August 14, 2010
WARNING: This is my last post, visa-vi it will be corny and mushy. If the movie Titanic or hallmark cards make you nauseas please do not continue.
Hello there everyone! No, I did not forget about you all, I’ve just been busy moving around (finally at my final place!). In my last days in the lab we had been working on DCPS, we sent in some samples for sequencing and unfortunately did not get anything successful. We also ran a couple more PCRs on DCPS and DCP2 and yesterday we sent in some more samples of DCPS for sequencing. The PCR on DCPS did work; I think that in the end we finally got that stage of DCPS down, though unfortunately the PCR on DCP2 did not work.
In the end I was able to understand how to get good PCR results on both DCPS and DCP1 and a good looking protein gel on DCP1. I was not able to successfully purify any proteins but even being able to successfully synthesize DCP1 makes me happy!
I would like to thank everyone in the Wilusz lab for helping me with this fellowship: Alan, Jeff, Fumi, Emily, Kevin, Ashley, Sai, Carolina, Alexa, Amber, Jerome, John, Stephanie, and Carol. I really enjoyed working in the lab for this fellowship and I am grateful for the opportunity to have done so. I would also like to thank Erin and Jess for letting me contribute to this awesome blog! And I would like to thank Alan for putting up with me all summer and for teaching me so much in the lab!
The end of each experience brings about a beginning to a new one. Most fear this transition because of the unknown obstacles that lie ahead, though with all the advice given to me from my friends in the Wilusz lab I feel confident and ready to take on new challenges. Thank you so much for everything!
And thank you to all the readers that have followed my experience in the lab! Research is something I recommend to everyone, it’s something new, interesting, and very rewarding to try. Farewell for now everyone.
August 9, 2010
Or however that song went. Howdy doody everyone? Hope everyone’s weekend was nice and safe! Sorry about the lack of post on Thursday, I was without internet so that made it kind of difficult to post–just sayin. Anyways, Alan and I have been trying to get DCP2 and DCPS before my last day and we just sent in DCPS for sequencing! DCP2 has been giving us some trouble and we haven’t been able to get a successful PCR off that one, though we have only used the new oligos we had ordered once. It would be uber sweet if we could get DCPS, since we were able to get a good protein gel on DCP1 back in June. But as the days start to wind down I think we will just try to get DCPS “even if it kills us” according to Alan.
Since my last day has been approaching I’ve been trying to work on some papers to turn into ASM to let them know how my “research experience” went and what I thought about it all. I also have to make an abstract to submit to the Spring 2011 ASM meeting and I hope it gets accepted because the meeting is in Louisiana! That would be cool beans!
Today was a rather fun day! The freezer needed defrosting so we got to clear it out and chisel out all of the ice! I knocked off one piece that was the size of my head! I was like WHOA! It was pretty epic and cold.and wet.
Ahem, well my last post will be this Thursday! So until next time
August 5, 2010
I am sad to report that my ten-week fellowship is coming to an end. With the end of the summer fellowship comes an end to my blogging experience. My project will continue during the fall semester; however, I will be a little less flexible with my time to report on account of homework and the like.
As of late in lab, I can pretty safely say that one point mutation is not the cause of temperature sensitivity in the rep gene in B. thailandensis. The best guess at this point is Ts can be attributed to a combination of mutations or quite possibly all five in the specific orientation are needed for the necessary tertiary structure of the Rep protein.
As far as the next step in my project: I have created and ordered new primers to change the fully mutated temperature sensitive plasmid back to the wild type rep sequence one point mutation at a time. This process is the reverse from the original method I employed. Stratagene’s QuikChange Multi Site-Directed Mutagenesis Kit will be used to convert one amino acid per PCR reaction from mutated to the wild type. (If you can recall: there are five different amino acid changes in the rep gene that were said to cause Ts in Burkholderia thailandensis) From this point on the entire process follows the same protocol as I have been doing the last ten weeks.
Once the PCR mutagenesis is complete, a digest and transformation into ultracompetent cells will take place. From here clones will be sequenced for verification of “mutation”. Plasmid DNA will be isolated and electroporated into B. thailandensis. Once colonies appear on selective media, clones will be patched for screening of temperature sensitivity.
If Ts is still seen in one or more of the clones different mutations will be changed back to try to further delineate the exact sequence necessary for Ts in B. thailandensis.
As well as taking this approach, I am also trying to create a library of combinations to burn the candle at both ends. Multiple plasmids containing different combinations of the five mutations were pooled and electroporated into B. thailandensis. Colonies will be patched and screened for Ts. If I am so fortunate to find clones that do in fact exhibit Ts, these clones will be sequenced to determine which combination of mutations leads to Ts in B. thailandensis.
Although I have not been successful in a “crossing the finish line” standpoint, I am hopeful that with time a more clearly defined sequence will be discovered. I know this is a tad cliché but, it’s like Aesop’s fables always told us, “slow and steady wins the race.”
With that I thank anyone who has been able to read and keep up with my posts and I wish you a happy end to summer and a hopeful end to the decade.
Go in peace,
August 3, 2010
Aloha everyone! No, I’m not in Hawaii; I just wanted to start with something new. It turns out that the re PCR of DCPS seems to have worked so we started the ligation process yesterday.
Today Alan and I did not go into the lab, but instead went to go to the Body World exhibit. The exhibit was so cool! And I was less creeped out then I thought I would be (double bonus)! It was all put together so artistically and you get so engrossed in it that you almost forget that all the displays were real people. It was interesting to learn and see all these things about the heart and body and even think, hey I learned that in biology! (Yes, I am that nerdy). I think the creepiest/coolest part there was the baby fetus exhibit. You could see what a fetus looks like at different stages of a pregnancy, and I was born almost two months early, so it was neat to see how small I was. Going from that small to over six feet tall must have knocked my parents off there cahoots! But Body World was truly amazing and I would like to thank Alan and his dad for letting me tag along!
Other than that, things have been relatively slow. My last day of the fellowship is next Friday, so next Thursday will be my last post. And I have no internet where I am staying at the moment, so I will try my very best to post this Thursday. Until next time, Aloha!
July 29, 2010
Yesterday was no ordinary day in the lab, well out of the lab to be exact. We all took a hike up Horsetooth for some lab bonding time. The view was beautiful! And the slight overcast was perfect to keep us cool. Jeff was uber giddy to bring his doggy Hank, and everyone made it up and down safe and sound (double points for rhyming-sort of). After the hike, Jeff and Carol were kind enough to treat us to Famous Dave’s BBQ. It was all so delicious, and Kevin, Fumi, and Emily were able to down a family feast at the expense of their health. At least they still have their pride. I got to try bread pudding for the first time, and might I say no amount of therapy will ever make that experience ok. It may be the fact I don’t really like the taste of soggy french toast, but it could also be the fact that it was coming out of my nose from laughing to hard. Either way, that might be the first and last time I have that eastern coast delicacy. A hard night’s sleep and a few ulcers later lead us to today.
Today in the lab Alan and I have restarted on DCPS and DCP2. We did an RT PCR on both and then a re PCR on both. As I wont be in tomorrow, Alan will run some gels tomorrow to see if we got the fragments of our enzymes or not. On that note I bid you all adieu, and have a great weekend!
July 27, 2010
Holy guacamole! Where did the time go!? Sorry about the skipped post everyone, but I’m finally at house 1 of 3 of the places I will be until the fall. I’m very thankful my friend Justine let me crash at her place for a week! Well now back to business!
So we got our DCPS sequenced and unfortunately it doesn’t seem there is a fit. We haven’t had the time to look at the sequence of XRN1, but that one’s pretty stubborn so I don’t have my hopes up for that. We ran a protein gel on our purification of DCP1 and LSM1 and there is a possibility something is there, but the band is really faint so we are going to have to find a way to concentrate it more.
And today I had a lab presentation which went pretty well.I think. I made these little cartoons and made some of the lab laugh pretty hard because one of my cartoons looked, well let’s just say I shouldn’t have added yellow in a certain place I did and it made it look like Danny the DNA was relieving himsel-oops. Hahahaha. I said a couple of wrong things, but eh, I tried my best and there were only a few sly remarks. Also, I brought caprisun and cookies (always a classic) which turned out to be a hit (score).
Well I am off to go finish organizing/unpacking! I hope everyone’s week is nice and I’ll post again Thursday!
July 24, 2010
This is Erin reporting the latest on the search the elusive temperature sensitive phenotype.
I knew it was too good to last. I had a sneaking suspicions my project was being too cooperative. I know this sounds cynical but I usually never go this long without weird or no results.
I have finally hit the point in my project where trial and error has become my only option.
When the mutation combinations were electroporated into B. thailandensis, plated, and patched for screening, neither combination yielded Ts phenotype.
The revertant plasmid and Ts control were transformed back into E. coli patched out and will be isolated for sequencing early next week. However, there is some funny business going on with the Ts phenotype.
Also, six plasmids were sent for sequencing that were mutated with multi-site mutagenesis with the last point mutation to be tested. Two out of six are positive for a D to G mutation at site 145 in the rep gene. This is exciting for me because I have not been able to isolate a plasmid with this single point mutation yet. This point mutation will also be screened for Ts in B. thailandensis early next week.
I wish I had more to post but for the time being I will just have to keep plugging along and see what the next new development is.
Thanks for reading and as always, enjoy.