iGEM 2021 — Team Warwick

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# Team:Warwick/Notebook

<!DOCTYPE html>

Notebook

# [![Brand logo](https://static.igem.org/mediawiki/2021/d/df/T--Warwick--images--logo.png)](https://2021.igem.org/Team%3AWarwick)

* Project
  + [Description](https://2021.igem.org/Team%3AWarwick/Description)
  + [Planning](https://2021.igem.org/Team%3AWarwick/Planning)
  + [Engineering](https://2021.igem.org/Team%3AWarwick/Engineering)
  + [Model](https://2021.igem.org/Team%3AWarwick/Model)
  + [Results](https://2021.igem.org/Team%3AWarwick/Results)
  + [Contributions](https://2021.igem.org/Team%3AWarwick/Contribution)
  + [Implementation](https://2021.igem.org/Team%3AWarwick/Implementation)
* Lab work
  + [Proof of concept](https://2021.igem.org/Team%3AWarwick/Proof_Of_Concept)
  + [Experiments](https://2021.igem.org/Team%3AWarwick/Experiments)
  + [Parts](https://2021.igem.org/Team%3AWarwick/Parts)
  + [Notebook](https://2021.igem.org/Team%3AWarwick/Notebook)
  + [Safety](https://2021.igem.org/Team%3AWarwick/Safety)
* Human practices
  + [Human practices](https://2021.igem.org/Team%3AWarwick/Human_Practices)
  + [Public engagement and education](https://2021.igem.org/Team%3AWarwick/Communication)
* Team
  + [Team](https://2021.igem.org/Team%3AWarwick/Team)
  + [Attributions](https://2021.igem.org/Team%3AWarwick/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AWarwick/Collaborations)
  + [Sponsors](https://2021.igem.org/Team%3AWarwick/Sponsors)
* Awards
  + [Model](https://2021.igem.org/Team%3AWarwick/Model)
  + [Human practices](https://2021.igem.org/Team%3AWarwick/Human_Practices)
  + [Sustainability](https://2021.igem.org/Team%3AWarwick/Sustainable)
  + [Entrepreneurship](https://2021.igem.org/Team%3AWarwick/Entrepreneurship)

## Lorem Ipsum

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![Sponsors](https://static.igem.org/mediawiki/2021/0/01/T--Warwick--images--sponsors.png)

© 2021 - Warwick iGEM CREscent, all rights reserved

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# Team:Warwick/Engineering

Engineering

# [![Brand logo](https://static.igem.org/mediawiki/2021/d/df/T--Warwick--images--logo.png)](https://2021.igem.org/Team%3AWarwick)

* Project
  + [Description](https://2021.igem.org/Team%3AWarwick/Description)
  + [Planning](https://2021.igem.org/Team%3AWarwick/Planning)
  + [Engineering](https://2021.igem.org/Team%3AWarwick/Engineering)
  + [Model](https://2021.igem.org/Team%3AWarwick/Model)
  + [Implementation](https://2021.igem.org/Team%3AWarwick/Implementation)
* Lab work
  + [Experiments](https://2021.igem.org/Team%3AWarwick/Experiments)
  + [Lab book](https://2021.igem.org/Team%3AWarwick/Lab_Book)
  + [Parts](https://2021.igem.org/Team%3AWarwick/Parts)
* Human practices
  + [Human practices](https://2021.igem.org/Team%3AWarwick/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3AWarwick/Communication)
* Team
  + [Meet the team](https://2021.igem.org/Team%3AWarwick/Team)
  + [Attributions](https://2021.igem.org/Team%3AWarwick/Attributions)
  + [Contributions](https://2021.igem.org/Team%3AWarwick/Contribution)
  + [Collaborations](https://2021.igem.org/Team%3AWarwick/Collaborations)
  + [Sponsors](https://2021.igem.org/Team%3AWarwick/Sponsors)
* Judging
  + [Awards](https://2021.igem.org/Team%3AWarwick/Awards)
  + [Medal Criteria](https://2021.igem.org/Team%3AWarwick/Medal_Critera)

### Engineering

* [Engineering Success](#engineering-success)
* [Initial research](#initial-research)
* [CRISPRa](#crispra)
* [The gRNA – recognising two things at once](#the-grna--recognising-two-things-at-once)
* [The plasmids](#the-plasmids)
* [Experimental layout](#experimental-layout)
* [Model of gRNA function – leakage, specificity and sensitivity](#model-of-grna-function--leakage-specificity-and-sensitivity)
* [References](#references)

## Summary

The engineering process was a lengthy one, and not without its problems. Unfortunately, we were not able to overcome all of them within the given timeframe but nonetheless we believe we have been successful in our dry design and partially successful in our wet work.

## Engineering Success

The process was, as every year, a lengthy one, and the core of our project. Our
lab team was thankfully able to enter the lab this summer and carry out a great
deal of work to validate our dry lab designs, which will be detailed below. We
are pleased to report that despite not having enough time to achieve our end
goals, we believe our project was at the very least partially successful.

## Initial research

After pinpointing the problem of Carbapenem-resistance Enterobacteriaceae (CRE),
we investigated several leads we had on detection methods. Ideas such as using
bait-prey interactions between biosensors proved far too complicated to design
and using specifically engineered bacteriophages to infect CRE with genes
encoding fluorescent reporters would have proven too lengthy and expensive.
Instead, our attention was drawn to an area previously explored by both teams
and academics from our university – the use of CRISPR as a detection tool by
engineering a guide RNA (gRNA) able to sense the presence of our target gene -
known as CRISPR activation, or CRISPRa for short. As for the latter, as
mentioned on the [Description page](https://2021.igem.org/Team%3AWarwick/Description), we settled upon the detection
of a particular Carbapenemase known as OXA-48 (Fig.1). This was because its
activity is slower than other Carbapenemases, which made it more difficult to
quickly detect using available tests such as the Carba NP test – a result
requires sampling and sequencing for a positive result.

![The overall structure of the OXA-48 Carbapenemase](https://static.igem.org/mediawiki/2021/4/41/T--Warwick--content--engineering--full_complex.png)

Fig. 1. The overall structure of the OXA-48 Carbapenemase. It is made up of 4 identical chains (each labelled in a different colour) which assemble to form the full protein.

Detecting DNA, however, has a chance of giving false positives as DNA can be
left over from dead bacteria after, for example, treatment of a surface with
biocides, resulting in unnecessary measures being taken. We therefore
endeavoured to detect the mRNA of OXA-48, due to its relatively short lifespan
being indicative of recently alive CPE within the sample (protein sequence
obtained from [1]).

## CRISPRa

![Plasmid map of our 'operator' plasmid](https://static.igem.org/mediawiki/2021/a/a6/T--Warwick--content--engineering--FIG_2.png)

Fig. 2. Plasmid map of our ‘operator’ plasmid, encoding our cgRNAs and the CRISPRa system.

Our CRISPRa system was engineered with all the controls in mind – each component
of the circuit is thus inducible. To make this easier, we settled upon using E.
coli cells of the MG1655-Z1 line, as they contain the elements TetR, LacI and
AraC, eliminating the need to introduce them ourselves (obtained from [2]).
Instead of using a normal Cas9 protein, however, we opted to use a deactivated
variant (dCas9), which has had its nuclease function abolished – the protein
would simply stay bound to the DNA sequence indicated in the gRNA.

The gRNA we engineered to sense the presence of the OXA-48 mRNA can be extended
to create a binding domain for the RNA-binding protein MS2 without compromising
its ability to bind to dCas9. MS2 would provide a scaffold for the binding of a
fusion protein composed of MCP and the transcriptional enhancer SoxS [3] –
effectively causing the upregulation of transcription downstream of the dCas9
binding site (protospacer adjacent motif or PAM). By placing the PAM just
upstream of the promoter of our reporter, we can cause its greatly increased
production.

## The gRNA – recognising two things at once

In a CRISPR system, a gRNA recognises its PAM sequence via Watson-Crick base
pairing in order to guide the Cas9 protein to its cleavage site. We added
another condition to this binding – the presence of our target mRNA – this
turned our gRNA into a conditional gRNA (cgRNA).

![Representation of our switch in an 'off' state](https://static.igem.org/mediawiki/2021/c/c6/T--Warwick--content--engineering--FIG_3.png)

Fig.3. Representation of our switch in an ‘off’ state.

As seen in the above figure, the cgRNA is made up of a number of distinct
domains. In its natively folded state, the dCas9 binding handle (grey) adopts a
conformation which prevents its binding to dCas9 – the system is thus in an
‘off’ state.

![Representation of our switch in an 'on' state](https://static.igem.org/mediawiki/2021/6/68/T--Warwick--content--engineering--FIG_4.png)

Fig. 4. Representation of our switch in an ‘on’ state.

However, when the OXA-48 mRNA is present (the trigger, in purple), the two RNA
molecules anneal, causing a significant conformational change in the secondary
structure of the cgRNA, allowing it to bind dCas9 and switching the system into
its ‘on’ state, allowing the complex to bind to the PAM and activate
transcription as detalied in the CRISPRa section above.

We simulated the secondary structure of our cgRNA in its natively folded form
and when annealed to the mRNA in NUPACK, as seen in Fig. 5.

![Diagrams of cgRNA structure](https://static.igem.org/mediawiki/2021/5/53/T--Warwick--content--engineering--FIG_5.png)

Fig. 5. (left) Diagram of the secondary structure of the cgRNA in its natively folded state. (right) Diagram of the secondary structure of the cgRNA when annealed to the target mRNA.

On the left of the figure, the dCas9 binding handle is misfolded, but the bonds
in the blocking domain, shown in blue, are not particularly stable – this is
indicative of the fact that the cgRNA can be brought out of this conformational
state. On the right of the figure, the conformation is drastically changed, and
the dCas9 binding handle (at the top in this figure) has folded correctly.

## The plasmids

All of these components and more were cloned into one of two plasmids, as seen
in the schematic in Fig.6. As for reporters, we chose to keep the original sfGFP
as well as introducing a new one – a fluorescent RNA aptamer known as iSpinach;
this RNA produces a strong green fluorescent signal when combined with the
non-toxic fluorogenic compound DFHBI.

![Schematic diagram of the two plasmids in our system](https://static.igem.org/mediawiki/2021/b/b6/T--Warwick--content--engineering--FIG_6.png)

Fig. 6. Schematic diagram of the two plasmids in our system.

Above is the schematic of the helper plasmid, encoding our trigger as well as
dCas9 and a chloramphenicol resistance gene to assist with selection. Below is
the schematic of a reporter plasmid, containing the two reporters – sfGFP and
iSpinach – as well as a cgRNA, the MCP-SoxS CRISPRa system, and a kanamycin gene
to assist with selection.

The design is modular to allow for a large amount of control (Fig. 7)– the
production of the cgRNA is inducible by arabinose (1) and the production of the
trigger is inducible with anhydrotetracycline (ATC, 2), while both are required
for the activation of dCas9 (3). Only when this occurs does MCP-SoxS (4)
exercise its enhancing activity upon the sfGFP promoter (5).

![Figure 7](https://static.igem.org/mediawiki/2021/9/91/T--Warwick--content--engineering--FIG_7.png)

Fig. 7. Schematic representation of the cgCRISPR sensing device.

## Experimental layout

The flow of our experiments can be seen in the figure below - the lab work was
split into four stages: foundation, improvement, engineering, and production.
Unfortunately, due to time constraints and unforeseen circumstances brought by
supply chain issues, we were only able to assemble the first reporter plasmids
in time, reaching the first iteration cycle.

![Representation of our lab plan. Top to bottom: foundation phase, improvement phase, engineering phase, production phase](https://static.igem.org/mediawiki/2021/5/5a/T--Warwick--content--engineering--FIG_8.png)

Fig. 8. Representation of our lab plan. Top to bottom: foundation phase, improvement phase, engineering phase, production phase.

We began by extracting the necessary components from existing plasmids
(PAJ-379-C1 and PAJ-390-C2 [4]) from Professor Alfonso Jaramillo’s lab at the
University of Warwick using PCR – this gave us the linearised backbones which we
would use in assembling the plasmids in the above sections. Our inserts were
provided by IDT and included the OXA-48 gene (sequence obtained from [1]). It
lacked a ribosome binding site as we did not want to proliferate Carbapenemase
resistance during our project. Other parts included the MCP-SoxS gene (sequence
obtained from [3]). We used Gibson and Goldengate assembly techniques to assemble
our helper and reporter plasmids, respectively.

Afterwards, MG1655-Z1 cells were transformed with the helper plasmid, giving
rise to the helper cell line. This cell line would be transformed with different
versions of the reporter plasmid containing different cgRNAs to screen for the
one which offered the greatest difference in fluorescence between a positive and
a negative result.

As alluded to in the diagram, as well as on our [Human Practices page](https://2021.igem.org/Team%3AWarwick/Human_Practices),
our system was designed to work in an in vitro scenario; the iSpinach aptamer
has been designed to function optimally in vitro. While we were not able to
pursue this cell-free application due to time and materiel constraints, we are
confident that a future iGEM team could validate the theoretical basis behind
this device working cell-free.

A detailed rundown and timeline of our lab work can be found on our [Experiments page](https://2021.igem.org/Team%3AWarwick/Experiments).

## Model of gRNA function – leakage, specificity and sensitivity

In order to find out which of our designed cgRNAs was most appropriate despite a
lack of data, we prepared a mathematical model using which we tested a promising
sequence against a number of cgRNAs with mismatching spacers. The results of the
first comparison can be observed in Fig.9 below – while there is a significant
level of activation, the longer sensing loop could cause unintended activation,
which is why we followed this up by testing a sequence with a shorter sensing
loop which would give more specificity to the target sequence.

![Graph of the results of the modelling of fluorescence over time](https://static.igem.org/mediawiki/2021/e/ea/T--Warwick--content--engineering--FIG_9.png)

Fig.9. Graph of the results of the modelling of fluorescence over time when using a cgRNA with a longer sensing loop (green), when compared against mismatching cgRNAs (orange, grey) and a negative (blue) and positive (yellow) control.

The comparison of the other cgRNA to a mismatch using our model (Fig.10) yielded
better results, however. There is a large difference in fluorescence between the
use of the cgRNA we tested when compared against both the negative control and
against a mismatching gRNA. Moreover, over time the fluorescence appears to
approach the value exhibited by the positive control! This was, to us,
indicative of the fact that the increased specificity had no negative effect on
activation.

![Graph of the results of the modelling of fluorescence over time](https://static.igem.org/mediawiki/2021/1/16/T--Warwick--content--engineering--FIG_10.png)

Fig.10. Graph of the results of the modelling of fluorescence over time when using a cgRNA with a shorter sensing loop (green), when compared against a mismatching cgRNA (grey) and a negative (blue) and positive (yellow) control.

We decided to investigate the sensitivity of this particular cgRNA as well and
simulated its activity with different ratios of cgRNA:trigger as evidenced in
Fig. 11. The results of this were satisfactory, as when compared to the absence
of the trigger, the 1:1 ratio yielded a large increase in observed fluorescence.

![Comparison of fluorescence using the cgRNA with the shorter sensing loop at different concentrations](https://static.igem.org/mediawiki/2021/7/72/T--Warwick--content--engineering--FIG_11.png)

Fig 11. Comparison of fluorescence using the cgRNA with the shorter sensing loop at different concentrations – no trigger (orange), 1:1 cgRNA:trigger (green), 2:1 cgRNA:trigger (black), 5:1 cgRNA:trigger (grey).

As expected though, as the ratio of cgRNA to trigger increased, less activation
was observed – this raises an issue when working with low concentrations of
OXA-48 mRNA in the sample; nonetheless, we believe that this gRNA would make a
fine candidate for further development.

See the reactions and values for our model below:

A pdf of the pages of the lab notebook used throughout the lab work

## References

[1] Mohammadi Bandari, N. Keyvani, H. Abootaleb, M. *Klebsiella pneumoniae strain TFH21 OXA family beta-lactamase (blaOXA) gene, partial cds*. Available at: <https://www.ncbi.nlm.nih.gov/nucleotide/MZ275246.1> [Accessed 19/10/2021]

[2] Sekar, K. Gentile, AM. Bostick, JW. Tyo, KE. *N-Terminal-Based Targeted, Inducible Protein Degradation in Escherichia coli*. PLoS One. 2016 Feb 22; 11(2):e0149746. doi: 10.1371/journal.pone.0149746. eCollection 2016. PONE-D-15-42990 [pii] PubMed 26900850. Available at: <https://www.addgene.org/65915/> [Accessed 20/10/2021]

[3] Dong, C. Fontana, J. Patel, A. et al. *Synthetic CRISPR-Cas gene activators for transcriptional reprogramming in bacteria*. Nat Commun 9, 2489 (2018). <https://doi.org/10.1038/s41467-018-04901-6>

[4] Gallzi, R., Duncan, J. N., Rostain, W., Quinn, C. M., Storch, M., Kushwaha, M., Jaramillo, A., *Engineered RNA-interacting CRISPR Guide RNAs for Genetic Sensing and Diagnostics*, The CRISPR Journal (2020), 3 (5), pp. 398-408, doi: <http://doi.org/10.1089/crispr.2020.0029>

![Sponsors](https://static.igem.org/mediawiki/2021/0/01/T--Warwick--images--sponsors.png)

© 2021 - Warwick iGEM CREscent, all rights reserved

Website content under [![Creative Commons License](https://static.igem.org/mediawiki/2021/4/45/T--Warwick--images--CC_BY_License.png)](http://creativecommons.org/licenses/by/4.0/)

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# Team:Warwick/Experiments

Experiments

# [![Brand logo](https://static.igem.org/mediawiki/2021/d/df/T--Warwick--images--logo.png)](https://2021.igem.org/Team%3AWarwick)

* Project
  + [Description](https://2021.igem.org/Team%3AWarwick/Description)
  + [Planning](https://2021.igem.org/Team%3AWarwick/Planning)
  + [Engineering](https://2021.igem.org/Team%3AWarwick/Engineering)
  + [Model](https://2021.igem.org/Team%3AWarwick/Model)
  + [Implementation](https://2021.igem.org/Team%3AWarwick/Implementation)
* Lab work
  + [Experiments](https://2021.igem.org/Team%3AWarwick/Experiments)
  + [Lab book](https://2021.igem.org/Team%3AWarwick/Lab_Book)
  + [Parts](https://2021.igem.org/Team%3AWarwick/Parts)
* Human practices
  + [Human practices](https://2021.igem.org/Team%3AWarwick/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3AWarwick/Communication)
* Team
  + [Meet the team](https://2021.igem.org/Team%3AWarwick/Team)
  + [Attributions](https://2021.igem.org/Team%3AWarwick/Attributions)
  + [Contributions](https://2021.igem.org/Team%3AWarwick/Contribution)
  + [Collaborations](https://2021.igem.org/Team%3AWarwick/Collaborations)
  + [Sponsors](https://2021.igem.org/Team%3AWarwick/Sponsors)
* Judging
  + [Awards](https://2021.igem.org/Team%3AWarwick/Awards)
  + [Medal Criteria](https://2021.igem.org/Team%3AWarwick/Medal_Critera)

## Summary

A concise timeline of our lab work – things didn’t always go to plan but we did our best to overcome any obstacles we came across!

### 12/08/2021

Obtained PAJ-390 and PAJ-379 containing MG1655-Z1 cells from Professor
Jaramillo’s lab.

Prepared plates with appropriate antibiotics for selection
(spectinomycin for all Z1 cells, chloramphenicol for PAJ-390, kanamycin for
PAJ-379).

Plated cells.

![Figure 1](https://static.igem.org/mediawiki/2021/4/41/T--Warwick--content--experiments--1.png)

### 13/08/2021

Cells have grown on the appropriate plates.

### 16/08/2021

Prepared liquid cultures of both lines for miniprep.

Prepared liquid cultures of the 390-containing line for electrocompetence induction.

### 17/08/2021

We induced competence in the cell line containing the 390 plasmid.

We miniprepped both plasmids – in hindsight the method was flawed as our yields were low.

### 18/08/2021

First, we plated the SAJ-351 cells which contained the 390 (dCas9) plasmids. We
also miniprepped the 379 (GFP) plasmid and obtained a concentration of 26.8
ng/µl.

![Figure 2](https://static.igem.org/mediawiki/2021/3/35/T--Warwick--content--experiments--2.png)

### 19/08/2021

Transformation failed, so we centrifuged a Falcon tube with the supposed transformants and resuspended the pellet in less media. We then plated the resulting concentrated culture

### 20/08/2021

There was no growth from the transformants.

### 23/08/2021

We tried to transform the SAJ-351 cells with the 379 plasmids.
The 379 plasmid contained resistance to kanamycin (Kan), so if the culture grew
on the Kan/Spec/Chlor plate then we would know the transformation had been
successful.

Unfortunately, there was no growth, so the transformation was
unsuccessful. However, the culture grew on the control plate without kanamycin,
so the cells were not killed in the procedure and the error lied with either the
transformation procedure, the concentration of the 379 plasmids, the competence
of the cells or the 379 plasmid itself.

![Figure 3](https://static.igem.org/mediawiki/2021/7/76/T--Warwick--content--experiments--3.png)

### 24/08/2021

The control grew, but the transformants didn’t. Therefore we made more competent cells from our existing culture, but we forgot to add antibiotics so the procedure didn’t work.

### 25/08/2021

We tried the transformation again but received the same
results (no growth). As we had carefully followed the procedures for both transformation and
competence, we decided the most likely cause for the lack of growth on the
Kan/Spec/Chlor plate was either the 379 plasmid itself or the concentration of
the 379 plasmids, as it was very low. Therefore, we re-did the 379 plasmid
miniprep and received a concentration of 40.7 ng/µl.

![Figure 4](https://static.igem.org/mediawiki/2021/2/24/T--Warwick--content--experiments--4.png)

We also ran a restriction digest on the 379 plasmids to see whether there were
any unexpected results. The results were inconclusive – after running the gel
for an additional 40 minutes on top of the required 40 minutes in the procedure,
there was still no visible ladder. We ran the gel again for another 20 minutes,
but unfortunately the agarose melted which meant we had to try again another
day.

### 26/08/2021

The transformation failed again.

### 31/08/2021

![Figure 5](https://static.igem.org/mediawiki/2021/f/f5/T--Warwick--content--experiments--5.png)

We repeated the restriction digest and it appears that it may have
worked as we had somewhat of a visible ladder, but the integrity of the gel was
weak, and it broke easily so we were not convinced of the results and planned to
run it for a third time in the future.

### 01/09/2021

We prepared new liquid cultures to repeat the miniprep.

### 02/09/2021

We again repeated the transformation of the SAJ-351 cells,
using the new miniprep of 379 plasmid. On the same day, we carried out yet another
miniprep of the 379 plasmids to increase the yield to 242.7 ng/µl as a backup in
case the transformation failed again.

### 03/09/2021

![Figure 6](https://static.igem.org/mediawiki/2021/1/18/T--Warwick--content--experiments--6.png)

However, the transformation was a success, creating a cell line which we called
PAJ! A single colony grew on the Kan/Spec/Chlor plate, meaning it contained the
plasmid with the kanamycin resistance gene. The success
of the transformation meant we did not need to repeat the restriction digest of
the 379 plasmid.

### 06/09/2021

![Figure 7](https://static.igem.org/mediawiki/2021/4/40/T--Warwick--content--experiments--7.png)

This single colony was replated on to obtain more of the PAJ cells, so we could
use them to prepare liquid cultures and run different experiments.

### 07/09/2021

Both cultures grew nicely, giving us confirmation that we had produced transformed cells.

### 09/09/2021

We obtained the chemicals necessary for inducing the PAJ circuit – ATC and IPTG.

### 13/09/2021

Our first attempt at PCR-extracting the backbone for the helper plasmid. This
failed as we used the improper polymerase, ordered primers for extraction as 3 3 kbp
fragments.

We also attempted our first run of the PAJ fluorescence experiment to check if the circuit
works. However, we did not successfully manage to produce GFP from the PAJ cells.

### 17/09/2021

![Figure 8](https://static.igem.org/mediawiki/2021/c/c1/T--Warwick--content--experiments--8.png)

We repeated the fluoresence experiment, using fresh liquid cultures and incubating these cultures
with ATC overnight to maximise production of GFP. We successfully proved that the PAJ circuit functions
by producing GFP from these cells.

### 20/09/2021

We successfully carried out PCR extraction of the 3 3 kbp fragments.

![Figure 9](https://static.igem.org/mediawiki/2021/3/32/T--Warwick--content--experiments--9.png)

### 21/09/2021

Our attempted purification of the PCR output failed. We realised it was
because the PCR had failed so there was nothing to purify.

### 22/09/2021

We carried out PCR and purification again but failed for the second time.

### 23/09/2021 - 24/09/2021

We spent a few days trying to debug the purification procedure and figure out
why it wasn’t working. One possibility was that the purification kit we used
was contaminated or faulty.

### 27/09/2021

We repeated the PCR for the third time.

### 28/09/2021

We reattempted the purification with a fresh kit and it worked beautifully.
We also obtained pure MG1655-Z1 cells.

### 29/09/2021

We attempted assembly of the helper plasmid and transformation into MG1655-Z1 cells.

### 30/09/2021

We found out that yesterday’s work failed.

### 31/09/2021

We reattempted PCR extraction with a new polymerase, allowing us to PCR
out the full 9 kb backbone

### 04/10/2021

Purification of the PCR output and assembly seem to have worked according to gel electrophoresis.
We also attempted transformation with the helper plasmid.

### 05/10/2021

Our transformation failed. We decided to use more competent cells to purify the
assembly output by transformation.

### 06/10/2021

Successful transformation of competent TOP10 *E. coli* with the helper plasmid
allowed to then prepare liquid culture for miniprep.

### 07/10/2021

We miniprepped the helper plasmid then prepared more competent MG1655-Z1 cells.

### 08/10/2021

We transformed our Z1 cells with the helper plasmid.

### 11/10/2021

The transformation was assessed as successful over the weekend. This allowed
us to extract backbones for the operator plasmids through PCR.

### 12/10/2021

We realised that a part of SoxS-MCP has not arrived from IDT with the rest of it.
However, we still managed to create competent Z1s containing helper plasmids.

### 18/10/2021

The SoxS part from IDT finally arrived. We attempted operator plasmid assembly
and transformation.

### 19/10/2021

We discovered that the transformation failed as the DNA was not concentrated
enough. We would have to go through more competent cells and miniprep again to
repeat, so unfortunately we ran out of time to complete our wet lab work.

The next stage would have been to transform the operator plasmid into our cells
and then carry out a fluoresence test to prove that the gRNA and dCas9 system
works according to plan and successfully produces a visual indicator in the
form of GFP. This would have provided us with real data which we could use to
obtain proof of concept. However, we did not have time, which is a shame but we
are still proud of all the progress we made in the lab over the summer.

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# Team:Warwick/Results

<!DOCTYPE html>

Results

# [![Brand logo](https://static.igem.org/mediawiki/2021/d/df/T--Warwick--images--logo.png)](https://2021.igem.org/Team%3AWarwick)

* Project
  + [Description](https://2021.igem.org/Team%3AWarwick/Description)
  + [Planning](https://2021.igem.org/Team%3AWarwick/Planning)
  + [Engineering](https://2021.igem.org/Team%3AWarwick/Engineering)
  + [Model](https://2021.igem.org/Team%3AWarwick/Model)
  + [Results](https://2021.igem.org/Team%3AWarwick/Results)
  + [Contributions](https://2021.igem.org/Team%3AWarwick/Contribution)
  + [Implementation](https://2021.igem.org/Team%3AWarwick/Implementation)
* Lab work
  + [Proof of concept](https://2021.igem.org/Team%3AWarwick/Proof_Of_Concept)
  + [Experiments](https://2021.igem.org/Team%3AWarwick/Experiments)
  + [Parts](https://2021.igem.org/Team%3AWarwick/Parts)
  + [Notebook](https://2021.igem.org/Team%3AWarwick/Notebook)
  + [Safety](https://2021.igem.org/Team%3AWarwick/Safety)
* Human practices
  + [Human practices](https://2021.igem.org/Team%3AWarwick/Human_Practices)
  + [Public engagement and education](https://2021.igem.org/Team%3AWarwick/Communication)
* Team
  + [Team](https://2021.igem.org/Team%3AWarwick/Team)
  + [Attributions](https://2021.igem.org/Team%3AWarwick/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AWarwick/Collaborations)
  + [Sponsors](https://2021.igem.org/Team%3AWarwick/Sponsors)
* Awards
  + [Model](https://2021.igem.org/Team%3AWarwick/Model)
  + [Human practices](https://2021.igem.org/Team%3AWarwick/Human_Practices)
  + [Sustainability](https://2021.igem.org/Team%3AWarwick/Sustainable)
  + [Entrepreneurship](https://2021.igem.org/Team%3AWarwick/Entrepreneurship)

## Lorem Ipsum

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Cras volutpat malesuada
dapibus. Integer aliquam eget risus a ornare. Sed rutrum felis ut eros varius,
sit amet placerat metus dignissim. Cras non mauris ultricies, pulvinar tellus
nec, suscipit tortor. Donec cursus euismod tellus, vitae pulvinar est faucibus
et. Suspendisse fringilla aliquet porta. Fusce nec tortor eget ante vehicula
placerat. Nulla dignissim quam nibh, ac tincidunt metus convallis eu. Sed nec
tellus lacinia, elementum libero a, iaculis justo. Proin interdum placerat elit,
et consectetur lectus feugiat non. In iaculis eros sit amet dui lobortis, eu
consectetur sapien vestibulum. In urna elit, semper non maximus ut, tempor non
diam. Nulla sodales lacus non dui mollis, vitae imperdiet magna pellentesque.
Pellentesque non hendrerit nisl.

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© 2021 - Warwick iGEM CREscent, all rights reserved

Website content under [![Creative Commons License](https://static.igem.org/mediawiki/2021/4/45/T--Warwick--images--CC_BY_License.png)](http://creativecommons.org/licenses/by/4.0/)

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