Team:BYU Provo/Notebook/CholeraDetection/Springexp/Period1/Dailylog

9/4/2013

BYU iGEM can count on every member, now that summer is over and all are again enrolled in classes. We took time at the beginning of class to list our main projects (aside from research itself) from here on out: they include cloning our parts into the iGEM backbone plasmid, filming a synthetic biology techniques video to collaborate with another team, finishing the website, and publishing our synthetic biology Children's Book and collecting data to demonstrate that children who read it and the included parental guide improve their understanding of science generally and synthetic biology in particular.

Kelton and I are responsible to see the book published, and the data collected. We set ourselves the following deadlines: Sep. 8th: Have in our possession the edited, shaded version of the book. Redge (the illustrator) confirmed that he would finish by Sunday the 8th. Sep. 11th: Submit the book for publishing. Sep. 20th: Distribute paper copies/pdf copies to elementary school teachers and parents. Children will be given a pre test before reading the book, and later a post test. The test results will allow us to quantifiably show that our book improves understanding of synthetic biology among young children. Sep. 25th: Compile and analyze the data.

Several weeks ago, I read a paper indicating that lysogenic lambda is induced to lysis by host cell recognition of AHL's, the molecules used in bacterial quorum sensing systems. I thought that our bacterial strain with the Lambda lysogen, TT9907, might recognize cholera's autoinducer, and perhaps that might trigger Lambda. I streaked a line of TT9907 next to cholera and then away from cholera. No bacteria grew next to cholera, but the strain did grow away from cholera! Now, we need to show that it is indeed lambda that is killing the cell and not some toxin from cholera.

We designed an experiment with 6 bacterial strains: 2 without the lambda lysogen, 1 with the lambda lysogen, and three that may have the lambda lysogen. The latter three strains are each from single colonies that grew following an electroporation procedure to get TT9907 to take up the plasmid pIG87. Amazingly, top agar lawn assays with a drop of hydrogen peroxide added to stress lambda into lysis show that after electroporation, there are no plaques. Either Lambda gets out during electroporation or TT9907 loses its susceptibility to lambda. Anyway, we took these six strains and streaked a line of them next to a patch of cholera and away from the patch. Depending on how the bacteria grow we'll be able to say if cholera (perhaps some toxin) kills TT9907 or lambda kills TT9907.

KK, KP

9/6/13 Our results from the side-by-side plate comparison of cholera with several strains of bacteria are encouraging! We grew a patch of V. cholerae for one week on LB plates. Then, we streaked a line of a bacteria adjacent to the patch and another away from the patch. We did this for six different strains on six different plates. None of the bacterial strains plated adjacent to cholera grew. All strains grew away from cholera. However, TT9907, the strain with lambda lysogen, showed plaques! There were distinct plaques on the line of bacteria leading away from Cholera. Through what we believe is a quorum sensing pathway, the lambda lysogen recognizes that E.Coli has detected cholera. It mobilizes excision from the genome, replicates, and lyses it's host.

We prepared top agar lawns of our 6 strains, with cholera plated in the middle, to show more clearly the result we saw today.

Clarice transformed the iGEM backbone into E.Coli and grew it up in an overnight. From those cells we isolated the plasmid, pIG91, in two Eppendorfs, at concentrations of 52 ng/uL and 82 ng/uL. Monday we will digest the plasmid and the SdiA gene to splice it in and submit to the registry.

KK, KP