Team:BYU Provo/Notebook/CholeraDetection/Summerexp/Period3/Dailylog

7/31/2013 By sad, frustrating experience, we’ve surmised that there is a problem with the patches of TT9901 and TT9907 to which we keep returning for experiments. We received these strains from the John Roth lab at UC Davis as three small glass stabs. Whenever we plate them on LB directly from the stabs, we always get spotty growth. We believe that we’ve been ignorantly selecting for host cells that are not susceptible to bacteriophage lambda. To correct this, we’ve selected 32 colonies from 3 patches: 15 colonies from one patch of TT9901, 15 colonies from one patch of 9907, and 2 colonies from our control strain that lacks the lambda lysogen, TT25281. We’ve started these as overnights. Tomorrow, we’ll freeze down one mL of each strain, and with the remaining culture, we’ll make top agar lawns using X1 top agar. In the middle of these lawns we’ll add a drop of hydrogen peroxide, which is known to stress cells enough to cause lambda to convert its lytic cycle. Any lawns that show plaques relative to a non-H202 control top agar lawn will be strains that we know have lambda and can become induced to lysis. 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

9/9/13

The children's book is shaded! We read it in class today and confirmed that everyone enjoyed it. Redge needs only to make a few grammatical edits. Hopefully tomorrow or Wednesday we'll get an appointment with Dr. Jaime Jensen, a member of the biology department here at BYU. Dr. Jensen earned her doctorate in Biological Science Education, and we hope that she will give us some good ideas on how to maximize the educational value of our book and its appending parental guide.

And, we have wonderful news from the top agar lawns we plated on Wednesday. Our results confirm that cholera does NOT kill bacteria that grow next to it, but DOES indeed stress bacteriophage lambda such that it lyses its host cell. See our photos to the right!

KK, KP

9/11/13

At 2:00 we joined Dr. Jaime Jensen for an insightful, productive and encouraging meeting about our book. Dr. Jensen has her Ph.D in biology education, and was able to offer some very good insights on our book, its parental guide, and the pre/post test questions we will administer. Unfortunately, we have a significant roadblock: our project has not been IRB approved. We weren't aware that in order to ask children to take a test on their understanding of synthetic biology and collect data there must be IRB approval. So, we've either got to luck out with an exempt status, or rethink collecting data from school classrooms. It would be less paperwork for us if we cleared a parental consent form with the IRB and visited a library, where we could ask parents to sign the form right there.

I found a protocol for purifying bacteriophage lambda! We hope to be able to do that tomorrow.

KK, KP

9/13/13

Yesterday, Thursday, we purified bacteriophage lambda, and today we set up spot tests to verify the presence of the phage.

Our purification protocol was as follows: we grew overnight cultures of TT9907 with lambda lysogen in a 30 degree Celsius incubator for 18 hours. After incubation, we heat shocked the culture in 2 mL aliquots at 43 degrees Celsius for 10 minutes. We recombined the aliquots and incubated them at 37 degrees Celsius for 6.5 hours to allow lambda to replicate. We again aliquoted the cultures into 1.5 mL aliquots, added 400 uL chloroform, vortexed, and allowed to sit for 10 minutes. After sitting, we centrifuged at 8000 rpm for 10 minutes to separate cellular debris from bacteriophage in the supernatant.

We added finishing touches to our book, including a dust jacket and a few blurbs on the back, and placed the order with an online publishing company! That will give us one hard copy; Monday at noon we also have a meeting with Giovanni Tata, the director of BYU Creative Works, to discuss publication of the book!!

KK, KP