Team:MSOE Milwaukee/Week10


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Week 10


As a team, we fixed a few of the protocols to add positive controls and more replicates at different concentrations for the xylose and eucalyptol procedures. We also recieved our order of xylose, eucalyptol, and our gene block parts. However, we discovered that we didn't have the Gibson Assembly master mix, which we will have to look into purchasing.


We began the procedure for making competent cells of the BL21 type. We also found a Gibson Assembly kit that we are ordering to assemble our gene blocks. The kit also contains super competent cells, which will be very useful in our future protocols. It is set to arrive tomorrow. When it does, we have to get the competent cells in the -80 as soon as we can. Hopefully this will help us with our transformation efficiency in later steps of our project. We also worked on some housekeeping activities, like updating our website, the safety information, and organizing our current materials.


We recieved our shipment of the Gibson assembly kit for our gene block assembly. We also finalized our plan for assembling the gene blocks, so we plan on beginning the protocol tomorrow. We also completed the competent cell protocol and now have a stock of potentially competent BL21 cells in the -80. We also completed protocols to determine whether BL21 cells would grow in xylose and what concentrations of Eucalyptol would inhibit cell growth. We added positive controls of LB broth to make sure our culture was viable and a negative control of water to ensure our samples weren't contaminated. We took OD readings every four hours for twelve hours after letting the samples incubate overnight to check for growth. More analysis needs to be done, but we initially found that the BL21 utilizes xylose for growth and found that Eucalyptol doesn't inhibit growth and the levels we tested. We hope to repeat the experiments to validate our results.


We began assembling our gene blocks using the Gibson Assembly protocol. We have a limited supply of our gene blocks, so we decided to verify our procedure by assembling blocks 15, 16, and 17. We completed the protocol for these blocks and then saved the samples in order to run a gel. The gel would then show us if the assembly was correct.


We ran a gel to ensure that our protocol was sucessful, and we saw a band in the correct location! This means that our protocol worked and our gene was assembled correctly, so we decided to continue with the procedure for the other gene blocks. We assembled the gene blocks in sets of two to four in order to keep our genes together. Next, we completed our PCR protocol to create more copies for testing of our genes. The PCR also added the prefix and the suffix, essentially making our genes into BioBrick parts! We hope to start characterizing these parts soon.


We ran a gel to ensure that our other gene blocks were assembled correctly. Our first gel was blank, but after completing the PCR protocol again, we found bands on our gel. This showed us that we assembled our gene blocks correctly and we have our BioBrick parts, ready for characterization.