Difference between revisions of "Team:UiOslo Norway/Human Practices"

We consider Human practices to be one of the most important and rewarding things about iGEM. During our project we gain a bigger perspective about synthetic biology. Our goal was to spread the knowledge about synthetic biology, but especially about our team’s project and about antibiotic resistance. We can't fight the battle alone.

Antibiotic resistance involves people all over the globe, and it is impossible to decrease the trend without as many as people being involved. We have been in touch with several experts in the field and we have held many lectures about our project to other students, both secondary school pupils and master students. We have been in several newspapers, attended a science festival, been in magazines and even on the radio. We think we did manage our goals.

You can read more about our work under Education, Public engagement and Integrated practices. We have learned so much doing this and we have met so many people on the way. Most important of all; it has been som much fun!

Increasing knowledge for primary and secondary school pupils
The 27th and the 28th of September "Ungforsk" was arranged at the University. "Ungforsk" roughly translated means young science and was arranged for the 20th time. Secondary school pupils get to visit the University were they can visit labs and learn more about science. We were so lucky that we got to hold a total of six presentations for those who came to visit. Ragnhild, Paul, Marthe and Torleif held those seminars, and they did a really good job.
Torleif also handed out our comic book to Torpa primary and secondary school. Let's hope they'll get more excited about syntethic biology.

Increasing knowledge for new students at the university
The 30th of August and the 6th of September we held lectures for new biology students at the University. We were raising awareness and talked about iGEM, but also presented our project and spread knowledge about antibiotic resistance. We held 3 lectures each day and 6 in total. The students seemed very interested about iGEM, so we hope next years team will have loads of interested students. Marthe, Hans, Paul and Camilla held those lectures.
Roza and Marthe also held a lecture at Sundvolden the 22nd of August.

Increasing knowledge for master students
Ragnhild held a lecture the 16th of August for master students. It was held at Kristine Bonnevies house at the University and there were also pizza afterwards.

We have done a lot to get public attention about iGEM, but also about us and antibiotic resistance. We have used social media, we have a blog, radio, magazines, different newspapers and even published a comic book to hand out. We have also gotten help from the headmaster to write about us in his blog, and we have also made it on different webpages.

Facebook and Instagram
We are very active on social media. On facebook we have over 560 followers and we are updating with professional articles and other things that are relevant. On Instagram we have about 300 followers. We share fun stuff as well as relevant articles and keeping people updated about our project.

Blog
We also have a blog which we try to update regularly. We share things about our project, but also conferences we've attendend, as well as other fun stuff to keep people interested. Visit our webpage here.

Radio
We have also been on the radio, on Østlandssending on NRK. NRK is Norway's national channel and everyone have free access to listen. You can listen to it here.

Magazines
We have been in the University magazine Apollon. You can read the article here.(It's in Norwegian though).

Newspapers
We have also been visible in different newspapers. Both on the internet and on paper. You can read about us here (Østlandets Arbeiderblad), here
(Romsdals Budstikke), and here (Porsgrunn Dagblad). You have to pay a fee to get to read the article.

Here is a list over activities, seminars we've held, articles about us, a radio interview and other things we have done to bring attention to our project.

Biotorsdag
24th of November

The Giant Jamboree, Boston, USA
26th of October – 29th of October

Cutting Edge Festival, Science Park
18th of October
Presentation and stand.

Comic book
September
We released our own comic book to raise awareness about synthethic biology!

Ungforsk
28th of Septemeber
Three presentations.

Ungforsk
27th of September
Three presentations.

Bjørnelab, Science Library
6th of September
Three presentations for new biologists students.

Bjørnelab, Science Library
30th of August
Three presentation for new biologists students.

Program seminar, Sundvolden
22nd of August
Presentation for new students.

Workshop, Youngstorget
22nd of August

Apollon Magazine
Bacteria edition
Article in the magazine.

Apollon
18th of August
Presentation

Presentation for master students
16th of August
Presentation.

Østlandssendingen, NRK Radio
1st of August
Talked about our project on the radio.

Porsgrunn Dagblad
28th of July
Article in the local newspapers.

Østlandets Blad
25th of July
Article in the local newspapers.

Romsdals Budstikke
25th of July
Article in the local newspapers.

Oppland Arbeiderblad
July
Article in the local newspapers.

The Nordic iGEM conference, Stockholm, Sweden
17th of June – 19th of June
Mini Jamboree, workshops and fun.

Headmaster's blog
Our headmasters wrote about us and our project on his blog.

NBS-nytt

GENialt

While we are attempting to fight antibiotic resistance with our novel diagnostic test, we acknowledge the fact that our test only works at a protein level. As the central dogma of molecular biology dictates, protein is the last instance in the information flow in a cell. The problem of resistance genes could of course also reside on a genetic level, upstream of actual enzymes. This is important, as a bacteria may very well have resistance genes without expressing them at a certain time. The expression could even be turned on by the presence of antibiotics (check for reference), which makes it important to not distribute antibiotics when such genes are present. All of these things points to a need for a diagnostic tool that not only detects enzymatic activity, but also the genes leading to said enzymatic activity. Such a tool would enable us to test for resistant microbes before they actually express their resistance. Further, earlier detection would lower the probability of spreading of the resistant bacteria, potentially protecting a lot of people from painful, dangerous infections.

This reasoning led us to CRISPR/Cas9. Cas9 is useful in this matter because of its ability to recognize specific nucleotide sequences. By now, it is known that by redesigning gRNA/tracrRNA/crRNA, one can design Cas9-systems that can recognize any sequence. Usually, this is used in combination with the native endonuclease activity of Cas9, enabling the researcher to cut DNA at specific locations and manipulate the target sequence. This differs from our approach. By taking a slightly unorthodox, but innovative approach to CRISPR/Cas9 usage, we want to interface it with our PhoneLab.

In order to combine CRISPR/Cas9 technology with our diagnostic tool, we imagine to preserve the “searching” capability of CRISPR/Cas9 systems, and replacing the “cutting” part with a detectable readout similar to what we have with the nitrocefin-based detection. This means that instead of cleaving the resistance genes, we want to couple the pairing of nucleotide targets and Cas9 to a colorimetric signal. By doing so, detection of genes could relatively easily be interfaced with the detection of ESBL activity in our PhoneLab, thus creating a more complete diagnostic tool for ESBL infections.

This could be achieved in several ways, but as a contribution to our project, we propose the following design of a CRISPR/Cas9 system to be used in PhoneLab.

First, we would need to design tracr/crRNA sequences compatible with known ESBL genes. These RNAs would be linked to a null-nuclease Cas9 (dCas9) - a Cas9 enzyme modified so that it binds tracr/crRNA and is guided to its targets,, but does not cleave the target genes. For each target gene, we propose to have two sets of tracr/crRNA, complementary to different, but adjacent parts of the same gene. Each of these RNA molecules would have a dCas9 partner, which again would be fused to half of a detectable protein partner in a split-enzyme assay. When the tracr/crRNA finds its goal, dCas9 would bring the two parts of the split-enzyme together, thus creating a detectable signal. So, in this system, two independent RNA/DNA-binding events would be required to take place, which lowers the probability of false positives.

The detectable protein partner of dCas9 is currently a matter of research for us, but one possibility is to use the subunits of galactosidase. LacZ alpha and -omega are two separate peptides that form the active form of galactosidase when in contact with each other. By fusing these two peptides to two separate dCas9 constructs recognizing adjacent parts of the same gene, the presence of said gene would be coupled to the forming of a functioning galactosidase. Thus, by adding for example liquid derivatives of X-gal, a visible color change could be achieved, similar to the one seen in our project with nitrocefin.

By designing different tracr/crRNA sequences, this design could potentially be altered to recognize any gene present in high enough numbers in a liquid sample.

To investigate how this would work with different classes of ESBL, we aligned the sequences found in clinical isolates of ESBL infections in Norway (information provided by Ørjan Samuelsen in Tromsø).

Although it includes relatively few sequences, this phylogeny suggests that the classes A, B C and D of ESBL are so different on a genetical level that the design we propose could probably be used to pin-point the class of ESBLs present in a sample, simply by designing crRNA/tracrRNA compatible to sequences unique to each class.

In summary: to expand our project, we want to create a potentially universal gene detection tool by utilizing parts from the iGEM registry, as there are several dCas9-constructs and LacZ peptides there. The set-up would include both bioinformatics, when designing the right RNA sequences, regular cloning, to fuse the LacZ peptides to dCas9 and protein expression to form the final constructs. If realized, this would enable us to efficiently test for both ESBL proteins and genes within minutes using our PhoneApp. Thus, we imagine to use CRISPR/Cas9 methods in a relatively new and creative way.

This outline is a result of conferring with supervisors Dirk Linke, Eric de Muinck and Paul Grini along with correspondance with Kevin M Esvelt from the Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA (corresponding author of the article “Cas9 as a versatile tool for engineering biology”, PubMed ID: 24076990).

Silver Text

siste avsnittet her

While we are attempting to fight antibiotic resistance with our novel diagnostic test, we acknowledge the fact that our test only works at a protein level. As the central dogma of molecular biology dictates, protein is the last instance in the information flow in a cell. The problem of resistance genes could of course also reside on a genetic level, upstream of actual enzymes. This is important, as a bacteria may very well have resistance genes without expressing them at a certain time. The expression could even be turned on by the presence of antibiotics (check for reference), which makes it important to not distribute antibiotics when such genes are present. All of these things points to a need for a diagnostic tool that not only detects enzymatic activity, but also the genes leading to said enzymatic activity. Such a tool would enable us to test for resistant microbes before they actually express their resistance. Further, earlier detection would lower the probability of spreading of the resistant bacteria, potentially protecting a lot of people from painful, dangerous infections.

This reasoning led us to CRISPR/Cas9. Cas9 is useful in this matter because of its ability to recognize specific nucleotide sequences. By now, it is known that by redesigning gRNA/tracrRNA/crRNA, one can design Cas9-systems that can recognize any sequence. Usually, this is used in combination with the native endonuclease activity of Cas9, enabling the researcher to cut DNA at specific locations and manipulate the target sequence. This differs from our approach. By taking a slightly unorthodox, but innovative approach to CRISPR/Cas9 usage, we want to interface it with our PhoneLab.

In order to combine CRISPR/Cas9 technology with our diagnostic tool, we imagine to preserve the “searching” capability of CRISPR/Cas9 systems, and replacing the “cutting” part with a detectable readout similar to what we have with the nitrocefin-based detection. This means that instead of cleaving the resistance genes, we want to couple the pairing of nucleotide targets and Cas9 to a colorimetric signal. By doing so, detection of genes could relatively easily be interfaced with the detection of ESBL activity in our PhoneLab, thus creating a more complete diagnostic tool for ESBL infections.

This could be achieved in several ways, but as a contribution to our project, we propose the following design of a CRISPR/Cas9 system to be used in PhoneLab.

First, we would need to design tracr/crRNA sequences compatible with known ESBL genes. These RNAs would be linked to a null-nuclease Cas9 (dCas9) - a Cas9 enzyme modified so that it binds tracr/crRNA and is guided to its targets,, but does not cleave the target genes. For each target gene, we propose to have two sets of tracr/crRNA, complementary to different, but adjacent parts of the same gene. Each of these RNA molecules would have a dCas9 partner, which again would be fused to half of a detectable protein partner in a split-enzyme assay. When the tracr/crRNA finds its goal, dCas9 would bring the two parts of the split-enzyme together, thus creating a detectable signal. So, in this system, two independent RNA/DNA-binding events would be required to take place, which lowers the probability of false positives.

The detectable protein partner of dCas9 is currently a matter of research for us, but one possibility is to use the subunits of galactosidase. LacZ alpha and -omega are two separate peptides that form the active form of galactosidase when in contact with each other. By fusing these two peptides to two separate dCas9 constructs recognizing adjacent parts of the same gene, the presence of said gene would be coupled to the forming of a functioning galactosidase. Thus, by adding for example liquid derivatives of X-gal, a visible color change could be achieved, similar to the one seen in our project with nitrocefin.

By designing different tracr/crRNA sequences, this design could potentially be altered to recognize any gene present in high enough numbers in a liquid sample.

To investigate how this would work with different classes of ESBL, we aligned the sequences found in clinical isolates of ESBL infections in Norway (information provided by Ørjan Samuelsen in Tromsø).

Although it includes relatively few sequences, this phylogeny suggests that the classes A, B C and D of ESBL are so different on a genetical level that the design we propose could probably be used to pin-point the class of ESBLs present in a sample, simply by designing crRNA/tracrRNA compatible to sequences unique to each class.

In summary: to expand our project, we want to create a potentially universal gene detection tool by utilizing parts from the iGEM registry, as there are several dCas9-constructs and LacZ peptides there. The set-up would include both bioinformatics, when designing the right RNA sequences, regular cloning, to fuse the LacZ peptides to dCas9 and protein expression to form the final constructs. If realized, this would enable us to efficiently test for both ESBL proteins and genes within minutes using our PhoneApp. Thus, we imagine to use CRISPR/Cas9 methods in a relatively new and creative way.

This outline is a result of conferring with supervisors Dirk Linke, Eric de Muinck and Paul Grini along with correspondance with Kevin M Esvelt from the Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA (corresponding author of the article “Cas9 as a versatile tool for engineering biology”, PubMed ID: 24076990).

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