Difference between revisions of "Team:UMaryland/Notebook"

This was our first week in lab! We took a week off after school to take a break and destress. Since our team is relatively new to lab techniques and our parts haven’t come in yet, we decided to spend this first week working on basic lab techniques and the Interlab Study. We attempted to amplify the A3, C3, and K3 backbones while we waited for parts to arrive.

Our first week went well and we were happy with the way we completed the Interlab study. This week, we wanted to get started on our plastic project and assembled the HlyBD into an iGEM backbone for our PETase secretion system. Transformations and overnights for the HlyBD part were successful. We sent out our HlyBD part to be sequenced. We still had more difficulty with working on amplifying the iGEM backbones and decided to work on doing some troubleshooting to determine where our process is going wrong.

We got back our sequencing results and found out that we did correctly assemble our plasmid. We attempted to troubleshoot the amplification of iGEM backbones. Unfortunately, our G-blocks were delayed again so we weren’t able to do too much wet lab work. We started planning our Mid-Atlantic Meetup and other outreach activities for our local community.

Another week while our G-block wasn’t able to be synthesized. We really do need to get this PETase G-block so while IDT is still attempting to synthesize it for us, we are reaching out to other teams that have worked on it in the past to see if we could possible get it from them instead. We worked on cleaning the lab and ordering new reagents. We also learned theory for many different lab techniques so that we can instantly learn how to troubleshoot the problems that occur in lab. Flowcharts are our friend!

Our part still hasn’t come in, so our lab work has slowed to a crawl. In the meantime, we worked on some outreach. Another student research team on campus came to tour our lab and we talked to them about synthetic biology and our project. We also started a journal club! We look at one or two different articles every week (typically in the field of synthetic biology). One student gives a presentation on the article and afterwards we discussed the implications that this could have on our project and what we learned from it. It went really well this first week and helped promote critical thinking. Hopefully, we can do things like this every week.

Again, we haven’t received all the parts that we need to produce our completed plasmid. In the meantime, we are working on producing bacterial cellulose for hardware for our plastic degradation. We also started brainstorming good designs for our hardware project. We started designing T-shirts and other graphics for our project to make it simpler to explain. This week, the lab members who weren’t in lab as frequently were concerned that they were losing understanding of our progress so we had a long lab meeting on the weekend to catch everyone up and update each other on all aspects of the project. The modeling team did a good job explaining what they discovered in terms of rate of plastic degradation. This encouraged us to allow at least a month for incubated PETase enzymes to degrade plastic and run experiments on it.

The door to our lab broke early this week so we were having problems getting in and out. Luckily, it was fixed quite fast. Our PETase Gblock finally came in so we can really get started on the primary plasmid for our project. Everyone was very excited for this. Also, our Mid-Atlantic Meetup was this Friday so we spent a lot of time preparing to explain our project and the modeling that we have done up to this point. It went really well and we could see a lot of different teams talking to each other and fostering connections and sharing knowledge. Holly from iGEM also came by and was an absolute delight to talk to. We learned a lot about other teams and their outreach in local communities.

This week we assembled our PETase Gblock and transformed it. We ran a gel on the PETase and sent it for sequencing. Unfortunately, it seems like the backbone annealed onto itself rather than assembling with the Gblock. We are going to run it again but attempt to further optimize the process so it hopefully doesn’t happen again. It’s starting to get to the point of summer where people are going to have to leave the housing near campus which will make it more difficult for students to come in and continue doing work.

Once again, we attempted to assemble the PETase from the Gblock and transformed and miniprepped before sending for sequencing. This time, it ended up working for us and we can now 3A assemble it with the cellulose-binding domain and our secretion system. The transformation of PETase with the HlyBD was successful so next week we will make overnights and send it for sequencing to confirm that it was assembled correctly.

We made overnights of our plasmid and sent it for sequencing. As we are getting closer to finishing up our plasmid, we started making plans for experiments to test the efficacy of our project when it comes to degradation. We also are now planning on making a way to detect plastic degradation through the use of other enzymes and plasmids. We looked at past iGEM projects for any ideas and started contacting other labs for certain plasmids to detect the byproducts of PET degradation. We also started working on a rough draft of a Jamboree presentation to display all the data and planning that has gone into our project so far. Professor Masai and Professor Jha were contacted regarding TPH enzymes and PcaU, respectively.

At this time, a bunch of lab members went on vacation since we didn’t have campus housing during August. Also, we only have two more weeks until school starts so we decided to give people a break and only a few people were in lab at that time. TPH plasmids arrived from Masai! Transformed and verified by SDS PAGE.

pNBP, transform pGLO and PcaU attempted, but failed

Initial PNPB results obtained and positive! PcaU g block arrived. Outreach trip to MRF at Shady Grove.

PcaU g block gibsoned into pGLO. Outreach trip to Anacostia Watershed.

PETase HLYBD sequenced and transformed into BL21 cells. Schedule for lab during classes discussed.

PcaU sequencing primers ordered, PcaU sequenced in pGLO backbone. Sequence confirmed PcaU transformed into BL21 DE3 cells. Preliminary experiments were conducted for the scale up of PETase CBD bioproduction.

Initial PcaU results for high and low range detection were carried out. PcaU detection protocol was discussed and finalized. PNPB assay performed testing the effect of cellulose binding on PETase CBD esterase activity. Another PNPB assay performed testing activity of PETase CBD HLYBD under varying induction conditions.

Completed PcaU tests and showed that PcaU could differentiate 1uM PCA concentrations. Expressed PETase HLYBD under the dual promoter system. The condition induced with both arabinose and IPTG showed signs of secretion, with a white substance accumulating on the sides of the tube. The substance was harvested for testing. All 2018 teams involved with PET degradation were contacted and we offered to test our PCA detection device on their degradation product. Makerere, Yale, and RHIT responded, however none were able to supply product at the time.

We spent the week preparing parts submissions. PcaU was assembled into C3 backbone and we removed an EcoR1 site for submission. We also ordered a g block just in case our work ran into difficulty. Fresh PETase enzyme was expressed and harvested. We began experimenting with various buffers to determine which would be best as TPH buffer, and ordered necessary reagents including ferrous ammonium sulfate and L-cysteine HCl. We tested PcaU sensitivity in DH5a vs BL21 cells and found that PcaU was functional in 5a cells but not as sensitive.

SEM images of PETase activity were obtained. It was at this point that we discovered the HlyA secretion system was not working for PETase. The modified PcaU sequence with removed EcoR1 was tested in pGLO and found to be equally sensitive as the original. Parts were shipped out. Over the weekend, TPH buffer was made and fresh cells expressing fresh TPH enzymes were sonicated.

We walked into lab on Monday to find out our freezer was broken, warm, and very smelly. Luckily our bacteria happened to be in another lab. On the weekend we performed tests using PCAU to detect TPH enzyme activity and then used PCAU to test a solution for the presence of TPA.