Team:Edinburgh/Project/Notebook

Team Edinburgh Finding NEMO

Experimental notebook

Due to the effects of the COVID-19 pandemic, this year our lab access was significantly reduced. Our available access to the lab was prevented due to lab-closures, then largely restricted in order to ensure the safety of ourselves and our colleagues. While this may have been the case, we were determined to produce some viable results for our system and so worked as hard as we could to make up for lost time. A testament to the practicality of our simplistic design, we were able to gather proof-of-concept results in only six short weeks! Below is a short summary of the work carried out each week and, if you would like to know more, a comprehensive lab diary in the form of a PDF below.

Week 1 : 14/09/20 - 19/09/20

Sequence design and culture setup

This week was predominantly about growing up E.coli cells to provide the necessary culture and cell-free extracts for future experiment. Much of the additional time was spent on sequence design for our first testable components - the T7 promoter, aptamer iSpinach, scaffold F30, transcription factor (TF) ArsR that specifically binds with As(III), and riboswitch that specifically binds with F(I).F(I).
Week 2 : 21/09/20 - 27/09/20

in-vitro transcription and aptamer fluorescence optimisation

The first experiments we conducted were for conformation of in-vitro transcription and aptamer fluorescence. The elements of our positive control scaffold (T7 promoter, F30 scaffold upstream and downstream, and iSpinach) were phosphorylated, annealed, and ligated to form the full-length construct. Then the in vitro transcription (IVT) reaction was performed to examine the fluorescent signal. Multiple modifications on the experimental condition were discussed and tested based on the previous protocol in the lab. Near the end of this week, the optimized protocol is proven to work. Additionally, the cell extract from E. coli K12 MG1655 was also collected for establishing the cell free system (CFS).
Week 3 : 28/09/20 - 05/10/2020

Testing optimised protocols and Fluoresence confirmations

This week the previous experiment on construct assembly and IVT were repeated following the optimized protocol. Two negative controls were considered to prove that the positive result is credible in the IVT reaction: the sample is absent in the first one and the T7 RNA polymerase (T7 RNAP) is omitted in the second one compared with the experimental group.
The result indicates the T7 RNAP is able to transcribe a designed DNA template from T7 promoter to generate the aptamer sequence iSpinach that can bind with fluorophore DFHBI to emit fluorescent signals that would be easily detected by necked eyes after about 20 min.
Week 4 : 05/10/20 - 09/10/20

Transcription factor testing

This week we moved to a more complicated model that introduces TF and riboswitch to detect the metal ions with negative control that didn’t add specific metal ions. The TF is provided from the CFS of E. coli K12 MG1655 obtained in week1&2 and Cupriavidus metallidurans CH34 extract given by Mengxi. F riboswitch was also tested in the system without cell extract. The fluorescence can be observed in both experimental and negative control groups, the difference cannot be observed with naked eyes.Therefore, the plate reader is required to provide precise data.
Week 5 : 12/10/20 - 18/10/20

Arsenic sensor optimisation, In-vitro transcription and collaboration

This week the IVT condition for ArsR downstream consturct has been modified again by increasing the T7 RNA polymerase concentration, however, there are no improvement in the results. A new ArsR sensing construct was ordered with an ArsR binding site upstream of the promoter. Designed bubble duplex for integrating upstream transcription pathway signal was assembled. Reverse aptamer sequence + Splint rolling circle transcription for signal amplification was also assembled. To collaborate with the Stanford team, we designed a construct integrating their toehold sequence and used sfGFP as the reporter. This is under construction this week. IVT and gel results suggest the complete structure of the bubble duplex might need alternative ligation protocol to avoid bubble pairing with other parts. The reverse aptamer + splint result suggested the T7 RNA polymerase can use short DNA oligo as template to initiation transcription, which is worth following up to potentially achieve isothermal amplification in the future.
Week 6 : 19/10/20 - 25/10/20

New Arsenic sensor testing, further collaboration efforts

This week was mainly about adjusting the protocol for ligation and PCR amplification of Stanford toehold construct. Although the protocol for the ligation has been adjusted, the ligation still did not seem to work. Will run another gel to double check the results later. ArsR binding site upstream construct has arrived and constructed this week. IVT for the new ArsR and Anderson promoter_F riboswitch were performed. The new ArsR construct gave better results (higher overall fluorescence and quicker significant difference) compared to the old. The new riboswitch did not give positive results, could be due to the metal concentration is not high enough, will try IVT with higher metal concentration later.
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