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Ran liquid cultures on B0015 (stop codon), I719005 (T7), J32015 (pelB), C0062 (luxR).

We created an in-depth plan for how we were going to assess our project. We divided the project up into manageable sections and assigned particular areas to people on our team. One area we decided on was protein engineering in which we hoped to engineer a more efficient cutinase protein. Minipreped J23101 and digested it.

Along with these parts, we also looked at a new miniprep kit that was given to us for free, titled Biobasic. We wanted to test it in comparison to the Invitrogen kit we currently use. Following the hydration we transformed the parts and left them overnight in the 37 °C room. Ran the digested J23101 on a gel.

Today we did a double digest of B0034 at the S and P sites and sequentially digested B0034 first at the E site using Buffer 4. We miniprepped samples using both the Invitrogen and Biobasic kits. Using the nanodrop spectroscopy, we determined that the Invitrogen kit was still better, so we continued to use that. We also began to look into Gibson assembly, and due to past iGEM inability to attain success, we requested the protocol from the iGEM Washington 2011 team. Minipreped part B0034 (RBS) and digested it in two ways, E+X and S+P.

Took cutinase sequence and put it into swiss model, gave us a model that was similar. Starting off the protein engineering side of the project we looked towards first replicating previously made mutations in the paper titled “Engineered Thermobifida fusca cutinase with increased activity on polyester substrates” referenced in our original paper, “Isolation of a Novel Cutinase Homolog with Polyethylene Terephthalate-Degrading Activity from Leaf-Branch Compost by Using a Metagenomic Approach.” We wished to replicate three mutations, T96A, V212A and Y127A. We wanted to verify that these mutations would produce similar increases in protein activity in our protein, LC Cutinase, as they did in the paper’s protein Tfu_0883. In order to do this we wished to compare best fit homology models generated for our protein and the papers. Using Swissmodel we generated 3VisB as a best fit for LC Cutinase and 1JFR as the best fit for the paper’s Tfu_0083.

We performed digests, double digests, and ligations. LC Cutinase was ordered at Genscript. We further looked over the the results of the homology models generated for the two proteins. We compared them in two ways: 1) Generating a multiple sequence alignment for both proteins and homology models and accessing the similarity of the mutation sites. 2) Loading the homology models in pymol and accessing the three dimensional similarity visually. Both methods gave us confidence in the mutations resulting in similar activity increase. J23100 and J23101 minipreps ran.

We performed gel extraction and redid double digest of parts that were mislabeled. We contacted Piedmont Plastics about specific kinds of PET. After looking over the results of the mutation verification with our mentors it was decided that we could expect with high confidence the mutations conferring similar results.  

Our team was finally back together, as a majority of people were absent for the latter part of last week. A few of us went over what was done last week, and how there was an error in running the gel extractions, so we made a decision to reattach the promoters to the B0034 RBS. Some of us began by running digestions of J23100, K206000, J23101, and R0010 at the E and S sites. B0034+C0012 was digested at X and P, J23101 was digested at S and P, and B0034 was digested at E and X. We also wanted to carry on some of the themes from last years project, and began trying to mutate K206000, or the pBAD promoter, to improve its function through random mutagenesis. Some of our team members also began preparing the E. Coli strain, numbered MG1655, by streaking the organisms on plates from the glycerol stock. After, we rehydrated MG1655 strain. We also liquid cultured and plated DH5α cells and tried to make competent cells for MG1655 strain, but put them in the 37 °C room. Some of our team members began to run error prone PCR (<a href="https://2012.igem.org/Team:UC_Davis/Notebook/Protocols">PCR Protocol</a>) on the K206000 part as well, to make a part family as well. In our discussion of how we were going to assay our samples, we also ordered pNPB, another sample used to determine degradation in comparison to PET. With the previous mutations confirmed we looked towards generating our own mutations. The first step was generating a 3D structure from LC Cutinase’s sequence (LC Cutinase being newly discovered has no known 3D model). We started this by using Swiss-model again to fit LC Cutinase’s sequence into the 3D model of 3VisB. This process took Swiss-model the most of the rest of the day to generate.  

When the team came into lab today, we used the spectrophotometer and the OD600 readings were over 1, which signifies we overgrew them. The error came from placing the cells in the 37 °C room instead of at room temperature. Oops. So we then remade the component cells for the MG1655 strain and extracted genomic DNA from the MG1655 and DH5α E. Coli strain. Luria Broth was made, and we incubated the starter culture and made inoue transformation buffer. We miniprepped the parts that were transformed yesterday, and sequentially digested B0034 and B0034+E0240 at the E site with plans for the X site to be digested on Wednesday. The PCR products from yesterday were ran on a gel to confirm the products, and then a purification was run, and the process was repeated twice. In the area of protein engineering we looked over the Swiss-Model generated 3D structure and confirmed its realism. The energy diagram showed that LC Cutinases sequence fit quite well into the model of 3VisB. We also performed an overlap of the two models, the newly generated LC Cutinase model and 3VisB’s model, in order to check how well they matched. We received an RMS value of .78A, quite good. Minipreps ran B0034, J23100, B0034+E0240, B0034+C0012, R0010, K20600

Today genomic DNA from DH5α and MG1655 was purified and we restreaked DH5α cells from yesterdays sample. The purified PCR from yesterday was also put on a gel and extracted. Some of our team members also began working on the modeling of the cutinase enzyme using computer software to see how we could optimize degradation. More research was done in regards to making sure we had all the materials and that the assays to be conducted were also looked into and finalized. A gel was also ran of the purified MG1655 and DH5α genome. Going further with our generated model of LC Cutinase we wished to create the two ligands with which it would interact, PET and pNPB. We did this in SMILES (a structural file generator). We then looked to fit these ligands into LC Cutinase’s active site in pymol and load that file in Foldit. Ran gels on minipreps and performed gel extractions and purification.

 

The day started off with us receiving the MG1655 strain we ordered from Yale University. Even though we already began to use samples from one of our mentors' labs, it was a good idea for us to make liquid cultures and plates of the cells in case something went wrong. The two enzymes required in the pathway for breaking down ethylene glycol also underwent PCR along with genomic data from the MG1655 and DH5α cells. Luria Broth (LB) was made for kanamycin plates as well. We preformed ligation and transformations of the genomic data into the DH5α cells, and successfully cultured competent cells. At the end of the day, we rehydrated primers for the mutated cutinase as well, with a hope that the mutations will improve the cutinase functioning. On the protein end of things we manually fit the ligands into the active site of LC Cutinase in pymol. However, upon loading these files in Foldit the protein did not wrap around the ligand as hoped. We then seeked out an alternative means in which to better fit or dock the ligands into the active site. Nano-dropped gel purifications.

We purified PCR of dehydrogenase. Competent cells failed, so we remade some more while making some modifications to the protocol. In addition we performed some gel DNA clean up, mini preps, digestions, ligations, and transformations. We decided on using Swissdock, a ligand to protein docking program, to fit our two ligands into LC Cutinase. This program ran a majority of the day but we received successful docking files for both ligands by the end of the day. Minipreps of J23100, J23101, K206000, R0010, B0034

We ran a gel and did some PCR cleanup. More work was done on SOEing. Did some digests, sent stuff for sequencing, and worked on reductase part construction. For the protein part of the project we moved forward with our successful docking files to load them into the Foldit program. However, we ran into technical difficulties with the requirements Foldit had for file types accepted. Ran gel digests/ purifications on J23100, J23101, K206000, R0010, B0034

On the protein engineering part we discussed with our professor the issues we had with the files. He gave us a helpful layout of the steps needed to convert files to the correct format as well as generate certain needed files. This took the rest of the day but we were able to load both ligands bound to LC Cutinase into Foldit successfully. Redigested B0034.

Ethylene glycol was ordered today. We did some more digest, double digest, and ligation. For the protein engineering part we now used Foldit to come up with our own mutations listed in the protein engineering page. We also further tested the previously made mutations in Foldit to helped substantiate our confidence in them further.  

We made forward and reverse primers so that the reductase could work in aerobic conditions. We performed ligations of K20600+B0034 (1), K20600+B0034 (3), reducates+psBIA3, vector controls (B0034 [1] and [3] and pSBIA3), and insert control K206000. For the protein part we had our mentors look over our generated mutations and decided upon seven different individual mutations.  

Today was the day scheduled for the iGEM meetup! After doing some stuff in the lab (find out what everyone did), our team left around 1 P.M. for Genentech Hall at the UC San Francisco campus. There we interacted with teams from UC Berkeley, UCSF, and Stanford-Brown and presented our respective projects. The advice and critiques were helpful to us as it gave us a different viewpoint of how we can improve our project. We also socialized outside following the presentations, and it was an interesting experience for our team as a whole. Today we finalized the generated protein mutations and used a website called PrimeX to generate the SDM primers for all ten mutations.  

The team split up again for more work and research. We finally got the strain we requested from Dr. Aguilar in Barcelona (Strain E-15 EG3), and we also received the primers for the glycoaldehyde reductase compatibility with an aerobic environment, so that was exciting. We similarly got a stock of ethylene glycol for which we can test the activity of the enzymes, and began culturing on it. We also continued our work on the SOEing procedure, trying to remove the Pstl restriction sites from the sequences after a brief mix up yesterday. We also got in contact with another professor here at UC Davis that does work in textiles and material sciences, and obtained some samples of PET that we can use in our assays of our enzyme.  

We skyped with the UC Merced team and looked at possible collaborations we could possibly have with them. It was also a chance for us to impart some of our knowledge to them, as this is their first year participating in the iGEM competition. We also spoke with the CEO of Micromidas Inc., a green technology startup based in Sacramento that does work in taking waste water and sludge, and through microbial processes, generating biodegradable plastics for marketing. This gave us some insight into the economic situation of the environmental market and how we could conduct an economic analysis of our project. We got the Cutinase. Transformed that into DH5α and MG1655 and we left that overnight. Ran a gel of many of our cutinase constructs, various backbones and both dehydrogenase and reductase.

Fast double digest worked! We ligated and transformed the dehydrogenase, reductase, an insert control and two vector controls. The competent cells did not turn out too successfully, as there were a limited number of cells that grew after a generic transformation to test them. Ran gels on J23101, B0034 and R0010+E0240 using fast digest enzymes.

Today was a busy day in the lab. We began constructing a genomic library for the Strain E-15 EG3, through using transposases. After getting the sequences in from Monday, there ended up be a base pair deletion and a point mutation in reductase, and there was a single base pair mutation in the dehydrogenase. The dehydrogenase was not as bad, as we can use site-directed mutagenesis (SDM) to fix the mutation. However, the reductase will need to be redone. Good news was that the SOEing worked, however! Nanodroped gel purification and made liquid cultures of Full Cutinase, Cutinase without His tag, K206000+B0034, B0015, B0034, R0010+E0240 and KAN.

Ran a gel of the PCR screen from yesterday. We began the second Tecan experiment – varying amount of ethylene glycol for the Strain E-15 EG3 to see the optimal concentration for growth. We also fragmented the Strain E-15 EG3 genome for genomic sequencing. Finally, we miniprepped B0034 + reductase, and B0034 + dehydrogenase, along with B0015 to build simple construct later on. Ran gel on Thursdays liquid cultures. First attempt on Gibson PCR and PCR screen, resulting gel showed weak bands and wrong lengths.

 

 

<br><br><b>Monday August 20th</b><br>

We redid the PCR screen for the part LacI part (J23101+B0034+C0012) as well as B0034+Kan, K206000+B0034+K936014 (Cutinase construct with inducible promoter), and B0034+K936014 (Cutinase construct with RBS). Using a fast digest, we ligated B0034+dehydrogenase along with B0034+reductase to build the simple construct. Also, we added a stop to the two enzymes in order to prepare and build the complex construct. The resulting nanodrop concentrations were lower than expected, so we decided to scale up the amount of DNA in the digestion reaction. Second attempt at Gibson PCR and PCR screen, bands at wrong lengths. Gel run and gel purification done on J23101+R0010+E0240 and B0034.

We began sequencing Strain E-15 EG3 today to see the different mutations that occurred from the University of Barcelona laboratory. It was actually easier to make a library than it sounds. We followed the non-PCR protocol, so it involved a lot of incubating and mixing. Literally, the hardest part was the math involved to find dilutions and concentration. In other news, we found out that the J23101 + B0034 + C0012 ligations worked! We are en route to our complex construction of the ethylene glycol side of things. We also met with our advisor, Dr. Facciotti, about the wiki to make some edits and additions. Sequencing for K206000+B0034+Full Cutinase sent in. Gel ran and gel purification done on B0034, KAN+B0015 and Full Cutinase.

We glycerol stocked the constructs that we PCR screened yesterday. We are excited for the end of our construction process! Continuing on with the results from the PCR screen pieces, we are ligating the promoters (J23101 and K206000) with B0034 + Reductase + B0034 + Dehydrogenase, R0010 with B0034 + Dehydrogenase + B0015, and B0034 with C0012. We’ll see how the ligations are when we transform them tomorrow. We got our sequencing data back, and it turns out that we have some mutations that we need to fix in the reductase and dehydrogenase enzymes. There was a mutation and a deletion in the reductase, and there was a mutation in the dehydrogenase. However, these will be fixed with some site-directed mutagenesis (SDM). PCR screen on KAN and Full Cutinase performed.

Time to transform! We transformed the ligations from yesterday, and we will hopefully see some colonies tomorrow. Later, we analyzed the sheared genomic sequence concentration with quantitative real-time PCR (qPCR). It is really nitpicky to do qPCR because of the sterilization required because we do not want bacteria to be introduced in and have their DNA be replicated through the thermal cycler. We also devised another experiment for directed evolution of the Strain E-15 EG3 to choose for efficient, rapid growth. We will grow the cells in 30mM of ethylene glycol and when they reach 0.2 OD, place 200µL of the cells into 5 mL of 30mM ethylene glycol.  Currently, the doubling time of Strain E-15 EG3 on ethylene glycol is about 6 hours, so we want to decrease this time to see colonies much more rapidly. Digestion performed on C0012, R0010+B0034+E0240, J23101, B0034+KAN+B0015 and B0034+Full Cutinase. First round of SDM performed for Cutinase mutants.

We analyzed our qPCR data and saw that we have a concentration of 5.617 pM of the sheared genomic DNA. We will be sending out our library ASAP for sequencing. We are also doing our first round of SDM today! We want to mutate the reductase to become more efficient under aerobic conditions by changing out two base pairs. The dilutions are tricky, but we got them down by the end of the day! We performed the SDM protocol on the J23101 + B0034 + Reductase + B0034 + Dehydrogenase, K206000 + B0034 + Reductase + B0034 + Dehydrogenase, and B0034 + Reducase + B0015. We also continued on with our Strain E-15 EG3 directed evolution, and we will almost every day. Digestion and gel extraction/purification run on J23101, B0034+KAN+B0015, B0034+Full Cutinase, C0012 and R0010+B0034+E0240. DPN1 added to first SDM PCR products and gel screen run of results.

The transformation of the SDM reaction with B0034 + Reductase + B0015 had no colonies so we are redoing the transformation. This time, instead of adding 1 µL of the vector, we will add 4 µL, 5 µL, and 6 µL. Hopefully this increases the amount of cells that take up the vector and therefore the mutated plasmid. The other two transformations were perfectly fine. We continued the SDM experiments on the DNA that came from the SDM of yesterday (pre-transformation) as well as B0034 + Dehydrogenase + B0015 to fix the dehydrogenase mutation. We ran the gels of the SDM and we did not see any bands before or after digestion with DpnI, which digests methylated parental DNA. However, we continued on with the transformations. Gel run on R0010+B0034+E0240 and B0034+Full Cutinase. Second round of SDM done. Transformations of first SDM PCR.

It turns out that we should not have done the second round of SDM on the DNA straight from the previous SDM reaction. The plasmid DNA must be methylated for the reaction to work, and the only way the DNA gets methylated is in the actual cells. Our new plan is to wait for the sequencing data to come back after each round of SDM to see if we have the correct sequences. Then we will take the correct ones and do a subsequent round of SDM. We redid the SDM with the B0034 + Reductase + B0015 and we finally finished the transformation at 9PM! We transformed with MG1655. Ran screen on second SDM run with and without DPN1.

 

We started the process for making some DH5 competent cells so that we have a ready stock on hand. We also revised the wiki and added some more information to the pages. We ran a gel on the PCR screen of the SDM reactions to fix the aerobic mutations on J23101 + B0034 + Reductase + B0034 + Dehydrogenase and K206000 + B0034 + Reductase + B0034 + Dehydrogenase and B0034 + Reductase + B0015. We only had bands show up correctly for the J23101 + B0034 + Reductase + B0034 + Dehydrogenase and K206000 + B0034 + Reductase + B0034 + Dehydrogenase, so we prepared liquid cultures for four colonies each in preparation for miniprepping and sequencing. Another PCR Screen was set up for R0010 + B0034 + Dehydrogenase + B0015, and these were the correct band sizes. Ran third round of SDM. Varied amounts of template, 50ng/ul and 20ng/ul. Also ran another segment SDM PCR on Y127A and T96A mutants, 50ng/ul. Conducted a PCR screen on parts J23101+B0034+KAN+B0015 and J23101+B0034+Full Cutinase. Added DPN1 to third SDM and ran screening gel. Third SDM products transformed.

 

<br><br><b>Monday September 3rd</b><br>

We did a SDM reaction on J23101 + B0034 + Reductase + B0034 + Dehydrogenase and K206000 + B0034 + Reductase + B0034 + Dehydrogenase, using multiple primer sets, including aerobic mutations (aer), dehydrogenase mutation (fix_deh), and the deletion in reductase (del_red). For B0034 + Reductase + B0015, we did a multiple SDM reaction using primers to fix all of the reductase mutations (aer), (del_red), and (mut_red). Re-did segment SDM on unsuccessful middle piece. Plate test showed plates were ok. DH5 alpha cells tested in transformation to check their ability. Transformed on DH5 alpha and MG6155. Liquid cultured J23101+B0034+KAN+B0015 and J23101+B0034+Full Cutinase.

Today, we began our SDM reactions by SOEing on the B0034 + Reductase + B0015, J23101 + B0034 + Reductase + B0034 + Dehydrogenase, and K206000 + B0034 + Reductase + B0034 + Dehydrogenase constructs. We are following the same protocol as before -- PCR then gel extraction and purification to select for the correct fragment lengths. Instead of using Taq and Pfu, we used only Pfu in a 1:1 substitution. We performed PCR screens on the SDM (multiple sets of primers) from yesterday of the J23101/K206000 + B0034 + Reductase + B0034 + Dehydrogenase, but we saw no bands. Therefore, we did another set of 12 PCR screens to see if there are any properly sized inserts in any of the colonies. We also did a multiple set SDM of the B0034 + Reductase + B0015 and transformed them. We will see how they turn out tomorrow. PCR screen on few successful transformations of mutants. Mutants F125Y, V212 and Y127A showed successful PCR screens. Performed liquid cultures on them. Ran digests on J23101 and B0034+KAN+B0015. Reattempted gibson with new primers.

We took the OD reading of the Strain E-15 EG3 and MG1655 and halted the culture at OD 0.2. The cells were taken to Arcadia for mutagenesis (EMS) and grown in LB overnight. A PCR screen was done for B0034+ Reductase+ B15 (aerobic), which showed successful band sizes and liquid cultured overnight. A gel was also run for the SOEing PCR, resulting in only several visible bands. The SOEing fragments were gel extracted but not ligated together because there were no complementary pieces. A new multiple set SDM was set up for J23101 + B0034 + Reductase + B0034 + Dehydrogenase, K206000 + B0034 + Reductase + B0034 + Dehydrogenase, and B0034 + Reductase + B0015 using a longer elongation time (10 minutes) and was transformed. Gibson PCR screen results had sizes expected. Gibson reaction itself was run afterward. Second Gibson PCR run using lower concentrations. Gibson reaction results showed no combination of pieces, unsuccessful. PCR screen on B0034+KAN+B0015+J23101 run and liquid cultures taken from successful screens.

We are trying the SOEing reactions again, in hopes that we get more bands. We went back to our stocks to make primer dilutions so that everything was at 10 uM, and again, we used 50 ng of template DNA. We had to re-transform the multiple set SDM reactions that we had from yesterday because they were accidentally vortexed and destroyed the competent cells. From yesterday, we are taking the mutants that grew overnight and plated 200 and 400 uL on ethylene glycol plates. We got our sequencing data back for the J23101 + B0034 + Reductase + B0034 + Dehydrogenase for the aerobic mutations. Minipreps done on B0034+KAN+B0015+J23101 and B0034. PCR screen done on Gibson PCR of all gibson backbones with varying concentrations. No results seen on gel. Fourth round of SDM ran using longer extension time.

We received our sample data back from sequencing for the J23101 + B0034 + Reductase + B0034 + Dehydrogenase on Friday and we did another round of SDM to fix the deletion that we also had. We used 50 ng of template again and followed the same protocol as before. We are also trying out a new method for SOEing, where we take a tiny amount of the gel via a P200 and then adding that to a PCR mix to amplify it. We also sent a million samples for sequencing. The different samples are the J23101 + B0034 + Reductase + B0034 + Dehydrogenase, K206000 + B0034 + Reductase + B0034 + Dehydrogenase, B0034 + Reductase + B0015, and R0010 + B0034 + Dehydrogenase + B0015. Hopefully these turn out well! DPN1 added to fourth SDM run and gel screen performed. Transformations performed. GFP full Lac Construct, J23101+B0034+Full Cutinase, V212A, F125Y, and Y127A sent for sequencing.

Today was full of analyzing sequences! The results were a mixed bag. Some constructs had one or two fixes, when we actually needed four to be fixed. We glycerol stocked more of the things that we sent to sequencing so that we had a culture that we could be more confident in, rather than on the replication plate. We miniprepped the things that we glycerol stocked. We also PCR screened the J23101 + B0034 + Reductase + B0034 + Dehydrogenase construct that was sequenced for the aerobic mutation and we added the deletion fixes. We will run the gel of it tomorrow. We also looked at all of our plates from the mutagenesis and marked which had growth, lawns, or no growth. Mutant sequences showed no mutations. Performed another transformation on fourth SDM PCRs using varying concentrations, spins some down, using various competent cells and using different resistance plates.  

More sequences were received of the multi-set SDM reactions for J23101 + B0034 + Reductase + B0034 + Dehydrogenase, K206000 + B0034 + Reductase + B0034 + Dehydrogenase , and B0034 + Reductase + B0015. These yielded only one set of mutation changes and therefore another SDM was set up for these samples to introduce one additional change. Our goal changed to mainly fix the reductase deletion and the dehydrogenase mutation. The additional change for the aerobic reductase mutation was set as a secondary priority after building the construct. The  SDM reactions were then digested and transformed, and will be prepared for further sequencing tomorrow. Another genomic library of the Strain E-15 EG3 was prepared in hopes to send off for genomic sequencing. The process was halted after the purification step and will be continued with the gel purification tomorrow. Growth seen on some plates. Screen run on successful transformation results. Tranformations on T96A and Y127A done.  

We miniprepped the J23101 + B0034 + Reductase + B0034 + Dehydrogenase with the aerobic mutations that we did a SDM reaction to fix the deletion. We also analyzed the sequences that we just received this morning. We began our replating of the EMS mutations. We scraped the lawns and then resuspended them in 500 uL of the buffer A. Then we took the OD and diluted it down twice to get a solution that would have 150 cells per 200 uL. We also transformed in the J23101 + B0034 + Reductase + B0034 + Dehydrogenase, K206000 + B0034 + Reductase + B0034 + Dehydrogenase, and R0010 + B0034 + Dehydrogenase + B0015. We will use these for testing in the Tecan later on. We sent more things off for sequencing. One of our members, Christine Olson, was so dedicated and stayed until 11 PM to do over one hundred PCR screens. What a dedicated iGEMer. From our genomic sequencing, we ran the qPCR to quantify our concentration. Successful screened mutants at this point were T96A, D98T, S101A, V212A and F125Y. All sent in for sequencing.  

We analyzed the qPCR results of the Strain E-15 EG3 library preparation for trial #3, and the genomic library yielded 220pM of DNA. This yield was not high enough to continue with genomic sequencing, so another library preparation needs to be set up.  A gel was run on the PCR screens done yesterday, but this did not give any bands. So, another PCR screen was set up directly from the previous rep plate. The EMS dilutions made yesterday were accidentally plated on LB carb plates, and not on ethylene glycol, so more dilutions were prepared today. A digestion was set up to ligate J23101 and K206000 together with one enzyme, dehydrogenase. However, it was digested at the wrong cut sites, and so the parts were redigested. The parts were gel purified afterwards to continue on with ligation on Monday. Gel screens run on T96A and Y127A successful transformed colonies. Succesful screens sent off for sequencing.

We digested B0034 + Dehydrogenase + B0015 to ligate it together with the J23101 and K206000. We want to do this so that we can see if just the expression of dehydrogenase will increase the success rate of the MG1655 and Strain E-15 EG3 living. We had to re-digest the pieces because the nanodrop of the gel purification was erratic. However, when we went to nanodrop the second round of digestions, the previous gel purifications were at a higher concentration than that of the second digestion. We ligated the pieces overnight at 16C. The concentration of the library for the genomic sequencing was too low, so we had to redo the preparation. We ran the qPCR overnight to see how much there is in the new prep. We also took the liquid cultures from yesterday and put them into glycerol stocks and minipreps. Successful sequence results were seen for T96A, D98T, S101A, F125Y and Y127A. pNPB assay performed on control (cells with no cutinase), J23101 and inducible construct all at varying cell concentrations.  

Today, we analyzed more sequences that we sent out yesterday. We also had to redo our digestions and ligations that we did a few days ago because it turns out that the purification was not done as well as it could have. Therefore we redid it in hopes of a better result. Lastly, we began a Tecan experiment where we test the optimal amount of arabinose for the induction of the K206000 + B0034 + Reductase + B0034 + Dehydrogenase in MG1655 and Strain E-15 EG3. We did a round of SDM on B0034 + Reductase + B0015 to get rid of the deletion in reductase. pNPB assay results looked promising. Rerun taking OD, using antibiotic LB, comparing induced/uninduced cells, comparing mutants and taking supernatant.  

<br><br>We finished the DpnI digest of the SDM from yesterday, and also digested fixed parts from previous rounds of SDM. We digested K206000 + B0034 + Reductase + B0034 + Dehydrogenase in both an aerobically-adapted and anaerobically-adapted form, J23101 + B0034 + Reductase + B0034 + Dehydrogenase in an anaerobic form, pelB + Cutinase + His, and K206000 + B0034 + pelB + Cutinase + His at Eco and Pst. We did this specific digestion so that we could put these parts (which are currently in a pSB1A3 backbone into the pSB1C3 backbone which is the requirement for BioBrick parts for this year. We ligated these parts together with the new backbone, and transformed them into DH5α. We transformed K206000 + B0034 + Dehydrogenase + B0015 and J23101 + B0034 + Dehydrogenase + B0015 into DH5α, MG1655, and Strain E-15 EG3. We continued the Tecan experiment from yesterday. We went through all of the EMS replates to see the amounts of colonies and documented how many there were. We also transformed the J23101/K206000 + B0034 + Dehydrogenase + B0015 constructs into MG1655, DH5α, and Strain E-15 EG3. Transformations of mutants into MG6155 done.

<br><br>The transformations were successful from yesterday and they had a good amount of colonies. We PCR screened the transformations. We began the liquid culture for the EMS tecan runs. We are beginning with the Strain E-15 EG3 with 0 minutes with EMS. We also transformed the some ligations that we did with the simple constructs and cutinase (separately) into pSB1C3. Mutant transformations screened and liquid cultures taken of successful screens. Another pNPB assay run using only 5 and 10ul of cells between control, induced, uninduced, J23101 and a second control.

Once again, there is too much background the take good data from our western blots. pNPB assay run using only blanks (no cells) and J23101 only in order to check for reasons of the strangely high degradation seen in blanks from last assay results. This assay showed normal blanks suggesting a one time screw up in the previous assay. Thus, another assay performed using 5 successful mutants, wild type control,J23101 and J23101 supernantant.

We took the OD of the Strain E-15 EG3 that we liquid cultured on the 21st. There was a spread, but none had reached 0.2 OD, so we let them grow another night. Digestion, Ligation and Transformation done on mutants to change backbone. Another pNPB assay run using cells and supernatant from controls and J23101.  

We took the OD of each of the Strain E-15 EG3 cultures again and they ranged from 0.08 to 1.5. We washed them in mineral solution and then took the OD again. Then we set up the Tecan to measure which of them have the best growth. We want to be able to see the variation in the Strain E-15 EG3 without any EMS and how the EMS changes the variation in the population. This plate serves as a control for the comparisons. We are also sequencing the B0034 + Reductase + B0015 to see if the SDM worked for the deletion fix. We also began a crude experiment to see if the K206000 + B0034 + Reductase + B0034 + Dehydrogenase versus the K206000 + B0034 + Dehydrogenase + B0015 versus the strain by itself. We want to be able to see if there is growth with one piece or only if there is both. We also did it with J23101 instead of the K206000. These tests were done in the MG1655, Strain E-15 EG3, and DH5α. We looked at the Tecan results that had just finished its run. We concluded that it was not done for a long enough time because of its growth curves. It had not made significant changes. We put it back in to grow longer in the Tecan.

<br><br>We made liquid cultures for multiple parts including PSB1C3, PelB+Cutinase+PSB1A3, Cutinase+Histag+PSB1A3, Cutinase +PSB1A3.  

<br><br><b>Thursday September 27th</b><br>

<br><br><b>Friday September 28th</b><br>

<b>Monday October 1st</b><br>

<br><br><b>Tuesday October 2nd</b><br>

<br><br><b>Wednesday October 3rd</b><br>

<ul style="font-size:10px;list-style-image:none;list-style-type:none;float:left;display:inline;color:#000000;"

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<li style="float:left;margin:0 10px;"><a

href="https://2012.igem.org/Team:UC_Davis"><p>Home</p><ul

style="text-indent:-15px;

list-style-image:none;list-style-type:none;color:#000000;"><li><a

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list-style-image:none;list-style-type:none;color:#000000;"><li><a

href="https://2012.igem.org/Team:UC_Davis/Team">Who we are</a>

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<li style="float:left ;margin:0 10px;"><a

href="https://2012.igem.org/Team:UC_Davis/Project "><p>Project</p></a>

<ul style="text-indent:-15px;list-style-image:none;list-style-type:none;color:#000000"><li><a

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href="https://2012.igem.org/Team:UC_Davis/Project">Project Overview</a>

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href="https://2012.igem.org/Team:UC_Davis/Project/Catalyst ">Module

Engineering</a></li><li><a style="color:#000000 "

href="https://2012.igem.org/Team:UC_Davis/Project/Protein_Engineering

">Protein Engineering</a></li>

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href="https://2012.igem.org/Team:UC_Davis/Project/Strain ">Chassis

Engineering</a></li>

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href="https://2012.igem.org/Team:UC_Davis/Project/Directed_Evolution ">

- Directed Evolution</a></li>

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href="https://2012.igem.org/Team:UC_Davis/Project/Our_Strain "> -

Rational Engineering</a></li>

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href="https://2012.igem.org/Team:UC_Davis/Criteria">Critera</a>

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href="https://2012.igem.org/Team:UC_Davis/Safety "> <p>Safety</p></a>

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<p>Notebook</p></a> <ul

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href="https://2012.igem.org/Team:UC_Davis/Notebook">Notebook</a>

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title="https://2012.igem.org/Team:UC_Davis/Data "> <p>Data </p></a> <ul

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"><li><a style="color:#000000 "

href="https://2012.igem.org/Team:UC_Davis/Data/Cutinase_Activity ">

Cutinase Activity</a> </li><li><a style="color:#000000 "

href="https://2012.igem.org/Team:UC_Davis/Data/Ethylene_Glycol ">

Ethylene Glycol</a> </li><li><a style="color:#000000 "

href="https://2012.igem.org/Team:UC_Davis/Data/Modeling ">

Modeling</a> </li><li><a style="color:#000000 "

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">Main iGEM</a> </li><li><a style="color:#000000 "

Practices</a></li> </ul>