Click on the picture to display team Heidelberg's collaboration page.
As fellow scientists exploring the field of targeted evolution, we helped the iGEM-team Heidelberg by conducting an experiment for them, to validate their results in another lab. Our colleagues used three different mutagenesis plasmids (MPs) created by A. H. Badran et al. in 2015: #1, #2 and #3, as they are named by the iGEM team. The MPs build up on each other in accession of their respective numbers.
#1 increases the mutation-rate through arabinose induced expression of dnaQ926, an ineffective E. coli DNA Pol III proofreading domain.
#2 also contains the gene encoding Dam methylase under arabinose induction, as well as the gene seqA under low-level expression. Dam methylase is crucial in mismatch-repair in E. coli, as it methylates the parental strand in DNA replication, making it distinguishable from the new daughter strand. If Dam methylase is overexpressed, the daughter strand is methylated too quickly, which impairs the mismatch-repair mechanism. seqA encodes a protein that delays the function of Dam methylase, which solves some issues of the arabinose induced promoter being not completely tight.
#3 contains the additional genes ugi, cda1 and emrR. ugi encodes cytidine deaminase, which catalyzes cytosine to tyrosine transitions and therefore causes base substitutions in the host’s genome. This transition goes through a deoxyuracil intermediate, which may be excised by uracil-DNA glycosylase during the native uracil-excision pathway. cda1 is a natural inhibitor of uracil-DNA glycosylase and therefore acts synergistically with ugi. emrR is a gene repressor for a number of different multidrug resistance pumps. The exact mechanism is unknown, but overexpression of emrR leads to a repression of those efflux pumps and therefore may lead to an increase of mutagenic pathway intermediates in the cell, increasing mutagenesis.
#1, #2 and #3 raise mutagenesis rates high enough, for E. coli to spontaneously evolve antibiotic resistances. If plated on several different antibiotics, under induction of the plasmids with arabinose, a portion of the cell population is supposed to survive with new resistances. This is what Heidelberg wants us to verify. Click here to read the protocol.
nsc = no single colonies
Note: Some plates showed no colonies at the time point that the pictures were taken. Empty plates were incubated longer and counted later.
Click on the picture to display team Munich's collaboration page.
In the beginning of October two of our team members, Michael and Julian, traveled to Munich to characterize our new basic part BBa_K2294007 using flow cytometry. Team Munich kindly provided us their flow cytometer machine as well as helpful advice. Their team member Robert even hosted us in his flat for one night. Thanks again, Rob! After two days of work Michael and Julian participated in a current and former Team Munich iGEM participant reunion before traveling back home.
Michael and Julian met current and former iGEM participants from team Munich
INSA-UPS France - Toulouse
Click on the picture to display team INSA-UPS France's collaboration page.
iGEM-team UPS-INSA Toulouse needed some help with the integration of their RFP containing plasmid. Luckily, our student team-leader is also a yeast expert. He used a high efficiency protocol which resolved their problem.
This is the Baltimore BioCrew fighting against plastic waste. Click on their picture to display their collaboration page.
As a potential application, our project could help clean up microplastics from the environment. Releasing GMOs and confronting the public with this topic bears several risks. We found out that the Baltimore BioCrew is working on a similar topic, sharing the same difficulties and questions. A delegation of our team flew to Baltimore and setup a meeting with the BBC. After a constructive talk and some European strudel we decided to help the Baltimore team and set up a survey to gain an insight on the thoughts of the population. Back in Europe we stayed in contact and supported the BBC whenever they had any further questions. We were very happy to support the BBC in their work and hope for ongoing collaborations in the next contests.