Notebook

Names of each student: Yuqi Song, Xiang Li(Alice), Chuqiang Peng Date: 9.27.23 Protocols Performed: Overnight culture E.coli JM109 in liquid LB medium. Purpose: To revive dormant E. coli and make more E.coli for the next step. Supplies used/Materials: E.coli JM109, liquid LB medium with AMP Results: Conclusions/Next Lab goals: Make solid LB medium and overnight E.coli on it. Extra notes: Names of each student: Yuqi Song, Xiang Li(Alice), Chuqiang Peng Date: 9.28.23 Protocols Performed: Overnight culture E.coli on LB plate Purpose: Prepare for next step Supplies used/Materials: Solid LB plate , E.coli Results: Conclusions/Next Lab goals: Culture E.coli in liquid LB Extra notes: Names of each student: Xiang Li(Alice) Date: 9.29.23 Protocols Performed: Culture E.coli in liquid LB Purpose: prepare for next step Supplies used/Materials: Liquid LB medium, E.coli Results: Conclusions/Next Lab goals: Culture E.coli in liquid LB with ethanol Extra notes: Names of each student: Chuqiang Peng Date: 9.30.23 Protocols Performed: 100μL culture was inoculated into a 10-ml screw-cap tube filled with 9.9 ml LB liquid medium and added 10μL ethanol. Overnight culture. Purpose: Increase the ethanol tolerance of E.coli Supplies used/Materials: 95%ethanol, E.coli, LB Results: Conclusions/Next Lab goals: Dilute the solution Extra notes: Names of each student: Yuqi Song Date: 10.1.23 Protocols Performed: 100μL culture was inoculated into a 10-ml screw-cap tube filled with 9.9 ml LB liquid medium and added 10μL ethanol. Overnight culture. Purpose: Increase the ethanol tolerance of E.coli Supplies used/Materials: 95%ethanol, E.coli, LB Results: Conclusions/Next Lab goals: overnight culture E.coli in LB plate. Extra notes:Names of each student: Kaia and Jasmine Date: 9/25/2023 Protocols Performed: Located the E.coli, JM109. Made LB agar and broth LB Broth: Weigh 5g peptone, 2g yeast, and 5g NaCl into a weigh boat. Pour the powders into a glass bottle containing 500mL of diH2O. Mix the mixture and label it “Knockout | iGEM | 7-12-2023 LB broth.” Agar: Weigh 5g peptone, 2g yeast, 5g NaCl, and 5g agar onto weigh boat. Add the powders into a glass jar containing 500mL of diH2O. Mix the mixture. Do NOT add antibiotic (ampicillin) yet. Label it “ knockout | iGEM | 7-12-2023 Agar without Ampicillin.” Place the two glass bottles into the autoclave for 30 min. Purpose: The LB agar will be used to culture the E.coli and will also be used to plate a few. Supplies used/Materials: Peptone, yeast, NaCl, weigh boat, agar, diH2O, 500mL glass bottle, autoclave. Results: N/A Conclusions/Next Lab goals: Pour the LB agar into the plates. Extra notes: A group of bottles with liquid in them Description automatically generated Names of each student: Tracy Date: 9/26/2023 Protocols Performed: Pouring LB into plate. Completely melt the LB agar on a hot plate. Place the plates upside down before pouring. Keep an open flame and place the plates and agar close to it. Do not open the plates until pouring time. Keeping close to the flame, use one hand to first run the agar bottle through the flame and the other hand to carefully open the culture plate’s lid. Pour until the agar is covering all the areas of the plate with no air bubbles. Repeat step 4 sixteen more times. Stay close and below the flame. Label the stack “iGEM Fall 2023 No Amp. KO 9/26/2023. Store in refrigerator for the next use. Purpose: To prep the culture experiment by pouring the LB agar that was previously made into the culture plates with no ampicillin. Supplies used/Materials: LB Agar, hot plate, open flame, lighter, 17 culture plates. Results: N/A Conclusions/Next Lab goals: Get started with SOC prep. Extra notes: Names of each student: Kaia and Jasmine Date: 9/27/2023 Protocols Performed: SOC media First prepare the solutions： Per liter: To 950 mL of deionized H20, add: Tryptone 20g Yeast extract 5g NaCI 0.5g 250 mM solution of KCI. KCI：1.86g Deionized H2O：100 mL 2 M MgCl2. MgCl2: 19g Deionized H2O：90ML 1 M solution of glucose. Glucose：18 g Deionized H2O：90ML Adjust the PH of all solutions to 7.0 and then sterilize all solutions by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle. Just before use, add 10 ml of a sterile solution of 250 mM KCl, 5 mL of a sterile solution of 2 M MgCl2 and 20 mL of a sterile 1 M solution of glucose. Purpose: SOC will be used run the plasmid and bacteria transformation. Supplies used/Materials: Tryptone, yeast, NaCl, KCl, MgCl2, glucose, diH2O, pH meter, autoclave Results: Conclusions/Next Lab goals: Begin heat shock. Extra notes: Names of each student: Nancy Date: 9/28/2023 Protocols Performed: Electroporation trial run Purpose: To test that the electroporation would be successful, a trial test was ran with a different E.coli strain. Supplies used/Materials: Results: Conclusions/Next Lab goals: Heatshock. Extra notes: Names of each student: Kaia and Jasmine Subteam name: Knockout Date: 10/4/2023 Protocols Performed: Heatshock Thaw chemically competent bacterial cell such as JM109 on ice. Combine 40 uL of JM109 with 1uL (1ng/ uL) of plasmid to 1.5mL eppendorf tube. Incubate cells on ice for 35 minutes. After ice incubation, place the samples into 42℃ water bath for 4 seconds. Put the samples back to ice for 2 minutes to recover. Quickly take the eppendorf tube out and immediately add 250uL of SOC medium. Place the eppendorf tube into 37℃ shaking water incubator for 1hr and 2omin at 250rpm. After shaking, streak 150uL of solution onto an LB agar plate with the respective antibiotic (ampicillin). Purpose: To separate the plasmid from E.coli Supplies used/Materials: Ice bath, eppendorf tube, ampicillin, SOC, shaking incubator, JM109, LB agar plate. Results: Conclusions/Next Lab goals: Extra notes: Names of each student: Elizabeth Plumart Date: 10.07.23 Protocols Performed: Transformation (heat shock) Purpose: Transform recET and ackA knockout into E. coli Results: Success Conclusions/Next Lab goals: Overnight culture for mini-prep Extra notes: Names of each student: Elizabeth Plumart Date: 09.18.23 Protocols Performed: Heat shock Purpose: Transform phaB, phaC, pChlamdy into E. coli Results: Success Conclusions/Next Lab goals: Overnight culture to make glycerol stocks, transform phaA, tdTomato, algae promoter, algae terminator into E. coli Extra notes: Names of each student: Elizabeth Plumart, Chloe Benjamin Date: 09.22 Protocols Performed: Overnight culture Purpose: Grow up colonies from transformation (phaB, phaC, pChlamdy) Results: Success Conclusions/Next Lab goals: Mini-prep Extra notes: Names of each student: Elizabeth Plumart Subteam name: Algae PHB Date: 09.23 Protocols Performed: Mini-prep Purpose: Extract plasmid (phaB, phaC, pChlamdy) from overnight culture Results: Success Plasmid/Gene, Colony Concentration (ng/ul) 260/280 pChlamdy, 1 47.8 1.92 pChlamdy, 2 56.4 1.92 pChlamdy, 3 48.7 1.93 phaC, 1 37.9 1.86 phaC, 2 51.5 1.90 phaC, 3 64.4 1.94 phaB, 1 505.7 (measured 3 times) 1.85 (260 = 10.114, 280 = 5.464) phaB, 2 48.3 1.93 phaB, 3 48.6 1.91 Conclusions/Next Lab goals: Transform kit plate parts, restriction digest & ligation to build expression construct Extra notes: Names of each student: Elizabeth Plumart, Chloe Benjamin Date: 09.25.23 Protocols Performed: Heat shock Purpose: Transform phaA, CaMV 35S, 3’ UTR, mCherry into E. coli Results: Success Conclusions/Next Lab goals: Overnight culture to make glycerol stocks, colony PCR on kit plate parts Extra notes: Names of each student: Chloe Benjamin Date: 09.26.23 Protocols Performed: overnight culture of E.coli plates (phaA, mCherry, CaMV 35S, 3’ UTR) Purpose: to prepare for the Miniprep and making glycerol stocks tomorrow Results: Success Conclusions/Next Lab goals: Colony PCR, Miniprep, Glycerol stocks, Restriction Digest to create algae construct, Ligation, Transform algae construct into E.coli Extra notes: It was decided that the colony pcr will be done on Wednesday, 9/27/23 instead. The shaker in nsc 318 was not working last night, so I put the overnight culture tubes in another incubator in nsc 318. The E.Coli plates were placed back in the fridge in nsc 110 and the purple box containing antibiotics were placed in the freezer in nsc 110. Names of each student: Xucheng Zhang Date: 09.27.23 Protocols Performed: colony pcr and gel electrophoresis Purpose: confirms E.coli has the genes mCherry (reporter gene), CaMV 35s(promoter), and 3’ UTR (terminator) Results: Success Conclusions/Next Lab goals: Miniprep Extra notes: Names of each student: Chloe Benjamin, Elizabeth Plumart Date: 09.27.23 Protocols Performed: miniprep Results: Success Plasmid/Gene, Colony Concentration (ng/ul) 260/280 phaA 1 80.4 1.92 phaA 2 96.8 1.93 phaA 3 90.3 1.92 CaMV 1 52.5 1.98 CaMV 2 57.1 1.98 CaMV 3 71.1 1.92 3’ UTR 1 71.5 1.95 3’ UTR 2 62.3 1.95 3’ UTR 3 57.3 1.96 mCherry 1 46.6 2.01 mCherry 2 65.3 1.99 mCherry 3 61.6 1.99 Conclusions/Next Lab goals: Miniprep Extra notes: Names of each student: John Biggs Date: 09.28.23 Protocols Performed: Restriction digest Restriction digest enzymes Gene Enzymes CaMV 35S (algae promoter) SpeI & PstI mCherry (reporter) XbaI & PstI phaA XbaI & PstI phaB XbaI & PstI phaC XbaI & PstI Purpose: Prepare construct for expression Results: phaA, phaB, phaC success, CaMV35S, mCherry fail Conclusions/Next Lab goals: The next step is completing the remaining restriction digests, gel extractions, and ligations to finish building the construct we want to express. Extra notes: Names of each student: Chloe Benjamin, Elizabeth Plumart Date: 10/2/23 Protocols Performed: Gel electrophoresis to verify if the restriction digests completed on 9/28/23 were done successfully Restriction digest enzymes Gene Enzymes CaMV 35S (algae promoter) SpeI & PstI mCherry (reporter) XbaI & PstI phaA XbaI & PstI phaB XbaI & PstI phaC XbaI & PstI Run out on gel, gel extraction on band. Purpose: to prepare for construct for expression Supplies used/Materials: Results: The DNA bands did not travel far enough down the gel, so we did not get successful results to analyze. Conclusions/Next Lab goals: The next goal is to repeat the restriction digests on 9/28/23. Extra notes: Names of each student: Xucheng Zhang Date: 10/3/23 Protocol performed: Restriction digests, gel electrophoresis, and gel extraction Restriction digest enzymes Gene Enzymes CaMV 35S (algae promoter) SpeI & PstI mCherry (reporter) XbaI & PstI phaA XbaI & PstI phaB XbaI & PstI phaC XbaI & PstI Gel extraction of each after gel electrophoresis should be done Purpose: to prepare for the expression of a PHB construct Supplies used/Material: Results: From left to right, the genes are in the following order starting at the 2nd well since the first well has the DNA ladder: CaMV 35S (promoter gene), mCherry (reporter gene), Conclusion/Next lab goals: Restriction digests of phAa and phAB were a success as seen in the gel electrophoresis results. However, restriction digests of the other genes will be redone. Extra notes: Names of each student: Chloe Benjamin Date: 10/3/23 Protocol performed: overnight culture to grow E.coli bacteria containing the genes, phAc and mcherry Purpose: to prepare for a miniprep as well prepare for the expression of a PHB construct Results: Conclusion/Next lab goals: Restriction digests Gel extraction Ligation Miniprep to isolate the plasmid containing the genes mcherry and phAc Names of each student: Xucheng Zhang Chloe Benjamin Elizabeth Plumart Date: 10/4/23 Protocol performed: Restriction digests and Gel extraction Ligation of phAa and phAb Miniprep to isolate plasmid containing the genes mcherry and phAc ~This will only be done if Eric cannot complete a gel extraction of mcherry and phAc due to DNA bands for both genes not being present on the gel. Restriction enzymes Gene Enzymes CaMV 35S (algae promoter) SpeI & PstI mCherry (reporter) XbaI & PstI phaA XbaI & PstI phaB XbaI & PstI phaC XbaI & PstI Ligate each CDS to 3’UTR. Purpose: to prepare for the expression of a PHB construct Results: mCherry, CA MV 35S, and terminator failed Conclusion/Next lab goals: Miniprep Names of each student: Xucheng Subteam name: Algae PHB Date: 10/5/23 Protocol performed: Measured results of mini prep completed on 10/4/23 Miniprep to isolate the plasmids containing the promoter and terminator Results: Promoter Results: P1 = Concentration: 3.8 ng/microliter and 260/280: 3.61 P2= Concentration: 7.4 ng/microliter and 260/280: 2.92 P3= Concentration: 15.3 ng/microliter and 260/280: 2.10 P4= Concentration: 4.0 ng/microliter and 260/280: 3.20 Terminator Results: T1= Concentration: 24.1 ng/microliter and 260/280: 2.06 T2=Concentration: 39.7 ng/microliter and 260/280: 1.67 T3=Concentration: 2.5 ng/microliter and 260/280: 3.77 T4=Concentration: 7.7 ng/microliter and 260/280: 2.10 phAc Results and mCherry Results Plasmid Gene,Colony Concentration 260/280 1-phaC 1 97 nanograms/microliter 1.87 1-phaC 2 57.6 nanograms/microliter 1.87 1-phaC 3 47.2 nanograms/microliter 1.84 2-phaC 1 91.1 nanograms/microliter 1.82 2-phaC 2 70.0 nanograms/microliter 1.94 Plasmid Gene,Colony Concentration 260/280 2-phaC 3: 78.5 nanograms/microliter 1.87 1-mcherry 1: 9.4 nanograms/microliter 1.71 1-mcherry 2: 8.1 nanograms/microliter 1.64 1-mcherry 3: 8.7 nanograms/microliter 1.77 Plasmid Gene,Colony Concentration 260/280 2-mCherry 1: 8.7 nanograms/microliter 1.58 2-mCherry 2: 7.9 nanograms/microliter 1.64 2-mCherry 3: 6.6 nanograms/microliter 1.63 Names of each student: Elizabeth Plumart Subteam name: Algae PHB Date: 10.07.23 Protocol performed: Transformation of mCherry, CA MV 35S, and terminator Results: Terminator failed (one colony that we can try), CA MV 35S and mCherry success Names of each student: Elizabeth Plumart, Chloe Benjamin Date: 10/09/23 Protocols Performed: Mini prep Restriction digest Restriction digest enzymes Gene Enzymes CaMV 35S (algae promoter) SpeI & PstI mCherry (reporter) XbaI & PstI Terminator EcoRI & XbaI Results: Mini prep- Success Plasmid/Gene, Colony Concentration (ng/ul) 260/280 CAMV 35S, 1 142.3 1.95 CAMV 35S, 2 122.3 1.94 mCherry, 1 135.4 1.92 mCherry, 2 131.5 1.92 terminator 128.2 1.92 Restriction digest- Success Names of each student: Xucheng Zhang, Chloe Benjamin Date: 10/10/23 Protocols Performed: Gel extraction, ligation, transformation Results: Gel extraction- success Ligation- success Transfo