25th March- First East Coast BioCrew meetup. Introduced members and researched previous iGEM projects to learn more about the competition.

1st April- Split into subgroups to brainstorm ideas for our project

15th April- Narrowed down idea: combat phosphorus pollution and air pollution-- amplifying or modifying plant organism to absorb air pollution and phosphorus

22nd April- Attended "Future of syntehtic Biology Conference"

20th May- Researched literature regarding rhizobium. Found glucose dehydrogenase (GDH) with cofactor Pyrroloquinline quinone (Pqq) had the ability to solubilize mineral phosphate.

3rd June- Started compiling information about Rhizobium tropici CIAT 899. Researched various medias R. tropici could grow in.

10th June- Planned for a two part system: one making the phosphate soluble and the other stimulating phosphate uptake. Researched systems for increasing phosphate uptake and found pstSCAB operon, which was used by previous iGEM teams.

17th June- Engineered gene constructs on Benchling and ordered them.

24th June- Spoke with Dr. Reid through zoom. Decided to make the project for private use after going through pros and cons. Began drafting emails to stakeholder groups.

1st July- Overview of media: ATCC 111 (soil), yeast mannitol (high Ca), yeast mannitol (low Ca). Began blue white screening with E. coli and empty plasmid.

8th July- Measured optical densities of R. tropici growing in three medias and found no significat growth rate differences. Blue white screening did not work as planned. Discussed future plans and modification to protocol.

15th July- Had succesful blue white screening. Made plans for plant experiments with root nodulation.

22nd July- GenSpace received R. tropici and empty plasmids.

29th July- Soybean plants grew, and we began the innoculation process. GenSpace performed PCR screening and discovered the genes were not inserted properly due to primer issues.

5th August- Set up Q5 PCR doubling this protocol (PCR using Q5) with tubes 1 and 2 as pBBR1 with PpqC primers and tubes 3 and 4 as pBBR1 with pstSCAB primers. We repeated the PCR and transformation.

11th August- Two team members attended the iGEM Mid-Atlantic Regional meetup and presented project, receiving feedback from other iGEMers.

12th August- Set up Q5 colony PCR. Sets 1 and 2 are pBBR2 that contain PqqC and gcd. Ran 20 clones from set 1 (1-1 through 1-20). Ran 8 clones from set 2 (2-1 through 2-8). Sets 3 and 4 are pBBR2 that contain pstSCAB. Ran 13 clones from set 3 (3-1 through 3-13). Ran 10 clones from set 4 (4-1 through 4-10). Positive clones appear to be 1-1, 1-2, 2-1, 3-1(?), 3-3 (looks small), 3-4 (looks small), 3-13, 4-1, and 4-7. Grew cultures overnight in LB+kanamycin (except for #3-4).

19th August- Ran PCR and gel electrophoresis with positive clones from last week. Plasmid Concetration; DNA recordings. Measured plasmid concentration with nanodropper. Set up PCR using 1 ul of each DNA as template using Q5 2X Master mix with primers M13F and M13R.

2nd September- Used electroporation to transofmr our R. tropici with Pqqc+gcd and pstSCAB genes.

4th September- GenSpace made 10% glycerol solution for bacterial washing before electroporation and innoculated transformed E. coli from BUGGS on to agar plates.

5th September- GenSpace isolated plasmid DNA from E. coli bacterial cultures with MiniPrep and pelleted R. tropici inEppendorf tubes for easier storage.

9th September- Began working on promotional video and judging form. Began work on parts registry.

10th September- GenSpace prepared for transforming electrocompetent R. tropici by making SOC medium, TSB Yeast medium plates, and preparing R. tropici for electroporation (competent cells stored in -80)

16th September- PCR of R. tropici in plasmid has been started. Plants have been growing quicker, temperature has increased since the plants were put in the incubator, the next measurement may not be ready in time for the wiki freeze. Genspace has made their competent cells, have made their first transformation.

23rd September- Began phosphate uptake plate assays. Met with the Chesapeake Bay Foundation-- will be writing a summary about what we learned: phosphate solubility relies on pH, when testing test in a range of pH conditions.

24th September- GenSpace removed the bacteria after two days of growth at 30 C and placed in minifridge for storage. Results - looking like some kind of contamination on plates 1 and 3. GenSpace Replated remainder of transformed Rhizobium on fresh TSB + Kanamycin plates, grew at 30C for 48 hrs, stored in minifridge.

30th September- Bacterial ODs are being taken again today for the model. Phosphate plate assays were not viable-- retesting with different bacterial dilutions and added step of drying plates under laminar flow hood. Phosphate colorimetric kit has arrived at Genspace.

1st October- GenSpace made 500 mL TSB Broth according to protocol and aliquoted 5 mL into 15 mL snap cap tubes. Inoculated: 3 colonies of PqqC + gcd, 3 empty pBBR2, and 3 colonies of pstSCAB.

4th October- GenSpace received package sent from BUGGS containing calcium phosphate tribasic, R. tropici, mannitol, P. fluorescens. Made 250 mL of tricalcium phosphate broth + kanamycin according to protocol. Measured OD values of GenSpace-grown Rhizobium. Inoculated GenSpace-grown R. tropici into tricalcium phosphate broth and incubated at 30 C. Inoculated BUGGS-grown R. tropici into TSB and incubated at 30 C.

7th October- GenSpace took OD of BUGGS R. tropici. Added amount from tryptic soy broth (TSB) to tricalcium phosphate broth (TCP). Added varying amounts to ensure that equal OD (0.881) was added. Found value by dividing lowest OD (0.881) by each sample's respective OD value. BUGGS ran protein gel on 8 colonies: 5 contained PqqC + gcd genes and 3 contained pstSCAB genes. Protein gel did not provide conclusive results.

10th October- Protein gel was run again, and results were...

11th October- GenSpace ran phosphate colorimetric assays, and results were....