [Yara]
* Produced liquid mCherry Rosetta culture from plates yesterday, and started to run our 8x8 well plate protocol overnight. [George,Oli]

##### --- **Thursday 4th of July**

* E.coli transformation into BL21 and Rosetta. Writing up protocols for restriction digest, gel excision, DNA extraction + ligation [Ruby, Becky]
* Ethics research and finishing protocol write up [Louise]
* Drafted emails for human practice interviews. Used flutter to continue with software development [Talia]
* Made a python script for detection of cas12a spacer sequences. Tested spacer sequences for potential DIVA opportunities and for false positives with the cow genome [George]
* Finished finding the cas12a spacer sequences [Tom, George]
* Modifying wiki page layouts [Nigella]
* Adding image transition to team page on wiki [Anouska]
* Lab protocol write ups from last week's lab work. Wrote an explanation about Cas spacer sequences [Yara]
* Found the surrounding sequences for the spacer sequences (50bp up and downstream of spacer sequence complement) [Yara, Nigella]
* Made the wet lab timeline colour-coded by project (see under lab protocols tab) [Ruby]
* 8x8 well test again. Performed Rosetta transformations with 5 different plasmids and a negative control. Plated onto 3 separate types of agar plates: Ampicillin, Chloramphenicol, and ampicillin + Chloramphenicol. [Oli, George]

##### --- **Friday 5th of July**

* Observed that the promoter seems to affect mCherry expression on ampillin + chloramphenicol plates. pX1800\_PJ23100-mCherry and pX1900\_PJ23100-mCherry didn’t produce any purple cultures on the plates, whilst pX1900\_T7-mCherry did. This implies high copy numbers affect the mCherry expression. 8x8 well plate again as well… [Oli, George]
* crRNA using introns only is completed, there is still some concern about where these sequences sit within the cow genome (cas13a). The bTB (cas12a) sequences have worked really well [Tom]
* MBP plates didn't grow except for 1- the DH5alpha cas-13 plate [Ruby, Becky]
* Made complete protein purification to analysis method (including nickel regeneration, making buffer, protein purification, qubit and western blot). Updated all protocols for the Wet lab section of the notebook [Ruby]
* Wrote up protocol and results for Rosetta experiments [George]
* Emailed companies about potential interviews [Becky]
* Set up social media for bovitect and created posts/content [Talia]
* Lab protocol write ups and making results tables for last week's experiments [Yara]
* Looking at safety forms and information on what is allowed [Louise]

![Becky handles an agar-filled petri dish inside a fume hood.](https://static.igem.wiki/teams/5124/notebook/w3b.webp)

Becky readying plates for over-night Rosetta growth.

![The wiki team are hard at work. Anouska is reclining on a low sofa, sticking out her tongue, clearly displeased about being photographed.](https://static.igem.wiki/teams/5124/notebook/w3c.webp)

Some wiki work with the rest of the group. Anouska is working, don't worry.

![A collection of small plastic tubular containers relax in a heated box.](https://static.igem.wiki/teams/5124/notebook/w3f.webp)

Thermocycling Rosetta and BL21 derived cas protein samples in preperation for a western blot!

![2 westernblots laid on tabletop](https://static.igem.wiki/teams/5124/notebook/cas-western-blots.webp)

Western blots indicating CAS presence

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**Week 4 [8/07/24 - 12/07/24]**

##### --- **Monday 8th of July**

* Analyzed data from last week. Data is inclusive, however we have shown, on Agar plates, that Rosetta transformed with high copy number promoters results in mCherry protein not being expressed when in the presence of ampicillin and chloramphenicol. [Oli, George]
* Rewrote all of the IHP documents and made the comprehensive guide to IHP [Ruby]
* Prepared sterile LB broth for induction of Cas later this week, and prepared Ampicillin LB-agar plates for transformation. Troubleshot crRNA sequences, hopefully no longer inverting weirdly [Tom]
* Transformed Cas13a, Cas12a, T7-mCherry, T7-Px1900 into Rosetta and BL21 E.Coli, aiming to produce a greater yield of Cas' [Becky, Yara, Tom]
* Finalising results tables of Qubit and Western Blots from Week 2 and 3 [Yara]
* Communicating with University of Warwick about the UK iGEM meet-up [Becky]
* Made liquid cultures for the maltose binding practical [George]

##### --- **Tuesday 9th of July**

* Plasmid Miniprep of cells that contain plasmids with MBP and Cas13 proteins (TBC using sequencing). Qubit of plasmids containing MBP and Cas13 proteins [Yara]
* Prepared Amp/Clam antibiotic aliquots for induction cultures tomorrow [Becky, Tom]
* Prepared IPEG solution for induction of T7 tomorrow, then started methods for probe testing. [Tom]
* Completed Fluorescein concentration curve and created graphs [Oli, George]

##### --- **Wednesday 10th of July**

* Made publishable graphs for fluorescein concentration curves from yesterday at different gains. Drafted COSSH form for RNAse and Dnase Alert kits [Oli]
* Inoculated induction culture media's (250 mL) with BL21 and Rosetta overnights (Cas12a, Cas13a, mCherry, PX1900 - note no PX1900 for Rosetta), monitored growth for 4 hrs and induced T7 expression at 0.6 OD. [Becky, Tom]
* Prepared for cell harvest on Thursday [Becky, Tom]
* Lab photography [Yara]
* Qubit of plasmids containing MBP and Cas13 proteins [Yara]
* Preparation of competent Rosetta cells
* Plasmid mini prep [Anouska, Nigella]
* Ethics resubmission [Anouska]

##### --- **Thursday 11th of July**

* Catching up and writing a section of 'All about bTB', basically consist of current knowledge, impact, statics, current test about bTB [Ellin]
* Harvested induced cells and freezed the pellets for weekend storage [Becky, Tom]
* Measured the fluorescence of simulated blood and PBS mixed with fluorescein. We have shown that the blood significantly blocks the signal [Oli, Tom]
* Preparation of competent Rosetta cells and storage in freezer – Day 2 [Yara]
* Editing photos from the lab [Yara]
* Made aliquots of Ampicillin for future use [Becky]
* Various tweaks to the Wiki, mostly involving the Teams page photo optimisation [Ben]

##### --- **Friday 12th of July**

* Found method of pairing flutter apps with Arduino via Bluetooth, for our hardware [Oli]
* Made publishable graphs from the blood vs PBS sensitivity tests [George]
* Planned out experiments for testing probes [George]
* More miscellaneous wiki tweaks [Ben]

![A bunch of large flasks with red and yellow liquid in the lab.](https://static.igem.wiki/teams/5124/notebook/week-3.png)

Cultivating and harvesting Rosetta and BL21!

![Three petri dishes are visible, all with small clusters of bacteria. The left-hand dish shows prominent fluorescent red colouring.](https://static.igem.wiki/teams/5124/notebook/w4a.webp)

mCherry is a Roestta culture showing up strongly in the dark.

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![Tom concentrates as he transfers a dark red substance from its large container to a smaller one, via a pipette.](https://static.igem.wiki/teams/5124/notebook/w4c.webp)

Tom pipetting simulated blood into flouroscein and PBS to measure amount of flourescence!

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**Week 5 [15/07/24 - 19/07/24]**

##### --- **Monday 15th of July**

* Set up a Planner page to keep track of progress [Oli], and tasks were added to monitor progress [everyone]
* More photos of the team at work were taken for the wiki, and the notebook page was brought up to date [Ben]
* Meeting [everyone]
* Finished off old protocols from last week, organised stuff for this week on Planer, prepared column buffers for tomorrow [Tom]
* Hardware design [Anouska, Oli]
* Transformation of pX1900 P\_J23100-mCherry E Coli [Oli]
* IHP question brainstorming [Anouska, Oli, George]
* Email and research about the use of blood [Anouska, George]
* Refining the protocol for testing the probes and carrying out the first part of the practical [George]
* Made up new nickel columns for protein purification [Louise, Yara]
* Planning engineering section of wiki [Ellin]
* Transforming Cas12a and Rosetta [Ellin, Nigella]
* Editing footer of wiki (linking wiki to Instagram and Email) [Nigella]

##### --- **Tuesday 16th of July**

* Protein purification of Cas12a, Cas13, PX1900 and mCherry expressed in both Rosetta and BL21 [Yara, Ellin, Ben]
* Testing 'cas' concentration needed for final experiment for both DNA and RNA [George, Anouska, Oli]
* Graphing 'cas' concentration experiment [George]
* More interview questions and wiki tweaking [Nigella]
* Overnight for our new competent Rosetta E coli [Oli]
* Hardware iteration design adding more epindorf openings (from 2 to 4) [Anouska]
* Lysed Rosetta and BL21 cells ready for protein purification [Tom, Louise]
* Remade contaminated Tris-HCl buffer solutions and researched methods for detecting RNA. [Tom]
* Resumed Flutter work, making a Doctor Who app (a test to work on my programming skills before tackling the cow app proper) [Ben]

##### --- **Wednesday 17th of July**

* Writing up a check in form for potentially bringing Cas out of the lab [Louise]
* Made amp plates [George]
* Changed the 'cas' concentration experiment graph into fluorescein concentration equivalents and performed statistical analysis on it [George]
* Group meeting catching everyone up with the different areas of the project [Everyone]
* Western blot of Cas12a, Cas13, PX1900 and mCherry expressed in both Rosetta and BL21 – due to use of incorrect antibody, need to repeat tomorrow [Yara, Ellin]
* Poster for Warwick meet-up [Oli, Anouska, Nigella, Talia]
* Completed the Doctor Who app and started laying the foundations for the Cow App in Flutter, which farmers will use to get test results [Ben]

##### --- **Thursday 18th of July**

* Finishing Warwick poster [Anouska, Oli, Nigella, Ruby]
* Writing up detailed notes of our lab progress so far [Anouska]
* Updated wet lab timeline [Ruby]
* Description of Chloramphenicol-Ampicillin experiments for the Wiki, along writing the next steps for the experiment going forward. [Oli]
* Engineering cycle [Ruby]
* Making competent Rosetta cells day 1 [George, Oli]
* Research into ethics of using blood further and research of making our own model blood [George]
* Second western blot [Ellin, Yara]
* Flutter work: making multiple (blank at this stage) pages of the Cow App, and decorating the home page [Ben]

##### --- **Friday 19th of July**

* Fixing menu bar on wiki [Anouska, Nigella]
* Protocol draft for photodiodes and fluorophore quencher system [Oli]
* Western blot of proteins in other BL21 fractions [Ellin, Yara]
* Finished Check in form for potentially taking the Cas proteins outside the lab [Louise]
* Started attributions page [Louise]
* Competent Rosetta E. coli prep [George, Oli]
* Carried out gel extraction, qubit quantification and started write up [Tom]
* Set up the camera page on the Cow App in Flutter [Ben]

![Three students stood looking at a door hinge](https://static.igem.wiki/teams/5124/notebook/5949938e-56f4-406a-a278-de3fff92b701.webp)

Trying to figure out how hinges function in order to draw then in CAD for our hardware design!

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![Students around a whiteboard having a meeting](https://static.igem.wiki/teams/5124/notebook/4e8e97cb-4c76-46a7-a126-8cd45bd7d089.webp)

Our weekly meeting catching everybody up to date on the progress of different parts of the project!

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![iGEM team member looking at test tubes](https://static.igem.wiki/teams/5124/notebook/protein-purification.webp)

Ben keeping a close eye on his protein purification.

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![top down image of 7 protein purification test tubes ](https://static.igem.wiki/teams/5124/notebook/more-protein-purification.webp)

More protein purification pictures

**Week 6 [22/07/24 - 26/07/24]**

##### --- **Monday 22nd of July**

* Concentration curves for Sulforhodamine 101 made and more concentrations of probes tested with optimal Rnase concentration found [George]
* Repeated PCR for Cas13a DNA test sequences and ran a 1.5% agarose gel, with blanks, to see if dimer primers where forming. Then gel extracted/PCR cleaned all samples. [Tom]
* Transformed Rosetta with Cas plasmids, mCherry, blank plasmid and nothing, to check for new rosetta contamination and for protein expression this week [Louise, Ellin]
* Lab photography [Yara]
* Lab protocol write up and presenting work in results tables [Yara]

##### --- **Tuesday 23rd of July**

* Team meeting with John, Mark, Chloe and Yik Ting
* Produced circuit diagrams for the photodiode detection system part of our hardware. Code for the voltage readings has been produced as well. [Oli]
* Graphed and completed the experiment of differing DNase concentration effects on the probes and completed the effects of varying probe concentration on RNase [George]
* Drawing up new hardware iteration of Fusion [Anouska]
* Research for Dr Rees interview [Anouska, Talia]

##### --- **Wednesday 24th of July**

* 3D printer Test [Anouska, Oli]
* Editing hardware on CAD [Anouska]
* Preparing for interview with Dr Rees [Talia, Anouska, George, Tom]
* Made week 6 update powerpoint for interview with david andrews [Ruby]
* Waiting on approval to use a physics lab to test parts of our hardware [Oli]
* Further work making the graphs better and creating a model using the graphs [George]
* Made up and sterilised LB media for our large induction cultures [Louise]
* Built a puzzle for team building purposes [Becky, Ben]
* Finally got Bluetooth page to the stage where the app could compile - however there were still errors present [Ben]
* Made up ITPG solution [Ellin,Tom]
* Carried out PCR clean up and ran another Tapestation gel column, then put results into a figure for wiki. [Tom]

##### --- **Thursday 25th of July**

* Team interview with David Andrews [Everyone]
* Updating wiki notebook [Anouska]
* Warwick meet-up [Ruby, Becky]
* Made up induction media and started incubating [Louise, Tom]
* Wrote summary of hardware concepts as well as what we want to achieve with our Arduino. Physics lab induction, just need to wait for components to arrive [Oli]
* Planned out my experiments for cas, probes and crRNA all working together [George, Tom]
* Making diagrams for how the test functions [George]
* Prepared T7 transcription reaction mixtures for tomorrow
* Induced expression of BL21 with Cas12a with IPEG [Ellin]

##### --- **Friday 26th of July**

* Warwick meet-up [Ruby, Becky]
* 3D Printer troubleshooting and ordered components for our hardware [Oli]
* Interview with Actiopohage (Cath Rees) [Talia, George, Tom]
* Triallled the full experiment with cas, probe, crRNA and target [Tom and George]
* Carried out transcription and Tapestation RNA gel, to confirm RNA was made [Tom]
* Making up buffer for nickel columns [Yara]
* Harvested cells for lysis to freeze over weekend [Louise]
* Extract, harvest, and freeze pellet [Ellin]

![Yik Ting looks bemused into a camera lens.](https://static.igem.wiki/teams/5124/notebook/jul2.webp)

A few members of the team are climbing fans. Here Yik Ting (one of our supervisors) is photographed during a session.

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![two students looking at a 3D printer](https://static.igem.wiki/teams/5124/notebook/4b3c720a-e170-4088-b9d4-dcd19b7a06e4.webp)

First time 3D printing one of our hardware prototypes!

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![Ruby displays our poster at the iGEM meetup.](https://static.igem.wiki/teams/5124/notebook/jul4.webp)

Ruby shows off our work so far at the iGEM meetup in Warwick!

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**Week 7 [29/07/24 - 2/08/24]**

##### --- **Monday 29th of July**

* Fixed circuit diagram (components now actually have a power supply 🤦‍♂️), 3D printer cleaning and troubleshooting again… [Oli]
* Emailed farmers and CHeCS about setting up interviews [Becky]
* Lysis of Cas 12a/13a BL21 large expressions [Louise, Ellin]
* Nickel column protein purification of Cas12a and Cas13a in BL21, couldnt continue purification as the columns were very clogged up, due to the use of large volumes of cell lysate [Yara]
* Continued the protein purification until 8 pm!! [Chloe]
* Lab and 3D printing photography [Yara]
* Emailed about the properties of Thermo Fisher's probes [George]
* Added protocols onto one note (8x8 well plate, ampicillin plates, making stock solutions of antibiotics) [George]
* Worked on diagrams to show how the project works [Ruby, George]

##### --- **Tuesday 30th of July**

* After small tweaks to the printer calibration, the phone is finally being printed [Oli]
* Update wet lab timeline [Yara]
* Western blot of Cas12a and Cas13a, cultured using IPTG and autoinduction respectively [Yara, Ruby]
* Finished graphics and description for the project, cas12a & cas13a [Ruby]
* Responded to check in form and did another for blood, plus emailing [Louise]
* Draft COSHH form for blood [Oli]
* Organised and graphed results of previous experiments [George]
* Research for the human practices section of the wiki [Becky]
* Transformation of BL21 with Cas 12a, 13a, and PX-1900 [Ellin]
* Ran RNAase contamination experiments on RNAase free water and elution buffer [Ruby]

##### --- **Wednesday 31th of July**

* Meeting deciding project direction after evil western blot with Chloe and Mark [Ruby, Becky, Lousie, Yara, Talia, Ellin, George]
* Update wet lab timeline [Yara]
* Physics lab photography [Yara]
* Wrote protocols for DNA/RNA contamination experiment [Ruby]
* Second protein purification of Cas12 using nickel column and purifying again with PD10 column [Ellin]
* Spoke to Dan and wrote up the ideas about amplifying signal or removing background noise [George]
* Wrote up protocols onto one note (Fluorescein concentration curve, Suflorhodamine concentration curve, making buffers, testing different enzyme concentrations for Rnase as well as the developments) [George]
* Moved 3D printer to cooler area and started test print again [Oli]
* Fluorescein photodiode and LED experiment: testing arrangements of LED to photodiode, as well as signal response from varying concentration of fluorescein [Oli]
* Transcription of Cas12a crRNA hsp20 sequence [Yara]
* Ran DNAase contamination experiments on 'RNAase free water', elution buffer and milliq [Ruby]
* Ran Sulphurholomine blood experiments- red blood is a no go [Ruby]

##### --- **Thursday 1st of August**

* Cleaned up successful 3D print. Added updates to Hardware section in the notebook. Learning AutoCAD to produce a frame for a gel-based BP-Filter [Oli]
* Did a tapestation of Cas12a crRNA hsp20 transcript, as well as transcription and tapestation of Cas13a crRNA BCL2\_a and Cas13a target BCL2\_a [Yara]
* Brainstormed ideas for our promotional video [Becky]
* Ran DNAase contamination experiments on milliq and autoclaved milliq [Ruby]
* Ran DNAase contamination experiments on milliq from downstairs, equilibriation buffer and highland mineral water [Ruby]
* Western blot [Ellin]

##### --- **Friday 2nd of August**

* Hardware experiments with perpendicular arrangement and bandpass filter [Oli]
* Ran RNAase contamination experiments on equilibriation buffer and milliq [Ruby]
* Organised team visit to farm with david andrews [Ruby]
* Team meeting/lab update [Ruby, Becky, Louise, Yara, Ellin, Oli]
* Finishing off western blot and clear out nickel columns [Ellin]
* Ran an agarose gel of the transcribed Cas13a crRNA BCL2\_a and Cas13a target BCL2\_a samples [Yara]
* Making buffers for next weeks experiments [Lousie, Becky]

![two students looking at a 3D printer](https://static.igem.wiki/teams/5124/notebook/w7-circuit-test.webp)

Testing our hardware circuits!

---

![A beaker glows softly, in the corner of the lab.](https://static.igem.wiki/teams/5124/notebook/transcription.webp)

Results from Cas12a crRNA transcription

---

![A white plastic box sits on a table, with a mobile phone sized slot cut in the end.](https://static.igem.wiki/teams/5124/notebook/jul8.webp)

To work with our app, we had a go at 3D printing a box to contain our samples to cut out external light and allow maximum contrast for photos. Here is the first prototype.

---

## August

---

**Week 8 [5/08/24 - 9/08/24]**

##### --- **Monday 5th of August**

* Lysis of BL21 with Cas13a [Louise]
* Protein purification [Nigella, Louise]
* Wrote up protocols onto one note (Testing different enzyme concentrations for Dnase, Testing different probe concentrations for Dnase and Rnase and trial of whole test put together as well as the developments) [George]
* Completed the blood COSHH form [George]
* Researched experiments for how to make plasma from blood [George]
* Remaking nickel columns [Becky, Yara]
* Transcription, tapestation and agarose gel write up [Yara]
* Went on the hunt for video equipment and sent out some emails. Drafted some initial ideas for the intro video. [Ben]
* Prep for farm trip tomorrow [Ellin]
* Going over and processing data of the 96 well plate contamination experiments [Ruby]

##### --- **Tuesday 6th of August**

* Wiki tweaking: fixing formatting issues [Nigella]
* Western blot [Yara, Talia, Ben, Nigella]
* Scouting locations for team/portrait photos and taking practise shots [Yara, Nigella]
* Updated wet lab timeline [George]
* Research on how to separate blood, SHERLOCK system and fluorophores [George]
* Went and interviewed David and the other staff at Warson beef [Louise, Ruby, Becky, Ellin]
* Collected dung and sputum samples [Louise, Ruby, Becky, Ellin]

##### --- **Wednesday 7th of August**

* Drafting email to local MPs requesting for interviews [Nigella]
* Had a gander around the campus to construct a storyboard for our promotional video [Ben, Becky]
* Helping out with promo video structure and script, looking through videos from previous years [Nigella]
* Filtered smaller chunks out of our purified proteins with size exclusion centrifuge columns [Louise, Ellin]
* SDS page and western blot [Ellin]
* Writing up protocol improvements [Louise]
* Talked to Dan Barber and Mark about how to improve tapestation results [Yara]
* Transcription of Cas13a crRNA/target HBEGF\_a, using nuclease free water. Tapestation performed using improved protocol, including running RNA control and original DNA template [Yara]
* Planning experiments with blood along with further research [George]

##### --- **Thursday 8th of August**

* Writing up results from yesterday's tapestation [Yara]
* Tweaking wet lab protocols, in preparation for their upload to the wiki [Yara]
* Completed warson beef interview page [Ruby]
* Emailed MPs [Ruby]
* Continued to write the script for the promotional video and learning to edit videos [Becky]

##### --- **Friday 9th of August**

* Emailed MP as actually in Honiton and Sidmouth constituency [Louise]
* Updating journals/wet lab timeline [Louise]
* Taking portrait photos of team members (Ben and Louise) for the wiki teams page [Yara, Nigella]
* Editing team members' portrait photos [Yara]

![iGEM student gives a thumbs-down outside a locked door.](https://static.igem.wiki/teams/5124/notebook/aug1.webp)

Ben's hunt for a recording studio to record our narration for the video was not entirely successful

---

![iGEM student walking through a forested area with camera slung over shoulder.](https://static.igem.wiki/teams/5124/notebook/yara-walking.webp)

Yara searching for a good spot for team photos.

---

![Cas13a purification western blot results.](https://static.igem.wiki/teams/5124/notebook/aug3.webp)

Ellin's western blot of Cas13a before, during, and after the purification.

---

**Week 9 [12/08/24 - 16/08/24]**

##### --- **Monday 12th of August**

* Interview with NFU [Ruby, Anouska, Nigella]
* Taking portrait photos of Ruby and Anouska for the wiki teams page [Yara, Nigella]
* Editing team members' portrait photos [Yara]
* Updating notebook [Anouska]
* DNase treatment and tapestation for Cas13a crRNA/target HBEGF\_a [Yara]
* Began work on 2-minute intro video [Ben]

##### --- **Tuesday 13th of August**

* Emailing potential interviewees [Nigella]
* Taking portrait photos of John, George, Ben and Nigella for the wiki teams page [Yara, Nigella]
* Taking photos of John in the lab [Yara]
* Lab photography of Yara [Anouska]
* DNA probe experiments [Ruby]
* Troubleshooting contamination with DNase probes [Rube, George]
* Finding methods for transporting pigs blood [George]
* Preliminary intro video work continues [Ben]

##### --- **Wednesday 14th of August**

* Protein purification with the AKTA machine [Louise, Ellin]
* Completing safety form [Louise]
* Finalising the transcription, tapestation and DNase write ups [Yara]
* Lab photography of George, Ruby, Ellin and Louise [Yara]
* Organised meeting with Ian Roome [Ruby]
* Meeting with Penn State iGEM team [Anouska, Nigella, Yara, Ben, Ruby]
* Promo video script writing [Nigella, Anouska, Ben]
* Emailed IDT about Dnase Alert Kit [George, Yara]
* Adapting and completing testing blood using a centrifuge/3D-Fuge to be able to show fluorescence through the blood more easily [Ruby, George]

##### --- **Thursday 15th of August**

* Pig blood collection from abattoir [Anouska, Ruby, Nigella]
* Even more video script writing [Ben]
* Did RNA probe re-runs with amended protocol [Ruby]
* Ran blanks for probe experiments [Ruby]
* Protein purification with the AKTA machine [Louise]
* SDS-page and western blot [Ellin]
* Testing absorbance of filtered horse blood [Anouska, Nigella, George]
* Running absorbance spectra for different dilutions of blood to find peaks to be able to measure the decrease in colour of the blood in other experiments [George]
* Further research and amendments to all blood protocols [George]

##### --- **Friday 16th of August**

* More promo video script writing [Nigella, Anouska, Louise, Ben]
* Emailing interviewees [Ellin, Nigella]
* Completed experiments involving heating and centrifuging blood [George]

![2 iGEM students standing over a camera looking at photos taken.](https://static.igem.wiki/teams/5124/notebook/photos.webp)

Ben and Yara looking through the photos for the teams page!

---

![Becky demonstrates that she has taken off her shoes.](https://static.igem.wiki/teams/5124/notebook/blood.webp)

Pig blood successfully acquired from the abattoir. Next mission: flouroscien experiments.

---

![Multiple members of igem team smiling into laptop camera waiting for teams meeting to start.](https://static.igem.wiki/teams/5124/notebook/pennstate.webp)

Getting ready to meet the Penn State iGEM team by video call!

---

**Week 10 [19/08/24 - 23/08/24]**

##### --- **Monday 19th of August**

* Caught up on transcription stuff, writing part registry description and Cas12/13a transformation. [Tom]
* All parts added to the registry! [Louise]
* Prep for auto induction and wet lab write up [Ellin]
* Test shots for video [Anouska, Nigella]
* Writing up proper description page for wiki [Nigella, Anouska]

##### --- **Tuesday 20th of August**

* Collated all RNA probe experimental data [Ruby]
* Wrote interview questions for Ian Roomes [Ruby, Becky, Anoushka]
* Research into re-design for a low-cost fluorometer [Oli]
* Produced LB for auto induction and finishing write up [Ellin]
* Prepared overnights, repeated transcription and prepared for lysis protocol for tomorrow. [Tom]
* Added features onto parts [Louise]
* Promo video filming [Nigella, Anouska, Becky, Ruby, Ben]
* Making graphics and animations for promo video [Anouska, Nigella]

##### --- **Wednesday 21th of August**

* Editing promo video, continuing making animations and graphics [Anouska, Nigella]* Making logo animation for wiki home page [Nigella]
  * Sending off more emails for interviews (DWT, WWF) [Nigella]
  * Contacting IDT for DNA probes [Ruby]
  * Made powerpoint for ian roomes interview [Ruby]
  * Wiki tweaks and Notebook page updates, particularly to images [Ben]
  * Writing the bronze contribution section of the wiki for RNA probe [Becky, Ruby]
  * Made up autoinduction media and inoculated with overnight Cas12a cultures. [Tom]
  * Prepared protocol and carried out plasmid cleave/blunting and then T7-transcription [Tom]
  * Added blurb text onto some of the registry pages [Louise]
  * Finalised Safety form [Louise]
  * Research into novel low-cost lock-in amplification method, after a meeting with Bio-photonics expert Julian Moger [Oli]

##### --- **Thursday 22th of August**

* Harvesting BL21 Cas 12 and started auto-induction of BL21 Cas 13 [Ellin]
* Interview with Ian Roome [Anouska, Becky, Ruby]
* Starting on IHP wiki page [Anouska]
* Finishing Promo Video [Nigella, Anouska]
* Writing the bronze contribution page of the wiki [Ruby, Becky]
* Began hardware write-up for the wiki. Further research into lock-in amplification. Reached to engineering department for Circuit board printing, which is also of interest to another non-iGEM physics researcher [Oli]
* RNA probe experiment reruns [Ruby]
* Carried out Dnase digest on T-7 transcription products, than ran digest on Tapestation [Tom]
* Restriction digest of crRNA and target plasmids, and agarose gel. [Tom]
* Finished experiments testing reducing bloods opacity including hand centrifuge and filtering [George]
* Graphed previous results from experiments testing reducing bloods opacity [George]

##### --- **Friday 23th of August**

* Tecan results & data processing [Ruby]
* Cell harvesting [Ruby, Becky]
* Hardware wiki writing. Waiting on response from engineering regarding PCB. Looking into other cheap optical detection methods. [Oli]
* Made in silica gel to compare to restriction digest. And repeated Dnase digest, and tape station. [Tom]
* Continued writing for crRNA registry entries [Louise]
* Research into how to make HEX (Hexochlorofluorescein) [George]
* Beginning to write up results for wiki [George]

![image of animation process using adobe express](https://static.igem.wiki/teams/5124/notebook/img-0641-2.webp)

Nigella making the opening logo animation for the wiki home page!

---

![igem team member sitting at table making animations using powerpoint](https://static.igem.wiki/teams/5124/notebook/animations.webp)

Anouska making the animated explanation of Cas proteins for our promo video

---

![ 2 igem team members helping set up another member for team photos](https://static.igem.wiki/teams/5124/notebook/yara-photoshoot.webp)

Yara and Ruby taking pictures of Nigella for the team wiki page

---

**Week 11 [26/08/24 - 30/08/24]**

##### --- **Monday 26th of August**

* Bank Holiday!

##### --- **Tuesday 27th of August**

* Creating English captions for promo video [Anouska]
* Submitting promo video [Nigella]
* Making sponsor card for wiki footer and making animated graphics for description page [Nigella]
* Writing up IHP page for wiki [Anouska, Ruby]
* Brainstorming adapted area framework [Ruby, Anouska]
* 3D printing components for our hardware. Got a quote for the PCB manufacturing [Oli]
* Using Compton scattering to determine what proportion of energy needs to be removed from measurements [Oli]
* Graphed results for reducing blood opacity and Rnase probes in different environments [George]
* Wrote up the protocol for the reduction of blood opacity [George]
* Produced new batches of buffer [Ellin, Louise]
* Tapestation results troubleshooting - Dnase digestion, metal bead extraction. Also conducted qubit quantification of DNA contents of samples. [Tom]

##### --- **Wednesday 28th of August**

* 3D printing components. PIC research as an alternative to our circuit. Decided on Fourier analysis to workout noise [Oli]
* Re-ran Dnase and metal bead extraction and ran a Tapestation results again [Tom]
* Graphed results for the excitation and emission spectra for fluorescein and DNase Alert probe [George]
* Writing results up for concentration curves [George]
* Ran fluorescence spectra [George]
* ICCD framework writing [Ruby]
* DNA probe initial experimentation with new probes [Ruby]
* Writing up interviews into IDCC framework [Anouska]
* Adding more to registry pages [Louise]

##### --- **Thursday 29th of August**

* Wrote protocol for full scale T7 transcription and clean up for tomorrow [Tom]
* Started getting images for registry ready [Tom]
* Formatting Home page on Wiki [Nigella]
* PCB Gerbers sent to manufacturer for printing, other relevant information regarding hardware component substitutes researched as well to reduce the cost of production [Oli]
* ICCD framework writing [Ruby]
* Interview presentation prep [Ruby]
* Harvesting BL21 cells containing cas [Becky, Ruby]
* Writing results for probe validity [George]
* Designing and coding results page for wiki [Anouska]
* Writing up the RNAse experiment for bronze contribution [Becky]

##### --- **Friday 30th of August**

* Curated fluorescein concentration equivalents graphs for the results section of the Wiki. Producing diagrams for our hardware to go on the wiki [Oli]
* Michelsen-Menten enzyme kinetic analysis for our RNA Probes [Oli, George]
* Writing more ICCD framework entries for IHP [Ruby]
* Meeting with David Cotton, dairy farmer [Ruby, Becky, Anouska, Tom]
* Carried out cleaving/blunting, T7 transcription. [Tom]
* Creating a rough flowchart for the wet lab [George]
* Writing results for sample opacity [George]
* Tweaks on whole of wiki [Nigella]
* Writing up silver HP [Anouska]

![image of animation process using adobe express](https://static.igem.wiki/teams/5124/notebook/animationevi.webp)

Nigella creating her trademark animated infographics for the description page

---

![person holding up small tube of clear blood](https://static.igem.wiki/teams/5124/notebook/blooddilute.webp)

Diluted blood, following heating protocol. This was tested for opacity by George, in tandem with the fluorescein detection experiment.

---

![image of 3D printed hardware design case](https://static.igem.wiki/teams/5124/notebook/hardwareweek11.webp)

Oli's wonderful 3D-printed casing, which will hold all electronic and optical components, plus cow sample

---

## September

---

**Week 12 [2/09/24 - 6/09/24]**

##### --- **Monday 2nd of September**

* Completed scaled up transcription protocol and conducted a Tapestation test. [Tom]
* Writing up silver HP for wiki [Anouska]
* Lysis of BL21 Cas 12 [Ellin and Louise]
* Fixing audio issues with promo video + wiki hardware and home page designing [Nigella]
* Wiki updates [Ben]

##### --- **Tuesday 3rdth of September**

* Attomarker interview [Anouska, Tom, George, Oli]
* Spoke with Ben Gardner with regards to lock-in amplification and other low cost methods of fluorescent detection. Researched the paper Ben gave me. Created order list for the circuits and obtained relevant existing materials to reduce strain on the budget. [Oli]
* Writing up Attomarker interview in IDCC framework [Anouska]
* Loading Cas12 onto His-Trap column for AKTA [Louise]
* AKTApure protein purification of Cas 12 [Ellin and Louise]
* Writing up the RNAse experiment for the bronze contribution [Becky]
* crRNA clean up experments and troubleshooting [Tom]
* Continuing wiki home + hardware page [Nigella, Ben]

##### --- **Wednesday 4th of September**

* Adding to description page, silver HP and IHP on wiki [Anouska]
* Hardware write for the description page of the wiki and the hardware page [Oli]
* Lysis of Cas13 [Louise]
* Loading Cas13 onto His-Trap column for AKTA [Louise]
* Western blot and qubit [Ellin]
* Writing up the RNAse experiment for the bronze contribution [Becky]
* crRNA clean up experments and troubleshooting [Tom]
* Added Flutter section to Hardware page, and populated it with images [Ben]
* Re-ran DNase experiments for differing enzyme concentrations [George]
* Created buffers for upcoming cas12a experiments [George and Louise]

##### --- **Thursday 5th of September**

* Hardware write-up for the wiki [Oli]
* Scripting/story boarding the presentation video [Becky, Talia]
* crRNA clean up, quantification and troubleshooting [George, Tom]
* 1st attempt doing a full test of Cas12a [George (mostly), Tom (helped)]
* Collecting fractions of Cas13 [Louise]
* Size exclusion AKTA purification of Cas 13a [Louise]
* Interview with Piers Pepperell [Anouska, Louise]
* Interview write up [Anouska]
* Fixed formatting problems with hardware page, and added more sections [Ben]
* Oxford meeting [Anouska, George, Tom and Becky]
* Re-ran DNase experiments for differing probe concentrations [George]

##### --- **Friday 6th of September**

* Western blot [Ellin, Becky]
* Began circuit assembly, waiting on a surface-mount capacitor but then it should soon be finished [Oli]
* Wrote up text for Hardware page [Oli, Ben]
* Completed maths for upcoming cas12a and cas13a [George]

![person holding up small tube of clear blood](https://static.igem.wiki/teams/5124/notebook/hardwareweek12.webp)

Our hardware, disconnected from any power supply.

---

![picture of hardware connected to software readout results](https://static.igem.wiki/teams/5124/icons/hardwareresults.webp)

The readout of background noise from our lock-in amplifier, as measured by Oli

---

![picture of western blot](https://static.igem.wiki/teams/5124/notebook/westernblot.webp)

Western blot after purification of Cas12a on AKTA pure by Ellin

---

**Week 13 [9/09/24 -13/09/24]**

##### --- **Monday 9th of September**

* Presentation video planning, wiki description editing [Nigella]
* Hardware assembly, wiki writing [Oli]
* HP page write-ups [Anouska]
* Filling in team page [Anouska]
* Presentation video script writing [Becky, Talia]
* More wiki tweaks [Ben]
* Further planning of Cas13a and Cas12a experiments [George and Tom]
* Run of a cas13a fluorophore cleavage experiment [George]

##### --- **Tuesday 10th of September**

* Full hardware assembly as well as mock-up for Paris [Oli]
* Visited David Andrews' farm to observe first day of bTB testing + interview [Nigella, Anouska, Becky, Ben]
* Adding new info from interview to wiki [Nigella]
* Presentation video script writing [Becky, Talia, Nigella]
* Transformations of cas samples into DH5 [Becky]
* Transcription of Cas13a (crRNA and target) and Cas12a (crRNA) sequences, then cleaned up and quantified. [Tom]
* Made ampicillin plates [George]

##### --- **Wednesday 11th of September**

* Got the hardware to pair with the firmware and software, was able to take measurements with it. Need to optimize the process for better readings. [Oli]
* HP wiki page [anouska]
* Ran experiments to see if ribolock is needed in cas13a reactions [George]
* Planning of controls experiment for cas13a [George, Tom]
* Graphing of DNase contamination data [George]

##### --- **Thursday 12th of September**

* More tables for the wiki [Oli]
* Safety page for the wiki [Louise]
* Minipreps and quantification of cas12a targets [Tom]
* Initial tests Cas12a [Tom]
* Hardware wiki page [anouska]
* Taking rest of the team pictures and editing [anouska]
* Making parts page for wiki and presentation video script writing [Nigella]
* Serial dilution of blood experiment [George]

##### --- **Friday 13th of September**

* Cas expression meeting [Everyone]
* Team page [anouska]
* Writing presentation video script + general wiki clean up [Nigella]

![image of bunch of tubes in tube rack](https://static.igem.wiki/teams/5124/notebook/cas13atrial.webp)

George trialling components used in cas13a system

---

![cows at farm waiting in line to be tested for bTB](https://static.igem.wiki/teams/5124/notebook/cows2.webp)

Nigella, Anouska, Ben and Becky visiting David Andrews's farm to observe bTB testing

---

![picture of hardware with LED flashes exciting the sample](https://static.igem.wiki/teams/5124/notebook/hardwareweek13.webp)

Picture of the hardware in action. The LED flashes at a fixed frequency, exciting the sample, which can then be measured using the lock-in amplifier

---

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

## Our Success

---

Through a series of design-engineer cycles, BoviTect has managed to **successfully** produce a positive fluorescent result from multiple bTB biomarkers. We have used synthetic biology to **successfully create a diagnostic tool for bTB**.The figure below demonstrates this; there is a clear and rapid fluorescent signal that is **significantly higher** than the fluorescence from the negative control in response to four of our selected bTB biomarker targets.

---

![targets (PLAUR, CXCL8, RGS16, NR4A1 and FOSB) in the Cas13a system compared to the negative control where Cas13a has been removed, with a threshold at 10 nM fluorescein which is 10x higher than the highest negative control.](https://static.igem.wiki/teams/5124/results/engineering-graph.webp)

*[Figure 1] This shows the targets (PLAUR, CXCL8, RGS16, NR4A1 and FOSB) in the Cas13a system compared to the negative control where Cas13a has been removed, with a threshold at 10 nM fluorescein concentration equivalents (nM) (dotted black line), units given in fluorescein concentration equivalents (nM) and R squared values for the Michaelis-Menten model the data is around. PLAUR and NR4A1 only shows the top error bars and CXCL8 shows only the bottom to make the data more clear to read. Only 2 repeats were able to be done and this is due to time and budget limitations.*

---

Figure 1 shows PLAUR and CXCL8 have surpassed the threshold value (>10-fold above negative control) and therefore are confirmed to be a positive result meaning our Cas13a system has multiple targets that can detect *Mycobacterium tuberculosis* biomarkers and report a significant positive fluorescent result. The threshold was picked as 10x the value of our highest negative control as our [stakeholders](https://2024.igem.wiki/exeter/human-practices) have stated that reducing the chance of false-negatives is one of the largest priorities. As well as this target NR4A1 and RGS16 also had a significant increase from the negative control, however did not surpass the threshold.

---

## **Design, Build, Test, Learn Cycles**

Our final working diagnotic tool is made up of many components which in turn were modified and tested in the process much like the Design, Build, Test, Learn process lays out. There were **three significant** design cycles that altered our engineering success the most, which are outlined below. We found that these cycles ended up being more like spirals, as the diagrams show. These have two or three design cycles feeding onto the next.

---

## **Sample Preparation Design Cycle**

![design engineer cycle visualised as a spiral containing two cycles of design, build, test, learn](https://static.igem.wiki/teams/5124/engineering/sample-preparation-design-cycle.webp)

### **Design 1**

Knowing that the eventual field-diagnostic would be working with biological fluids such as blood, we needed to assess whether the fluorescent signal would be blockedby the sample needed for the diagnostics test. We therefore designed a series of experiments to assess various methods of sample preparation in order to reduce sample opacity and decrease background noise.

### **Build 1**

In order to abide by the 3Rs[1] as a key part of our scientific values in [Human Practices](https://2024.igem.wiki/exeter/human-practices), we began by testing on synthetic blood as the first R is Replacement. In this sense we were trying to replace the need for using real animal blood by using synthetic blood.

We trialed different methods to reduce the opacity by the blood including filtering and the use of a "hand centrifuge"[4].

We trialed a hand centrifuge instead of an electric one because we wanted to try and make our test more accessible in the field where we believed electricity may not be available.

### **Test 1**

To mimic the signal we expected from our diagnostic, we spiked synthetic blood samples with fluorescein and measured fluorescencewith a fluorometer (Tecan Infinite m200 Pro) at excitation 488 nm and emission 519 nm which were then converted into fluorescein concentration equivalents.

![picture of blood samples in a block](https://static.igem.wiki/teams/5124/engineering/blood-picture.webp)

*[Figure] Our blood samples when conducting opacity tests.*

### **Learn 1**

From this we learnt that synthetic blood was not a suitable substitute to replace biological material as it lacked many of the key similarities to real blood, such as coagulants and serum.

However, we did learn that the background noise was very high and this interfered with the fluorescent reading, therefore we would need to find a solution to reduce opacity to allow our test the highest sensitivity possible as this was an important factor from our [stakeholders](https://2024.igem.wiki/exeter/human-practices) and key to integrating human practices into our research process.

We also learnt from our [Human Practices](https://2024.igem.wiki/exeter/human-practices) that according to veterinary practitioner Piers Pepperell, an electric centrifuge, or other methods that involve electricity, were feasible as there is often electricity available in the locations that bTB testing occurs.

### **Design 2**

We redesigned our sample tests but this time using real blood. We ensured to keep this in line with our scientific values and the 3Rs[1] by following the principle of reduction. To do this we bought the minimum amount of pigs blood which was a waste product from an abattoir to trial. Buying from an abattoir also meant that this was a non-hazardous material as it was already deemed food-grade. Cattle blood was not used as it is not availble for sale in the UK due to biohazard restrictions.

### **Build 2**

We didn’t try heating and coagulation with synthetic blood because we knew there weren't any coagulants in the synthetic blood, but now we had real blood we could trial methods such as coagulating the blood through waiting and heating as well as repeating filtering and centrifuging on real blood.

Also, as informed by [Human Practices](https://2024.igem.wiki/exeter/human-practices) we now were trying to an electric powered centrifuge.

However we had to make some adaptations, for example using a 5 μm filter size for the first filter and then decreasing the filter size for each subsequent filter to 1.2 μm, 0.8 μm and 0.2 μm filter due to the red blood cells size blocking the smaller filters.

### **Test 2**

We conducted these different tests on the pigs blood and did a fluorescence intensity scan at excitation 488 nm and emission 519 nm as decided on by previous experiments. These results were converted into percentage change of the fluorescein concentration equivalent when compared to a benchmark of untreated blood with 10 nM fluorescein. This data is availble on the [measurement page](https://2024.igem.wiki/exeter/measurement).

Our method for testing the heating of blood was drawn from Arizti-Sanz, 2020 [2]

### **Learn 2**

Analysis of our results showed that heating was the most effective at reducing the absorbency of the blood so that we can get a clear fluorescent result to the bTB targets. Our eventual diagnostic device would be required to include a heating elements for blood sample processing.

Click for References

[1]The 3Rs | NC3Rs [Internet]. Nc3rs.org.uk. The 3Rs | NC3Rs; 2017 [cited 2024 Sep 4]. Available from: https://nc3rs.org.uk/who-we-are/3rs

[2]Arizti-Sanz J, Freije CA, Stanton AC, Petros BA, Boehm CK, Siddiqui S, et al. Streamlined inactivation, amplification, and Cas13-based detection of SARS-CoV-2. Nature Communications [Internet]. 2020 Nov 20 [cited 2024 Oct 1];11(1). Available from: https://www.nature.com/articles/s41467-020-19097-x#Sec8

---

## **RNA Transcription Design Cycle**

![design engineer cycle visualised as a spiral containing two cycles of design, build, test, learn](https://static.igem.wiki/teams/5124/engineering/rna-transcription-design-cycle.webp)

### **Design 1**

Potential targets from either the *Mycobacterium tuberculosis* genome (for direct bTB detection) or the *Bos taurus* genome (sequenced from a Hereford cow, for identification of immune resonse biomarkers) were identified from literature[5,6]. These genes were processed by our custom python scripts (for either Cas12a or Cas13a respectively). Using string manipulation the scripts found all possible spacer sequences within genes/mRNA, that satisfy the Cas12a PAM site and Cas13a PFS site.

These spacer sequences were appended to the crRNA for the respective Cas protein, to make the full sgRNA sequence. The sgRNA sequences which had the correct hairpin folding were selected, and DNA templates for the following were synthesised by IDT: Cas12a sgRNA, Cas13a sgRNA, Cas13a targets and Cas12a target DNA was also ordered.

### **Build 1**

We planned to amplify the 250 ng of each DNA template for the target sequences using PCR, so there would be enough template DNA to run a T7 *in vitro* transcription reaction of Cas13a targets. We also planned to order enough linear sgRNA DNA template oligomers to transcribe the sgRNA.

Therefore PCR amplification and T7 transcription was carried out on relevent sequences, as can be seen on the sgRNA tab of our [Results](https://2024.igem.wiki/exeter/results) page.

![abstract representation of DNA sequences synthesised by IDT](https://static.igem.wiki/teams/5124/results/screenshot-2024-10-01-at-10-11-03.png)

*[Figure 2] Abstract representation of DNA sequences synthesised by IDT.*

*A) Template DNA to be used in transcription of Cas13a and Cas12a sgRNA. The sequence contains a T7 promoter, however unlike the target sequences does not contain a forward M13 primer. Since transcription needs to end abruptly after the spacer sequence, to ensure specific folding of the RNA, requried to form a complex with the Cas13a and Cas12a proteins.*

*B) Template DNA for Cas13a target, including a T7 promoter and M13 primer binding sites for amplification, via the polymerase chain reaction.*

*C) DNA sequence to be used as the Cas12a target direclty, including M13 primers binding sites for amplification, via the polymerase chain reaction.*

### **Test 1**

A High Sensitivity DNA Qubit quantification was carried out on the PCR product, which showed all sampled had a yield below &lt0.01 µg mL.

Nevertheless a T7 *in vitro* transcription was attempted, however no RNA was detected on a HSRNA Screentape (Agilent Tapestation 4200) - likely because there was not enough DNA template.

### **Learn 1**

It became apparent that our transcription protocols were optimised for larger amounts of template DNA than we had, and due to budgetary constraints we could not order in enough linear DNA to carry out the transcription reactions.

At this point there was also concerns about removal of DNA from our transcription product, as template DNA would trigger the Cas12a system, leading to false positives in our final testing.

### **Design 2**

The template DNA for; Cas12a sgRNA, Cas13a sgRNA, Cas13a targets and DNA for Cas12a targets, were re-designed and re-synthesised by IDT to include Type IIS restriction sites, so they could be cloned into plasmids.

In order to ensure transcription ended abruptly a Type IIS endonuclease site was added just after the sgRNA sequence ended, this in theory would cause T7 RNA polymerase to run off our linearised DNA templates. Klenow was used to blunt the ends of the linearised plasmids and restore any cleaved spacer nucleotides, and to prevent the plasmids from reforming.

### **Build 2**

Our supervisor then cloned our DNA fragments into a high copy number plasmid, pX1800, then transformed the plasmids into DH5α *E.coli*, which in turn were grown and a plasmid extraction was carried out.

The plasmids were cleaved and blunted in a combined reaction, then transcribed using a T7 in vitro transcription reaction. We also compared different ways of cleaning up the DNA templates; using a DNase digestion or a DNase digestion followed by a AMPure bead extraction (to remove the DNase). This data can be viewed on the sgRNA section of our [Results](https://2024.igem.wiki/exeter/results) page.

![First successful transcription results, of Cas13a targets and Cas12a sgRNA, measured on a HSRNA tape (with Agilent Tapestation 4200)](https://static.igem.wiki/teams/5124/results/sgrna-results-pic-2.png)

*[Figure 3] First successful transcription results, of Cas13a targets and Cas12a sgRNA, measured on a HSRNA tape (with Agilent Tapestation 4200)*

*A) Shows tape columns: B1- #8 CXCL8 target, C1 - #9 RGS16 target, D1 – #11 EthA\_b sgRNA, E1 – #15 RD4\_c. Blue box surrounds bands where desired peaks are observed.*

*B) Shows the normalised sample intensity graph of #9 RGS16 target. Due to a slightly degraded lower marker, a peak at 45 bp is observable. A 219 bp peak, close to our desired 153 bp (and due to the unreliability of the degraded sample buffer, this is likely our desired product).*

*C) Shows the normalised sample intensity graph of #15 RD4\_c. Due to the degraded lower marker, and the small size of the transcribed fragment, the desired product peak at 38 bp (44 bp expected) is adjoined to the lower marker. The 124 bp peak is likely from the transcription of a plasmid templates that has not been cleaved properly (127 bp expected).*

### **Test 2**

Transcribed samples were ran on a RNA Screentape using an Agilent Tapestation 4200, showing RNA of our desired size was present. Both DNase digestion and the DNase digestion followed by a AMPure bead extraction, had similar results.

Our final Cas13a testing resulted in a functioning detection system, which reinforces the information of the Tapestation as the sgRNA and targets must be functioning as intended.

![graph showing first successful transcription results, of Cas13a targets and Cas12a sgRNA, measured on a HSRNA tape](https://static.igem.wiki/teams/5124/engineering/engineering-sgran-results-pic.webp)

*[Figure 4] Shows the Agilent Tapestation 4200 RNA ScreenTape results from final T7 in vitro transcription. Where; Cas13a sgRNA sequences (1,2,4,5), Cas13a target sequences (6,7,8,9,10) and Cas12a sgRNA sequences (11,12,13,14,15) were tested. Against a contorl, where the T7 polymerase was not included in the reaction mixture. This figure includes an image of the RNA ScreenTape and a representative normalised sample intensity sample graph.*

*A)Shows the tape gel – showing uniformity among varients of each RNA component.*

### **Learn 2**

Dnase digestion followed by a denaturation cycle was used, due to budgetary restraints concerning AMPure bead use.

We found we could produce high quality sgRNA and target RNA for our Cas13a test, using our transcription protocol.

---

## **Cas13a Expression and Purification Design Cycle**

![design engineer cycle visualised as a spiral containing three cycles of design, build, test, learn](https://static.igem.wiki/teams/5124/engineering/cas13a-expression-and-purification-design-cycle.webp)

### **Design 1**

The coding sequence for LwCas13a was taken from the plasmid pC029-Lw2Cas13a from *Leptotrichia wadei F0279* (Addgene plasmid #91919)[1] and codon optimised for expression in *E. coli* using the IDT tool. Any forbidden restriction enzyme sites were removed. A 6xHis tag and TEV protease cleavage site was added at the N-terminal, Type IIS cloning prefix and suffixes were added and the complete sequence was synthesised as a g-Block by IDT. This plasmid was made with the intention to transform it into BL21(DE3) and Rosetta.

### **Build 1**

This sequence was cloned into a medium copy plasmid (origin of replication from pBR322[2]) carrying an ampicillin selection marker with a composite part (IPTG inducible T7 promoter system) comprising the T7 promoter, lac operator and the RBS from bacteriophage T7 gene 10 and the transcription terminator from bacteriophage T7 RNA polymerase.

Plasmids were transformed and the protein expressed. As a positive control for protein expression we also transformed a plasmid coding for His-Tagged mCherry. As a negative control for protein expression we transformed an empty plasmid pX1900.

*E.coli strain*: BL21(DE3) and Rosetta.

Induction method: Addition of 100mM IPTG (37°C overnight) to 250mL cultures.

Purification: Gravity Nickel Affinity column only, washed with 20mM Imidazole buffer, eluted with 250mM Imidazole buffer.

### **Test 1**

**Western Blot Result:**

![Western blot result showing some expression of Cas12a in BL21(DE3) and Cas13a in Rosetta](https://static.igem.wiki/teams/5124/engineering/getimage.webp)

*[Figure 5]: A western blot of purified proteins from gravity nickel affinity column. First 4 proteins produced in BL21(DE3) and others produced in Rosetta.*

### **Learn 1**

Cas13a did express in Rosetta but with a low yield. Rosetta transformation efficiency was low (Cas12a did transform and express in BL21(DE3)), and competent cell preparation proved difficult.

### **Design 2**

Decided to continue with BL21(DE3) rather than Rosetta due to the better transformation results. Changed to autoinduction media [3]to see if that gave better expression results.

### **Build 2**

Transformed and expressed again.

*E.coli strain:* BL21(DE3) only

Induction method: Autoinduction media (20°C for 24 hours) to 1L cultures.

Purification: Gravity Nickel Affinity column only but poured fresh columns but still washed with 20mM Imidazole buffer, eluted with 250mM Imidazole buffer.

### **Test 2**

**Western to show BL21(DE3) in autoinduction:**

![Western blot result showing much stronger bands of expression of Cas12a and Cas13a in BL21(DE3) in autoinduction media](https://static.igem.wiki/teams/5124/engineering/screenshot-2024-10-01-at-10-47-22.webp)

*Figure 6: Western blots of proteins from gravity nickel affinity column. All proteins produced in BL21(DE3) and lysate is before purification and elutions are after purification. Concentration of Cas13a elution (measured via Qubit 2.0 Fluorometer): 612 µg/ml*

![SDS-PAGE of steps through purifcation stage. Many bands showing not a very pure result.](https://static.igem.wiki/teams/5124/results/getimage-1.webp)

*Figure 7: SDS-PAGE of samples collected at every step during purification. Multiple bands indicate impurities at every step, showing that protein purification was not successful.*

Concentration of Cas13a elution sample (measured via Qubit 2.0 Fluorometer): 612 µg/ml

Concentration of Cas13a: 612 x 0.0626 = 38.3112 = 38.3 µg/ml (3 s.f.)

Concentration of Cas 13a was calculated by importing each image using a custom Matlab script, convert to a numerical matrix of RGB intensities, and counting blue pixels given a certain threshold (Red <=100, Green <=100, Blue >= 100). Final values were plotted, the full process is available on the Protein Purification section of our [Results](https://2024.igem.wiki/exeter/results) page.

### **Learn 2**

Yield increased of Cas13a but columns got clogged up in larger flask sizes - back to 250mL just multiple.

End results still have poor purity, so now we have better yield, need to improve the purification process. For this we talked to experts in the area from our university.

### **Design 3**

To increase purity we decided to use a pressurised 1 mL His-Trap column before further purifcation off all pooled protein containg fractions using size exclusion chromatography (AKTA Pure).

### **Build 3**

Transformed and expressed again.

*E.coli strain:* BL21(DE3) only

Induction method: Autoinduction media (20°C for 24 hours) to 250mL cultures.

Purification: Supernatant was loaded onto a His-Trap FF crude 1mL column (Cytiva) Eluted across a gradient of 20mM to 250mM Imidazole in 15 column volumes. Protein containing fractions were identified and concentrated to 2mL.

This was loaded onto a Superdex 200 10/300 120mL (Cytiva) (shortened to SEC) Eluted with 1 column volume of 20mM Tris-HCl, 500mM NaCl, 5% glycerol, pH 7.5.

Protein containing fractions were identified and pooled to give our final usable samples of Cas13a.

### **Test 3**

The figures below show the progression of protein purification after sequential purification steps, with an eventual purity of 37.45% being achieved. Although this was a lower purity than we had hoped for, subseqeunt experiments determined it was sufficient for our diagnostic system to be sucessful.

![SDS-PAGE gel showing fractions from His-Trap column, with fractions 6-11 showing darker bands of Cas-13a.](https://static.igem.wiki/teams/5124/results/getimage-2.webp)

*Figure 8: SDS-PAGE gels of the fractions collected from the His-Trap columns, with the most concentrated protein containing fractions collected and pooled.*

![Western blot showing fractions from His-Trap column, with fractions 33-36 showing darker bands of Cas-13a.](https://static.igem.wiki/teams/5124/results/getimage-3.webp)

*Figure 9: Western Blots of the fractions collected from the Superdex Size Exclusion Gel column, with the most concentrated protein containing fractions collected and pooled.*

![SDS-PAGE gel and western blot showing the stages through the process of purification, with fewer bands at each stage showing increasing purity.](https://static.igem.wiki/teams/5124/results/getimage-4.webp)

*Figure 10: SDS-PAGE gel and Western Blot of the different stages of the purification process.*

Concentration of Cas13a elution sample (measured via Qubit 2.0 Fluorometer): 183 µg/ml

Concentration of Cas13a: 183 x 0.3745 = 68.5335 = 68.5 µg/ml (3 s.f.)

Concentration of Cas 13a was calculated by importing each image using a custom Matlab script, convert to a numerical matrix of RGB intensities, and count blue pixels given a certain threshold (Red <=100, Green <=100, Blue >= 100). Final values were plotted, *refer to figue 8.*

![Graph showing increase in purity between His-Trap and SEC from 6.26% Cas-13a to 37.45%.](https://static.igem.wiki/teams/5124/engineering/getimage-5.webp)

*Figure 11: A - SDS-PAGE showing lanes after the His-Trap and SEC. B - graph showing percentage of Cas13a protein after the His-Trap and after the SEC. There is a significant increase in the percentage of Cas13a in the overall protein content.*

### **Learn 3**

We used this Cas13a sample in our test of the overall system, and got a positive result for some of the targets, and a negative result for the negative controls, meaning the test worked! Purity could still be improved for a real life test. Cas13a band on the SDS-PAGE look clearer and show lower levels of impurities than the first iteration. Lower concentration of protein samples indicates more unwanted proteins has been filtered out.

Click for References

[1]Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ, et al. RNA targeting with CRISPR-Cas13. Nature. 2017 Oct 12; 550(7675):280-4.

[2]Sutcliffe JG. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979; 43 Pt 1:77-90.

[3]Studier FW. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif. 2005 May; 41(1):207-34

[4]Byagathvalli G, Pomerantz A, Sinha S, Standeven J, Bhamla MS. A 3D-printed hand-powered centrifuge for molecular biology. PLoS Biol. 2019 May 21;17(5)

[5]McLoughlin KE, Correia CN, Browne JA, Magee DA, Nalpas NC, Rue-Albrecht K, et al. RNA-Seq Transcriptome Analysis of Peripheral Blood From Cattle Infected With Mycobacterium bovis Across an Experimental Time Course. Frontiers in Veterinary Science. 2021; 8:662002.

[6]Taylor GM, Worth DR, Palmer S, Jahans K, Hewinson RG. Rapid detection of Mycobacterium bovis DNA in cattle lymph nodes with visible lesions using PCR. BMC Vet Res. 2007 Jun 13; 3:12.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

Here you can find the protocols we engineered and followed to conduct our lab experiments. That way, they are easily accessible for anyone who wishes to replicate our research! **Note: Protocols may have to be adapted if using different bacteria strains.**

### Contents

---

Plasmid to Protein Experiment Pipeline:

[Transformation Protocol](#transformation-pdf)

[Liquid Culture Preparation Protocol](#liquid-culture-preperation-pdf)

[Plasmid Extraction Protocol](#plasmid-extraction-pdf)

[DNA Quantification using Qubit Fluorometer](#dna-quantification-using-qubit-fluorometer-pdf)

[Restriction Digest Protocol](#restriction-digest-pdf)

[Running an Agarose Gel Protocol](#running-an-agarose-gel-pdf)

[Autoinduction Media for Overexpression of Protein Protocol](#protein-expression-pdf)

[IPTG for Overexpression of Protein Protocol](#iptg-induction-pdf)

[Lysis of Expressed Bacteria Protocol](#lysis-of-expressed-bacteria-pdf)

Protein Purification, Quantification and Identification Protocols:

[Column Recharging Protocol](#column-recharging-pdf)

[Buffer Preparation Protocol](#buffer-preparation-pdf)

[Protein Purification Protocol](#protein-purification-pdf)

[Protein Qubit Protocol](#protein-qubit-pdf)

[Western Blot Protocol](#western-blot-pdf)

Fluorescent Probes:

[Environmental RNase Contamination Protocol](#environmental-rnase-contamination-pdf)

[Testing different enzyme concentrations for RNase Alert](#enzyme-rnase-pdf)

[Testing different probe concentrations for RNase Alert](#probe-rnase-pdf)

[Testing different enzyme concentrations for DNase Alert](#enzyme-dnase-pdf)

[Testing different probe concentrations for DNase Alert](#probe-dnase-pdf)

[Fluorescein Concentration Curves Protocol](#concentration-curves-pdf)

Transcription Protocols:

[PCR Amplification of Targets Protocol](#pcr-target-amplification-protocol)

Other Protocols:

[MBP Restriction Digest Protocol](#mbp-restriction-digest-pdf)

[Gel Excision and Ligation Protocol](#gel-excision-and-ligation-pdf)

[Reducing Blood Opacity Protocol](#blood-opacity-pdf)

[Reducing Synthetic Blood Opacity Protocol](#synthetic-blood-opacity-pdf)

[Cas13a Final Proof of Concept Protocol](#cas-final-proof-pdf)

# Plasmid to Protein Experiment Pipeline

## Transformation Protocol ---

Transformation is the process of adding desired plasmids into a chemically competent cell. The bacterial cell is incubated with the plasmids on ice, transferred to intense heat for a short amount of time (to make the cell membrane semi permeable) then put on ice again to help the cells recover with the plasmids inside them.

## Liquid Culture Preparation Protocol ---

When colonies have correctly grown on plate, a liquid culture needs to be grown up of a single colony to create a monoclonal culture for plasmid extraction and growth of further cultures for protein expression or similar uses.

## Plasmid Extraction Protocol ---

To confirm that the correct plasmids have been added to the cells, the plasmids are extracted again to run on an agarose gel with a ladder to see if the plasmids are the correct size. This is also a way to multiply and extract more plasmids if running out.

## DNA Quantification using Qubit Fluorometer ---

Once plasmids have been extracted, the concentration of DNA extracted needs to be quantified, so the Qubit Fluorometer is used, alongside standards to acquire the concentration.

## Restriction Digest Protocol ---

Restriction digest is the process of cutting up the plasmids before and after (at the promoter and terminator) the desired assembled genes, to create fragments that can be identified in an agarose gel. The plasmids are cut by enzymes, such as EcoRI and PstI.

## Running an Agarose Gel Protocol ---

An agarose gel is a way to separate DNA fragments by size, like the desired genes and plasmid backbone. The fragments formed are negatively charged, so when they are run with a current, the smaller fragments move faster through the gel towards the positive terminal. The result is bands that should be in line with the expected size of the fragment on the ladder.

## Autoinduction Media for overexpression of Protein Protocol ---

Once the plasmids have been added into the bacteria, they need to be activated to express the proteins. This is done through using autoinduction media, which uses glucose to grow up the concentration of cells, then lactose to induce high expression of the gene, and produce lots of desired protein.

## IPTG Induction for overexpression of Protein Protocol ---

Once the plasmids have been added into the bacteria, they need to be activated to express the proteins. This is done through addition of the chemical IPTG to induce high expression of the gene, and produce lots of desired protein.

## Lysis of Expressed Bacteria Protocol ---

To harvest the protein, the cells must be broken down both chemically and mechanically to extract all the protein but also not destroy it. The cell lysis digests all the parts of the cell that are not required like the cell membrane and DNA and should leave the protein intact.

---

# Protein Purification, Quantification and Identification Protocols

## Column Recharging Protocol ---

Nickle columns are used in protein purification due to the ability of histidine-tagged proteins to bind to nickel reversibly. However, between uses for different proteins, nicked columns need to be 'recharged' by stripping and reattaching nickel ions to the silica beads.

## Buffer Preparation Protocol ---

Proteins with histidine tags that bind to nickel bind reversibly- a pH controlled buffer containing imidazol can be used to displace the his-tags. When a high concentration of imidazole is added, the nickel ion binds to the imidazole instead of the histidines. When performing a protein purification using a nickel column, some non-desirable proteins may loosely bind to the nickel beads due to having naturally occurring histidine. Therefore, two buffers (containing a lower and higher concentration of imidazol respectively) are prepared- one to displace loosely bound background proteins, and one to elute the desired protein from the column.

## Protein Purification Protocol ---

The amino acid histidine has an affinity for nickel, meaning that proteins containing a histidine (his) tag will reversibly bind to nickel. Nickle columns contain nickel bound to silica beads, which cell lysate or other protein-containing solutions can be ran through to separate the protein from the solution. The column can be washed (removing loosely bound non-target proteins) then eluted, producing (hopefully) pure fractions of the desired protein for further analytical work.

## Protein Qubit Protocol ---

A protein qubit is an analytical technique used to determine the concentration of protein in a sample by using target-selective dyes that fluoresce when bound to protein. Using the standards, a standard curve is established by which the samples are compared.

## Western Blot Protocol ---

Western Blots are used to visualize, distinguish, and quantify proteins in a sample, so effectively to make sure the protein of interest is present. Proteins are separated by size via gel electrophoresis, before being transferred to a membrane and incubated with antibodies specific to the protein of interest. A secondary antibody is added alongside staining to visualise the protein- the thicker the band, the more present.

---

# Fluorescent Probe Protocols

## Environmental RNase Contamination Protocol ---

## Testing Different Enzyme Concentrations for RNase Alert ---

## Testing Different Probe Concentrations for RNase Alert ---

## Testing Different Enzyme Concentrations for DNase Alert ---

## Testing Different Probe Concentrations for DNase Alert ---

## Fluorescein Concentration Curves Protocol ---

---

# Transcription Protocols

## PCR Amplification of Targets and T7 *in vitro* Transcription Protocols ---

---

# Other Protocols

## MBP Restriction Digestion Protocol ---

Restriction digests use naturally occurring enzymes that cleave DNA at specific sequences. This produces DNA fragments with compatible 'sticky' ends to be inserted into the plasmid (vector), in this case into our genes of interest (Cas12a and Cas13a). Before this occurs, the fragments of DNA are separated by weight into bands via gel electrophoresis in an agarose gel, ready for excision and ligation.

## Gel Excision, DNA Extraction and Ligation Protocol ---

After completing an agarose gel using restriction digest DNA fragments, bands can be physically removed from the gel to break it down and allow DNA to be extracted for ligation. Ligation is the process of re-forming of parts of DNA by inserting a gene into 1 complete plasmid.

## Reducing Blood Opacity Protocols ---

## Reducing Synthetic Blood Opacity Protocol ---

## Cas13a Final Proof of Concept Protocol ---

This experiment was conducted as a final proof of concept for our Cas13a-based diagnostic test. The results of this experiment can be viewed [here](https://2024.igem.wiki/exeter/results). This protocol could be adapted for any complimentary crRNA and target pair.

{% endblock %}

| Gene | Description[1] |
| --- | --- |
| CXCL8 | Chemokine ligand 8, involved in infection response and tissue injury. |
| FOSB | FBJ murine osteosarcoma viral oncogene homologue B, plays a role in regulating cell proliferation, differentiation and transformation. |
| NR4A1 | Nuclear receptor subfamily 4, group A, member 1, plays a role in inflammation and apoptosis. |
| PLAUR | Plasminogen activator, urokinase receptor, a biomarker of inflammation. |
| RGS16 | Regulator of G-protein signalling 16, linked to many different disease states. |