Notebook Documents
Ethanol Team Notebook
Knockout Team Notebook
PHB Team Notebook (Part I)
PHB Team Notebook (Part II)
What should this page contain?
- Include pictures of your teammates, don't forget instructors and advisors!
- You can add a small biography or a few words from each team member, to tell us what you like, and what motivated you to participate in iGEM.
- Take team pictures! Show us your school, your lab and little bit of your city.
- Remember that image galleries can help you showcase many pictures while saving space.
Important: Your wiki pages will be archived at the end of the iGEM season and this content will remain online. Please keep this in mind as you post photos and personal information on this page.
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