Team:NYMU-Taipei/Notebook

Notebook

We looked back to numerous igem teams project in the past few years and tried to choose our topic. We then reached a consensus to do a project that will help improve the situation of COVID-19, which is ravaging the world. After tons of discussion and document reading, we planned to fight the threatening corona virus by targeting the spike protein on it’s surface.

We came up with several ideas to avoid being infected by SARS-CoV2, such as FRET assay for detection, using a protease for inactivation, and some applications that may help our design. Divided into small groups, we started to look deep into our plan and design every part in detail. Lots of paper research are done in this phase and an initial prototype of our project is formed.

Lab doings

We did a comprehensive clean up of our lab, including sterilizing containers we might use, throwing away expired solutions from the fridge and putting on new bench paper. Made account and collected virus genome data from database.
Other issues

We started to look for the sequences we may need to purchase form addgene and learn how to design primers for later plasmid construction.

Lab doings

We made some LB plates with different antibiotics and started to get familiar with the instruments in the lab
Other issues

We listed the plasmid we may construct and decide which method to connect the DNA fragments (Restriction enzyme ligation or Gibson assembly)

Lab doings

We did LB plate planting and plasmid extraction to the bacteria with plasmids we bought from addgene, then use restriction enzymes to check if we got the right plasmid. We also made a list of what restriction enzymes and DNA markers we have in the lab. Made preliminary results from virus sequence analysis.
Other issues

We communicated with NCKU igem team and are looking forward to have further discussion and exchanges later in the summer.

Lab doings

Continued to do plasmid extraction and store them in the fridge for later PCR. Modeled virus protein structural sequence to see important mutation sites in 3D.
Other issues

We had a vigorous discussion about which kit and what tag(SUMO or his tag) we are going to use for the protein purification of our fusion protein constructs. Also, we decided what linker to use for our fusion protein after a series of paper research.

Lab doings

We cut the plasmid (Scarlet I-FRET 10-Venus) with restriction enzymes and then collected the sequence of Venus fp and Scarlet-I fp by gel electrophoresis and gel extraction. The results of gel extraction is known by nanodrop spectroscopy.
Other issues

We spent a great afternoon having an interview with doctor Zhan and learnt a lot from his experience and advices.

Lab doings

Keep doing plasmid extraction (because some of the plasmids remain having low concentration) and restriction enzyme digestion as last week.
Other issues

Some of us visited a professor in Chung Xing University to get information about the use of chitin tag and how to utilize the chitin tag to let our construct bind to the contact lens’ surface. However, The professor advised us not to use chitin tag and introduced another method using merely his tag to achieve our goal.

Lab doings

PCR progress: Venus FP, ACE2, bacterial expression vector
Other issues

We started to construct our wiki using hackMD website. The rough framework of our wiki is formed.

Lab doings

PCR progress: Venus and Scarlet FP, ACE2 Gibson assembly progress: ACE2 RBD
Other issues

We had an online seminar with NCKU igem team, at which exchange our thoughts and discuss about the challenges we met and find a solution. We also discussed about our collaboration about human practice related to contact lens (which is a part we have in common).

Lab doings

PCR progress: pepN(failed), spike protein(low yield) Transformation: ACE2( to BL21 strain).
Other issues

We had another online seminar with CSMU igem team and had a good time sharing our thoughts and advices about each other’s’ project. We also had a lab collaboration(PCR double check) scheduled.

Lab doings

PCR progress: pepN(failed), spike protein(low yield) Transformation: ACE2( to BL21 strain)
Other issues

We visited Prevision contact lens factory and demonstrated our design to them.

Lab doings

PCR progress: pepN(failed), spike protein(low yield)
Other issues

We started our collaboration events with NCKU igem team, propagating citizens not to discard contact lens carelessly after use.(Slogan: Throw contact lens properly, marines life live more happily) Also, we started to draw up the framework of our team promotion video.

Lab doings

PCR progress: pepN(failed), spike protein(low yield).
Other issues

We find out that making contact lens as our only product may be lacking persuasion, so we discussed if there are other kinds of application our construct can be used. Also, the protease we chosen (pepN) was found not able to cut spike protein, so we immediately initiated a new search for a replacement.

Lab doings

PCR progress: ACE2, Venus & Scarlet FP, Spike protein(low yield).
Other issues

We’ve finalized our team promotion video and determined our project name “Off The Crown”. We also decided which track of igem we are participating in. We came up with another protease(pepP) ordered the primers to construct its plasmid. Furthermore, in case the spike protein is too big for the protease to cut, we extracted the four pepP cleavage sites on spike protein and created fragments that has 35bp of DNA front and behind the cleavage site.

Lab doings

We’ve finalized our team promotion video and determined our project name “Off The Crown”. We also decided which track of igem we are participating in. We came up with another protease(pepP) ordered the primers to construct its plasmid. Furthermore, in case the spike protein is too big for the protease to cut, we extracted the four pepP cleavage sites on spike protein and created fragments that has 35bp of DNA front and behind the cleavage site..
Other issues

The second plan of our application is determined (detection box DeBux), and we started to design the structure of the box and preparing to use 3D printing to form its structure.

Lab doings

Started to construct the gel we are going to stick our fusion protein to(Using poly HEMA). Gibson assembly progress: pepP only(success) Transformation: pepP only(to BL21 strain).
Other issues

Wiki description page and home page design finished. Criteria “excellence in another area” done (collaboration with NCKU igem team). Integrated human practice recorded and organized.

Lab doings

PCR progress: cut 1-4 spike fragment & vector, P+A pepP, ACE2, & vector Gibson assembly progress: spike cut 3&4 Protein purification: pepP, RFP, ACE2 Perform oxidization to our gel(KMnO4).
Other issues

Start writing our judging form.

Lab doings

Gibson assembly progress: spike cut 1-3, PepP+ACE2(success) Protein purification: pepP, RFP, Spike cut4, ACE2 Test the ability of our gel to bind to his tagged protein. pepP cleavage test(use SDS PAGE and Western Blot to check the results.
Other issues

Lab doings

SDS-Page: spike cut 1-4, pepP, RFP Protein purification: pepP, RFP, Spike cut 1-4 Make hydrogel and oxidize the gel. Test the ability of our gel to bind to GFP with his tag.
Other issues

Complete our wiki.