Notebook

July Notes

Week of 07/01/2024

• Wet Lab:
  • Made all of the ingredients for BG-11 media
• Dry Lab:
  • Started research into transformation of L. Fusiformis
  • Discovered transformation protocol from Lumen Biosciences
• Human Practice:
  • Reached out and interviewed Olivia Mayer and Lydia Mapstone for community outreach
  • Reached out and interviewed Mckenna Hink for advice for working with Limnospira fusiformis
  • Reached out and interviewed Naama Aviram to further understand Cas10 mechanisms

Week of 07/08/2024

• Dry Lab:
  • Researched native promoters and terminators to be used in L. Fusiformis
  • Started development of BLACKBIRD to create optimized inserts
  • Discovered the Cyanogate system, which contained a variety of parts for use in cyanobacteria
• Wet Lab:
  • Made BG-11 media
  • Made LB+Amp media/plates for plasmids (pDV002, pDV044, pDV052)
  • Streaked plasmids on plates to grow up in incubator
  • Picked colonies to grow up in liquid culture, checked densities

Week of 07/15/2024

• Dry Lab:
  • Discovered the MoClo toolkit through our research of cyanogate
  • Learned how the Type IIS restrictions enzymes are used in Golden Gate reactions, which sets the foundation for learning how the MoClo toolkit works
  • Split our workload between the Lumen Biosciences protocol and the MoClo toolkit
  • Decided to work in parallel with other cyanobacteria with shorter doubling times, notably UTEX 2973
• Wet Lab:
  • Continued growing plasmids to check densities
  • Received A.platensis and figured out how to store
  • Started growing A.platensis in BG-11 in incubator
  • Made glycerol stocks and snap froze plasmids
  • Spun down/froze plasmids for miniprep

Week of 07/22/2024

• Dry Lab:
  • Started in silico modeling of Cyanogate system
  • Created basic constructs, including level 0 parts with existing inserts and optimized inserts
• Wet Lab:
  • Miniprepped plasmids, got strange results
  • Made LB+Cam media
  • Inoculated old plasmids (pDV002, pDV044, pDV052) & new plasmids (pRK24, pRL528)
  • Checked densities of plasmids
  • Plasmids spun down into glycerol stocks, others spun down for minipreps
  • Miniprepped twice – still not getting good results
  • Streaked more plates for pRK24 & pRL528
  • Made more LB+Cam plates
  • Miniprepped pDV002, pDV044, pDV052, still bad results
  • Inoculated pRK24, pLR528, checked OD’s
  • Made three more plates of L. fusiformis
• Education:
  • Girls in Engineering Presentation
  • Interviewed Ravendran Vasudevan for MoClo related questions

Week of 07/29/2024

• Dry Lab:
  • Split into groups: Single Plasmid, Double Plasmid, Argonaute, Limnospira
• Wet Lab:
  • Made more LB+Amp plates/liquid media
  • Streaked pDV052, pDV044, pDV002 plates to grow overnight
  • Miniprepped pDV002, got first good results
  • Inoculated pDV052 & pDV044 overnight
  • Made new BG-11 media
  • Miniprepped pDV044 & pDV052, still bad results
  • Plated parts of MoClo kit
  • Made spectinomycin media for plates/liquid culture
  • Took single colonies from MoClo plates and inoculated them, passaged them at the end of the day
  • Increased A.platensis growing temperature from 23℃ to 37℃
  • Diluted primers & made working stocks of rest for wet lab
  • Plated multiple CyanoGate parts
  • Passaged MoClo parts at the end of the day for minipreps
• Human Practice:
  • Interviewed Shan Huang for Argonaute related questions
  • Interviewed Alistair McCormick for MoClo related questions

September Notes

Week of 09/02/2024

• Wet Lab:
  • For single-plasmid team, troubleshooted the PCR protocol using OneTaq 2X Master Mix
  • Ran an experiment using un-optimized PET28 GFP plasmid using both flanking and GFP-specific primers, with a subsequent gel electrophoresis
  • Took an inventory of the mini-prepped parts that we had, and grew out any parts that we did not already have saved in its eluted form
  • Received and rehydrated our optimized inserts for both Argonaute and Single Plasmid team
  • Did a Golden Gate Assembly reaction using the optimized inserts
  • For Argonaute team, inoculated CbAgo, Down homology arm (HA), Chi site, and C products
  • Performed a chemi-competent transformation
  • Ran a touchdown colony PCR using OneTaq with optimized inserts, with a subsequent gel electrophoresis and PCR clean up (for Argonaute PCR products)
  • Discovered that the recipe for X-Gal plates was wrong, found another recipe that used a much higher concentration of X-Gal that would hopefully increase the efficiency of the blue-white screening process
• Other:
  • Promotional project video filmed and edited

Week of 09/09/2024

• Wet Lab:
  • Using the colony PCR results from last week, ran a gel electrophoresis
  • Was getting subpar results on our gels, with little to no visualization for the fragments
  • Troubleshooted our gel electrophoresis, ran many diagnostic gels testing the following variables:
    • Viability of the loading dyes
    • Amount of sample to be loaded
    • Type of MasterMix used (OneTaq or Q5)
  • Eventually was able to reach a point of visualization for our PCR products on our gels, but was unable to get the right sizes
  • Troubleshooted the rest of our pipeline: colony PCR and Golden Gate Assembly
  • For Golden Gate Assembly, tested if the molar ratio of the insert made a difference (tested 3:1 and 4:1 as opposed to 2:1 for the Argonaute inserts)
  • From doing a Golden Gate Assembly with the backbone and no insert, discovered that the backbone was religating back to itself and some of the colonies did not contain the LacZ fragment at all
  • Started running inverse PCR on double plasmid backbone pC1.509

Week of 09/16/2024

• Wet Lab:
  • Ran a Q5 PCR for a Cpf1 mini-prepped sample, then ran a gel
  • Came to the discovery that Top10 cells were resistant to the antibiotic Streptomycin, which was our antibiotic resistance marker
  • Started to use DH5-𝝰 cells instead for transformation
  • Ran a Q5 PCR on several samples of the pICH41308 backbone in order to see which samples contained the LacZ fragment
  • Plated double plasmid parts and checked that all the other ones were already mini prepped
  • Inoculated double plasmid parts to be miniprepped the next day
  • Miniprepped double plasmid parts – got good graphs & values
  • Reamplified PCR of inverse backbone pC1.509 for double plasmid system from last week
  • Planned to send out an order for sequencing to Berkeley of our Cpf1 and empty backbone PCR products
  • Started a Level 1 Golden Gate Assembly reaction for the antibiotic-resistance level 0 part

Week of 09/23/2024

• Wet Lab:
  • Ran THE experiment to test if our restriction cutter and ligase worked
  • Experiment was golden gating 3 separate samples:
    • 1 - T4 buffer, backbone, insert, water
    • 2 - T4 buffer, backbone, insert, restriction cutter, water
    • 3 - T4 buffer, backbone, insert, restriction cutter, ligase, water
  • Saw growth on 2 which was not expected, so we decided that our cells could be bad, the plates could be bad, our restriction enzyme could be bad (prob not tho cus it’s new), or our strep could be bad
  • Found another box of DH5a comp cells
  • Plated DH5a comp. Cells onto LB + x-gal + spec, both boxes to test if either of them grew on spec
  • Both ended up growing, so we determined our plates were prob bad
  • Need to make new plates and ordered new spec as well
  • Came in too late for chemicomp protocol, OD600 needed to be around 0.55 and was 1.0 when checked
  • Optimal growth period is 8 hrs in starting culture, then 10 hrs for inoculations
  • Started to grow up DH5a cells from original bottle to make our own comp cells
  • For double plasmid team, ran our first golden gate reaction
  • Prepared for transformation after golden gate
  • Miniprepped more double plasmid system parts
  • Ran a gel on all of our inverse PCR results and picked the best one for gel extraction DNA clean up protocol
  • Performed another inverse PCR on the double plasmid backbone to get rid of the extra bands we’ve been seeing on the gels (increased binding temperature of the primers by a couple degrees)
  • More argonaute primer, optimized CPF1, and optimized GFP dilutions done