Team:UMaryland/Notebook
Notebook
2 June 2021
Today, we embarked on our iGEM research journey with our first day of our cloning boot camp at the University of Maryland’s Institute of Bioscience and Biotechnology Research (IBBR). With Anjali, Brittanni, and Priya new to synthetic biology, our advisor, Dr. Ed Eisenstein, is helping get up to speed with the basics of molecular biology needed for iGEM. Our goal over the next few weeks is to better understand the cloning process by cloning a gene for the super folder variant of GFP using Restriction Enzyme Ligation (REL), Gibson Assembly (GA), and Golden Gate Assembly (GG). For our first day, we made up luria broth media, made antibiotic solutions (ampicillin, kanamycin, chloramphenicol), and practiced streaking plates.
3 June 2021
In order to learn more about the restriction enzyme ligation cloning strategy, today Anjali, Priya, and Brittanni digested our pET-28 vector with the restriction enzymes NcoI and XhoI. We also went over the steps of the ProMega Purification protocol.
4 June 2021
Today, Anjali, Brittanni, and Priya learned how to prepare glycerol for future glycerol stocks. We also reviewed our boot camp cloning game plan. For the REL approach, we digested the pET-28+S vector with NcoI and XhoI and amplified the sfGFP with primers sfGFPmod-NcoI-F and sfGFPmod-XhoI-R for later ligation. For the GA approach, we digested the pET-28+S vector with NcoI and SacI and amplified the sfGFP in two steps. For the GG approach, we used the pBuild vector and utilized sfGFP amplified in two steps.
7 June 2021
Today, Anjali, Priya, and Brittanni learned how to do PCR amplification. We did a PCR amplification of sfGFP with the primers sfGFPmod-NcoI-F and sfGFPmox-XhoI-R. We also learned how to make agarose gel and run it using gel electrophoresis. After running the gel, we learned how to analyze expected banding patterns, cutting. We also learned how to use the NanoDrop Spectrophotometer, which is used to measure the concentration of nucleic acids in a sample.
8 June 2021
Anjali, Brittanni, and Priya conducted the second round of PCR amplification required for the Gibson Assembly and Golden Gate cloning approaches. We then did a restriction digest from the REL approach and viewed the results (REL, GG round #2, GA round #2) on gel electrophoresis. We then transformed products from each approach into NEB5-alpha cells and let them incubate for ~16 hours.
9 June 2021
When analyzing the results with Dr. E, Anjali, Britanni, and Priya found that while REL and GA approaches were successful, the GG approach was unsuccessful as this was the only approach without clones. For the plates where we did have clones, we picked colonies and inoculation liquid cultures.
10 June 2021
After taking out the overnight liquid cultures from the incubating shaking table, Anjali, Brittanni, and Priya conducted a restriction digest screen of the liquid cultures from the day before. To do this, we purified the DNA from the cells with the Wizard Prep protocol, conducted a gel electrophoresis (cut out bands of interest), and conducted a Miniprep DNA purification. The gel allowed us to validate that the inserts matched our expectations. After purifying the DNA and measuring its concentration, we found that its concentration was not high enough to do sequencing. As a result, we concentrated the DNA with a speed vacuum and then sent our DNA for sequencing with Macrogen.
11 June 2021 - 30 June 2021
After successfully completing our SynBio Boot Camp, Anjali, Priya, and Britanni spent their time outside of the wet lab, focusing on developing their project design, genetic constructs, planning outreach, and raising funds. On 21 June 2021, Anjali, Brittanni, and Priya also presented a lab meeting about the Boot Camp results to the advisors, Dr, Eisenstein and Dr. Kahn, as well as the other members of our team. We conducted a more thorough literature review to determine which of the genes from M. phosphovorus we wanted to test. During this time, Anjali, Priya, and Brittanni (with the editing help from Cole Dwiggins and Matthew McHugh) also wrote and submitted the iGEM Safety & Security Grant and Impact Grant.
2 July 2021 - 6 July 2021
Although most of our molecular biology work will be done at IBBR, Priya and Brittanni went down to the College Park iGEM lab on 2 July 2021 to get materials and the space organized from later biochemical experiments. Anjali continued working on designing the genetic constructs for our project. Priya and Britannni worked without iGEM mentor, Yan, to develop a schematic for a phosphorus-detecting biosensor.
8 July 2021
In addition to continuing to plan our genetic constructs and cloning schedule, Anjali, Priya, and Brittanni also met with Dwight Dotterer, the Nutrient Management Program administrator at the Maryland Department of Agriculture. At this meeting we learned a lot about the scope of the problem and good targets for our design!
9 July 2021
Today, Anjali, Priya, and Brittanni met with Dr. Jenna Rickus, one of the advisors from the 2016 Purdue iGEM Team. This was a great way to interface with the past iGEM team whose work inspired us!
10 July 2021 - 21 July 2021
Anjali and Priya continued to work on the genetic constructs. We finalized a plan for the gene combinations we wanted to test. For the phosphate transport genes, we want to test Pit A alone, Pit B and C, and then Pit A, B, and C. For the polyphosphate kinases, we want to test PPK1, PPK2, and PPK1 and PPK2. For the phosphate release, we want to test PPX (exopolyphosphatase). Since our ideal end goal is to take the best gene combinations out of the test batch and implement them into one E. coli (a three plasmid transformation), we decided to make each construct a different antibiotic resistance (chloramphenicol, ampicillin, or kanamycin). Since we want to make an efficient phosphorus sequestration unit, we also decided to implement different gene regulations on each gene group. The Pit genes will be regulated by the inducible pLac promoter. The PPK genes will be regulated by the constitutive weak Anderson promoter. The PPX gene will be regulated by the inducible pBAD promoter. We finalized our gBlock designs for the Pit Genes and PPK genes, where we designed modular gBlock with the promoter and terminator gBlock being the same for each gene group and a separate gBlock for each optimized (to E. coli) gene sequence.
26 July 2021 - 29 July 2021
While we waited for our gBlocks to arrive to begin the cloning for our project, Anjali, Priya, and Brittanni helped Dr. Eisenstein with an iGEM InterLab Study for the Engineering Committee. During this week, we learned how to make chemically competent cells, transformed the test plasmids into the cells, plated them, and grew overnight liquid cultures for Restriction DIgest screening and analysis.
2 August 2021
With eight of our gBlocks arriving at IBBR, Anjali, Brittanni, and Priya resuspended them to a final concentration 25 ng/uL. Since we want to clone each gene group into a different antibiotic resistance, we needed to obtain/make chloramphenicol, ampicillin, and kanamycin plasmid backbones. Due to issues with our iGEM distribution kit, we decided to use pSB1C3-mRFP1 chloramphenicol-resistance plasmid that Dr. Eisenstein had in his lab to make the PPK gene constructs. We did a restriction digest of pSB1C3-mRFP1 with the BioBrick prefix/suffix restriction sites, EcoRI and PstI.
3 August 2021
Since the plasmid yield after restriction digest was low, we did another restriction digest of the pSB1C3-mRFP with EcoRI and PstI, this time allowing incubation rounds to be 3 hours each instead of 1.5 hours.
4 August 2021
Anjali, Priya, and Britanni ran a gel electrophoresis of the digested pSB1C3-mRFP1. The banding on the gel matched our expected banding patterns. We also performed a Gibson Assembly reaction to combine the promoter, PPK1, and terminator gBlocks. In this GA reaction, we used 80 ng of backbone and did a 3-fold excess of the pmol/rxn. We transformed the PPK1-pSB1C3 plasmid into NEB5-alpha cells and incubated them on CmR-resistant plates.
5 August 2021
Today, Anjali, Priya, and Britanni checked the plates for colonies and found colonies on both plates! We then inoculated LB-CmR liquid cultures with picked colonies for overnight incubation and stored the plates in the cold room. Since we want the Pit gene group and the PPX gene group to be in ampicillin-resistant and kanamycin-resistant backbones, respectively (and we did not have pSB1A3 or pSB1K3 plasmids available), we decided to make these backbones from the pSB1C3-mRFP1 backbone. We did a restriction digest pSB1C3-mRFP1 for KAN or AMP gene insert amplification by cutting the CmR gene out with EcoRV and AatII. We then ran a gel electrophoresis, and cut/purified the backbone fragment desired. Today we also ordered VR and VF2 iGEM sequencing primers and another set of primers to amplify KAN/AMP genes out of a vector with those resistances into our digested pSB1C3-mRFP1. Today, UMaryland iGEM also attended the 2021 Mid-Atlantic Meetup hosted by William & Mary. We got to present our project and learn more about others’ cool projects!
6 August 2021
Anjali, Priya, and Brittanni did the wizard DNA prep for the 8 overnight PPK1 liquid cultures and subsequently did a restriction digest screening with NotI (we used this restriction enzyme because it was a unique, dual cutter). The gel banding matched our expectations, giving us the confidence to send out our samples for sequencing.
9 August 2021 - 10 August 2021
Because our necessary primer orders did not come in yet, we did more project planning and outreach planning.
11 August 2021
With our primers coming, Anjali, Priya, and Brittanni set up a pre-mix reaction for pSB1C3-PPK1 sequencing. We also amplified the KAN and AMP genes out of the pET-28 and pET-18 vectors, respectively. We then conducted a gel electrophoresis of the PCR round #1 products to determine whether the actual gene matched the expected gene sizes, which they did. With it, we did a Gibson Assembly of the KAN or AMP genes with pSB1C3-mRFP1-digested-EcoRV/AatII. Since we ran out of time to do a transformation, we stored the GA product in the cold room.
12 August 2021
Today, Anjali, Priya, and Brittanni transformed the KAN-pSB1C3 and AMP-pSB1C3 into NEB5-alpha cells and plated them onto plates with the correct antibiotic resistance. We also did a Gibson Assembly of the promoter, terminator, and PPK2 genes with the pSB1C3 backbone, and stored the GA product in the cold room.
13 August 2021
Today, the trio checked the plates from the day before, where we saw many colonies; however, very few were red. Our positive clones should have been red since they were ligated into a digested pSB1C3-mRFP1 backbone. However, at this time we did not realize this and did an overnight liquid inoculation of non-red colones (which were likely the original pET-28 or pET-18 vectors). We also did a PCR amplification to form two ends of pSB1C3, one with the CmR gene and the other with the ori region. Afterward, we ran a gel electrophoresis of the PCR products, cut/purified them, and measured their concentration. We also transformed the GA PPK2 product into NEB5-alpha cells on CmR plates.
14 August 2021
Today, the trio took out the PPK2 plates, where we found colonies on both of them. We parafilmed them and stored them in the cold room. We also did the Wizard DNA purification of the KAN-pSB1C3 and AMP-pSB1C3 liquid cultures.
16 August 2021
The trio ran a restriction enzyme map with pSB1C3-mRFP1 (undigested), KAN-pSB1C3, and AMP-pSB1C3. We also did an overnight liquid culture inoculation of PPK2. We also re-inoculated the liquid cultures for the KAN-pSB1C3 and AMP-pSB1C3 samples because we did not pick the red colones expressing RFP before.
17 August 2021
Today, the trio did wizard prep of the PPK2 liquid cultures. We measured the pSB1C3-PPK2 concentrations and did a restriction digest screening of PPK2. We also conducted a restriction digest screening of AMP-pSB1C3 and KAN-pSB1C3. After testing a couple of the liquid cultures from these samples, the yield was also low. Not much DNA was recovered from the 2.5mL samples. Instead, we plan to prepare DNA to cut out. To do this, we will grow the colonies in 10 mL of CmR-LB in a Falcon tube. We had Dr. E help us streak out the LB-KAN plates.
22 August 2021-25 August 2021
Priya and Anjali did PCR amplification for the 2 ends (antibiotic and origin regions) of AMP-pSB1C3 and KAN-pSB1C3. We ran a gel electrophoresis and cut/purified the samples, where we found the concentrations were too low. There was something wrong with the KAN-pSB1C3 plasmid, causing us to have to troubleshoot. The AMP-pSB1C3 had okay concentrations, allowing us to use it for Pit Gene transformation.
26 August 2021
Anjali and Brittanni used the AMP-pSB1C3 backbone to do Gibson Assembly with the Pit A, Pit B, or Pit C genes. We transformed these cells into XL1-Blue Cells, since this strain over-expresses the lac repressor and this gene group is under regulatory control of the pLac promoter. In order to troubleshoot the KAN-pSB1C3 genes, we ran a gel and analyzed the results.
27 August 2021
Anjali took the Pit gene plates out of the incubator and found that there were colonies on all plates. She then did an inoculation of the liquid cultures.
28 August 2021
Priya and Brittanni came into lab to do the Wizard DNA prep of the Pit liquid cultures.
31 August 2021
On this day, Matt, Pavan, and Yasi began by outlining our desired accomplishments with the bioreactor and determined how to achieve those set goals through the engineering design process monitored through a gantt chart. The team began the engineering cycle by individually researching mechanisms that a bioreactor is composed of and by reviewing bioreactor work done by previous year iGem teams. The team began brainstorming how different parts of the reactor could be developed and reached the agreement that the basis of the final bioreactor will consist of some form of agitator and a mesh component to limit the E coli infused silica beads..
2 September 2021
Mattew, Yasaman, and Pavan each proposed comprehensive designs for the bioreactor that varied in how the silica beads would be housed and could be transported to extract the captured phosphorus. Three different methods of agitation were also suggested: DC motor, shaker table, and magnetic stir bar.
3 September 2021
The three bioreactor designs that were individually brainstormed were presented to Dr. Khan and Dr. E for critiquing. Dr. Khan emphasized the importance of combining as many of the procedures required for the cleaning of the water and gathering of the phosphorus into one vessel to improve the ease of use of the machinery.
7 September 2021
Similarities of bioreactors and fermenters were studied to evaluate how we can incorporate similar structures in our solution for the bioreactor. Pavan, Yasaman, and Matthew evaluated the suggestions Dr. E and Dr. Khan had the individual designs and had a group brainstorm session to combine the most promising ideas for the bioreactor.
10 September 2021
Yasaman, Matthew, and Pavan began imagining different casings that can be constructed to house the silicon beads in the bioreactor. From sheet designs to multiple cylindrical columns were considered. The exposure of the polluted water to the surface area of the silicon beads as well as how the mesh casing would impact water flow. Then the group thought of how each of these factors could be modeled with MATLAB.
11 September 2021
Since we did not have a ready-to-use KAN-pSB1C3 plasmid, we tried redoing the Gibson Assembly with the KAN gene and the pSB1C3-mRFP-digested-EcoRV/AatII backbone. We then transformed the GA product into NEB5-alpha cells.
12 September 2021
We measured the concentration of the Pit genes from the plasmid DNA recovered by Priya and Brittanni. We also set up pre-mix reactions to send the Pit genes for sequencing.
14 September 2021
Yasaman wrote down some preliminary equations that could be used to evaluate the efficiency of cleaning polluted water with silicon beads in different mesh casings and bead density per square inch of surface area. The group’s modeling plans were presented to Dr. Khan to evaluate the legitimacy of the calculations. Dr. Khan stated that we should incorporate a limiting factor into our modeling of the bioreactor, such as water flow and dynamics.
18 September 2021
Our sequencing results indicated that the Pit genes did not uptake into the plasmid, but rather re-circularized the NotI sites. We tried redoing the Pit A Gibson Assembly and transformation into XL-1 Blue cells onto AMP plates. We then conducted a restriction digest of AMP-pSB1C3 with EcoRV and AatII to redo the KAN-pSB1C3 plasmid. We then ran a gel electrophoresis, where we found the gel matched the expected banding patterns.
21 September 2021
Pavan, Yasaman, and Matthew found resources on the dynamic flow of fluids in relation to a propellers design ie. plane propeller or boat propeller. They also looked into how previous research papers conducted fluid dynamic analysis.
25 September 2021
In order to do a biochemical analysis of the PPK genes, we re-did the PPK1 and PPk2 transformation into NEB5-alpha cells on CmR plates to take to the College Park Lab, where Dr. Kahn would help us with the polyphosphate quantification experiments.
26 September 2021
A bill of materials was created to order the parts required to complete the necessary prototyping of the bioreactor’s agitator as either a shaker table or DC motor (demonstrated by a continuous servo motor).
28 September 2021
Pavan, Matthew, and Yasaman created a list of questions they had about the project and the necessary components needed to model the dynamics of a bioreactor.
29 September 2021
Pavan met up with Dr. Kenneth Kiger, a professor in the mechanical engineering department at the University of Maryland, to ask for guidance on the modeling of the project's fluid dynamics. He stated that we need to evaluate the effective density of the bead, the stirring action required, and the transport/diffusion of the phosphorus. He told the group to look into “well-stirred reactors”.
6 October 2021
Today, Warren, Anjali, and Priya met with Dr. Toor, who is a UMD professor and extension specialist of Nutrient Management, Soil Quality, and Water Quality.
7 October 2021
Matthew and Pavan began modeling the bioreactor by creating multiple MATLAB simulations that modeled the relationship between the volume of polluted water, the total surface area of the silica beads, and the period of time the solution is agitated.
8 October 2021
The trio grew more PPX-KAN for later analysis. We also inoculated 10 clones from Pit A, Pit B, and Pit C in order to run more restriction digest screens to determine where the issue in our transformation was.
9 October 2021
The trio took out Pit A, Pit C overnight liquid cultures and did the STET protocol to prepare for restriction digestion. After running the screening, we found that the banding suggested that the genes did not uptake into the plasmid. In order to try to troubleshoot this, we ordered more primers to try and amplify the existing gBlocks.
17 October 2021
Pavan and Yasaman programmed the continuous servo motor to rotate as needed to simulate a DC motor for their experiments. Yasaman began on the CAD modeling of the bioreactor.
18 October 2021
Pavan tested whether a higher concentration of tea was collected in a water bottle when the tea bags were agitated using a shaker table v.s. the servo motor.
19 October 2021
Matthew and Yasaman finalized the CAD model of the bioreactor from the data that was collected from their experiments and research.