Line 637: | Line 637: | ||
Lab work: | Lab work: | ||
<ul> | <ul> | ||
− | <li> | + | <li>Miniprep of Promoters + CYT/HAO</li> |
− | <li> | + | <li>Preculture of SOD/HAO biobrick</li> |
+ | <li>Biobrick Formation via 3A Assembly into pSB1k3: arab/HAO + arab/CYT; VhB/HAO + VhB/CYT</li> | ||
+ | <li>Restriction Enzyme Digest</li> | ||
+ | <ul>300 ng of insert 1 (digested with 0.5uL of E and S), 300 ng of insert 2 (digested with 0.5 uL of X and P), 100 ng of vector (digested with 0.5 uL of E, P & DpnI)</ul> | ||
+ | <ul>2 uL Cutsmart, 20uL total rxn volume for master mix</ul> | ||
+ | <ul>Incubated at 37 degrees C for 30 min, heat kill at 80 degrees C for 20 min, ligation overnight</ul> | ||
</ul> | </ul> | ||
</span> | </span> |
Revision as of 02:07, 3 October 2017
Lab work:
- Media Creation:
- Ampicillin stock solution created
- Chloramphenicol stock solution created
- 50% glycerol stock solution created
- LB Broth created
- Transformation Buffer made and filter sterilized
- MgCl2, CaCl2, MnCl2, KCl, and PIPES stock solutions created
- Created LB plates with no antibiotics
- Created LB media with chloramphenicol
- Created LB plates with ampicillin
- Created Stop Buffer
- E. coli DH5 alpha pre-cultured overnight in 3 mL of LB with MgCl2
- Another 500 mL LB+MgCl2 created as backup
Lab work:
N. europaea and Pc. denitrificans in starting culture
Lab work:
- Completed competent cell preparation for DH5 alpha
- E. Coli DH5 pre-cultured in 3 mL LB
- Competent DH5 alpha 50% glycerol stocks made and stored in -80 degree C freezer
- 1 mL of the rest of the pre-culture inoculated in 500 mL LB+MgCl2 and incubated in 18C shaker in PLSB lab.
- Creation of another 500 mL LB+MgCl2 as a new backup media.
- Another pre-culture of E. Coli DH5 was also incubated at 37C in the gilmer lab in case the main culture at PLSB was contaminated expectedly.
- OD values of the E. Coli DH5 culture incubated at 37C shaker after overnight in the morning
- The DH5 cell culture was then centrifuged and the pellets were resuspended twice in transformation buffer. DMSO was added to the cell media and the cell culture was aliquoted into 500 uL. The aliquots were freezed with liquid nitrogen and stored in -80C.
- The cells were thawed on ice and the DNA from the competent cell kit was centrifuged. The plasmid backbone from the kit was transformed into the DH5 cells and incubated and spread around plates for overnight culture.
- Competent Cell Check: First set of plates didn’t meet our expectation in terms of colony density, transformation of the same set of competent cells was executed again
- Blank Value = 0.018
- 08:16 Value = 0.047
- 09:00 Value = 0.051
- 10:00 Value = 0.057
- 11:00 Value = 0.030
- 12:00 Value = 0.073
- 12:30 Value = 0.116
- Second transformation attempt with the same competent cell stock was successful with ideal results
- Competent cell check calculations
- First day of Interlab Lab Study
- Positive and negative controls and 1-6 test plasmids transformed in E. Coli DH5 alpha
- 16 chloramphenicol plates were used to culture different concentrations of E. Coli cells, 8 with 200 pg/uL and 8 with 20 pg/uL
- Plates had many colonies on them
- We picked two colonies of each device and inoculated them, then put in 37 degrees C 220 rpm shaking incubator
- More Agar plates with chloramphenicol supplement were created
- The calibration of the plate reader was done with the measurement of OD600 reference point and fluorescein standard curve
- The different bacteria strains were pipetted into the well plate and the OD level of the cells were measured
- Due to the time constraint, the cells were chilled at -20 degrees C overnight and not diluted to OD=0.02 yet
- As the chill was later determined to have possible effects on the accuracy of the measurements, the bacteria strains were re-cultured overnight and will be diluted and incubated for the second time on the next day
- 10 DNA plasmids from the DNA distribution kit were transformed into competent E. Coli DH5 alpha cells
- LB made for transformed E. coli culture which was used to inoculate the cells in preparation for glycerol stocks tomorrow
- N. europaea media stocks made for culturing
- May be irregularities in the composition of the media, as it took very long for pH to increase, also N. europaea Broth step #2 had a unusually low initial pH
- Prepared for miniprep procedure
- Created N. europaea growth media
- Miniprep completed
- N. europaea culturing begins
- Transformations in preculture
- Made Pc. denitrificans plates and Pc. denitrificans glycerol stocks, replated transformed E. coli
- Plating N. europaea
- Glycerol stocks of 50% N. europaea
- Plating Pc. denitrificans
- Competent Cell Prep for Pc. denitrificans
- Made agar (1L) for Pc. denitrificans: plated onto 40 plates, 100 mg ampicillin added to the 1L culture
- Made liquid media for Pc. denitrificans then autoclaved
- Plated 10 plates, 5 from one 3.5 mL aliquot, 5 from the other
- Put in incubator at 37 degrees C then moved to incubator at 26 degrees C
- 6x plates of Pc. denitrificans incubating at 37 degrees C
- 4x plates of Pc. denitrificans incubating at 26 degrees C
- Failed: no cell pellet retrieved after after the transformation buffer was added
- Starting Culture Pc. denitrificans
- Re-grow N. europaea from glycerol stocks
- 3 mL culture in falcon tubes with Pc. denitrificans from glycerol stocks
- 3 mL culture in falcon tubes with N. europaea from glycerol stocks in shaking incubator
- Pc. Denitrificans had no growth on most plates
- One plate with growth had two phenotypes of colonies
- Made Several Pc. Denitrificans plates with no antibiotics to dry overnight
- Made N. europaea and Pc. denitrificans plates from glycerol stocks
- Made Pc. denitrificans liquid precultures
- Made SGM17 Media
- Made N. europaea, Pc. denitrificans, and Kanamycin LB plates
- Begin assembly plans for promoter characterization with GFP generators
- 3 mL of Pc. denitrificans preculture was added to 500 mL of the Pc.denitrificans broth and put in the shaking incubator (37 degrees C, 220 rpm)
- Gram Stains performed on the two phenotype plate
- Transformation of VhB, Sod, Nar to competent E.coli DH5 alpha and plated on LB plates with ampicillin
- Plated competent E.coli on 10 LB kanamycin
- Nanodropped BBa_E0240, concentration: 100.9 ng/uL
- Lighter colonies- gram positive (left); darker colonies- gram negative(right)
- Pc. denitrificans Electroporation Transformation:
- Pc. denitrificans heat shock transformation and plated competent cells
- Created N. europaea and E. coli liquid cultures
- Pc. denitrificans liquid culture measured OD600 of 0.945, 0.355, and 0.365
- Transferred liquid culture into 1/100 dilutions in SGM17 media without Glycine
- Created a 250 mL aliquot with 2% glycine supplement
- SGM17 + Glycine Pc. denitrificans liquid culture had not changed color while the SGM17 cultures did
- N. europaea liquid culture has turbidity
- N. europaea plates are not growing colonies
- Created 50% glycerol stocks of DH5 alpha E. coli with VhB, Nar, and Sod
- Pc. denitrificans liquid culture started
- OD 600 for SGM17 + glycine is 0, 0.012, 0- no growth
- OD 600 for SGM17 varied from -1.577 to 2.659; SGM17 media may be making OD readings unreliable
- Miniprep for three promoters (VhB, Sod, Nar) completed and stored at -20 degrees C
- 50% Glycerol stocks made from remaining liquid culture
- Competent Pc. denitrificanscells made
- Transformed DH5 E. coli with Pc. denitrificans Tat w/ GFP, N. europaea Tat w/ GFP, RFP and plated transformed cells
- Performed PCR on BBa_E0240 to amplify 4 uL of DNA
- Pc. denitrificans liquid culture in SGM17 + glycine: Centrifuged, washed with sucrose washing solution, pellet resuspended
- Anneal temp was set at 55 degrees C and Elongation/melt temp at 95 degrees C, 30 cycles
- Plated N. europaea on N. europaea and LB plates
- Made liquid precultures of N. europaea tat w/gfp, Pc. denitrificans tat w/ gfp, RFP in DH5 E. coli
- Electroporation Transformation of Pc. denitrificans
- 6 transformations with Nar (3 with glycine, 3 without glycine)
- Plated transformations on SR plates with ampicillin
- Completed miniprep of DH5 alpha with Pc. denitrificans tat w /gfp, N. europaea tat w/ gfp, RFP
- Made 6 glycerol stocks each for Pc. denitrificans tat w /gfp, N. europaea tat w/ gfp, RFP
- Made LB plates with no antibiotics
Lab work:
- Made SR plates and media
- Biobrick Formation of TAT reporter circuits: Restriction Digests
- Biobrick Formation of TAT reporter circuits: Gel Separation
- Biobrick Formation of TAT reporter circuits: Ligation Reaction
- Started Pc. denitrificans liquid cultures
- Digested 2.2 uL of N. europaea TAT DNA with E and S
- Digested 2.0 uL of Pc. denitrificans TAT DNA with E and S
- Digested two separate reactions of 1.2 uL RFP Part DNA with E and X
- 4 uL loading dye for 20 uL restriction digest
- Used 0.5 uL ladder and 4 uL dye, with rest molecular grade water for 20 uL total
- Left to Right: Ladder, Empty, Pc. denitrificans TAT, Pc. denitrificans TAT undigested, N. europaea TAT, N. europaea TAT undigested, RFP, RFP, RFP undigested
- Performed using 4.5 uL of insert, 3 uL of the vector, 10.5 uL water, 0.75 uL T4 ligase, 2 uL 10X buffer
- Repeat Biobrick transformation of TAT reporter circuits
- 5uL of DNA, 50 uL of competent cells
- Performed using two different protocols (with and without SOC media)
- Calibrated dissolved oxygen probe
- Replated N. europaea tat and Pc. denitrificans tat both with RFP
- Replated BBa_E0240 from glycerol stock to miniprep again
- Transform Nar into Pc. denitrificans cells with electroporation
- HAO and CYT plasmids transformed into E. Coli DH5a and placed in the 37 degree C incubator
- BBa_E0240 plated
- Made negative controls for all liquid media
- Pc. denitrificans with Nar grown without glycine had growth, Pc. denitrificans with Nar grown with glycine had no growth
- Biobrick Formation of GFP and Promoters: Restriction Enzyme Digest
- Biobrick Formation of GFP and Promoters: Gel Electrophoresis
- HAOB transformed into E. coli DH5
- Started preculture of Pc. denitrificans
- Started precultures for Pc. denitrificans TAT and N. europaea TAT transformations
- Insertion of 4 promoters (SOD, VhB, pBAD & T7) into GFP Generator backbone
- GFP part digested with E & X (4 replicates); Promoters each digested with E & S
- Promoters ran off gel
- Created three gels with six wells each
- Miniprepped HAO and CYT
- Started preculture HAOB
- Made 50% glycerol stocks with remaining preculture
- Nanodrop: HAO-628.9 ng/uL; CYT-450.4 ng/uL
- Prepared two 0.8% agarose gels and one 2.0% agarose gel
- Measured OD to create Pc. denitrificans growth curve, ran into issues with nanodrop
- Created 4% Nusieve GTG agarose gel
- Miniprepped HaoB, Pc. denitrificans TAT, and N. europaea TAT
- Seaplaque gel: ladder, uncut gfp, 4x cut GFP
- Nusieve gel: ladder, uncut VhB, cut VhB, uncut Sod, cut Sod, uncut T7, cut T7
- Miniprep RFP from the liquid E. coli culture
- Plated Sod and VhB from glycerol stock
- Prepared preculture for GFP using LB + Chloramphenicol
- Transformed Pc. denitrificans TAT GFP + RFP and N. europaea TAT GFP + RFP into E. coli
- Miniprepped GFP
- Ligated GFP vector with arabinose, Sod, and VhB inserts
- Created LB+AMP, LB+Chloramphenicol, Pc. denitrificans Media
- Sequenced Pc. denitrificans TAT with RFP and N. europaea TAT with RFP
- Miniprepped and made glycerol stocks for Sod, VhB, Pc. denitrificans TAT with GFP and RFP,N. europaea TAT with GFP and RFP
- Transformed and plated Sod with GFP, arabinose with GFP, VhB with GFP, and control in E. coli DH5
- Created Pc. denitrificans growth curve
- Biobrick formation of inserts into standard vectors: Restriction Enzyme Digest
- Biobrick formation of inserts into standard vectors: Gel Electrophoresis
- Biobrick formation of inserts into standard vectors: Ligation
- Inserts: Pc. denitrificans TAT GFP + RFP (500 ng); N. europaea TAT GFP + RFP (500 ng)
- Vector: pSB1C3 (250 ng)
- Enzyme Digest: Inserts and vectors cut with E & P (0.5uL each)
- Ladder, spacer, Pc. denitrificans TAT (undigested), Pc. denitrificans TAT, spacer, N. europaea TAT (undigested), N. europaea TAT
- Digested with 1uL Vector, 1.8 uL insert
- Created control with vector and no insert
- Incubated at room temperature overnight
- Transformation and plating of TAT sequences from the previous day in E. coli
- Created SeaPlaque gels
- Miniprepped SOD+GFP, Arabinose+GFP, VhB+GFP
- Biobrick formation of HAO and CYT with promoters: Restriction Enzyme Digest
- Biobrick formation of HAO and CYT with promoters: Gel Electrophoresis
- Inserts: VhB, SOD & arabinose promoters (500ng each)
- Vectors: Cytochromes, HAO full operon (250ng each)
- Enzyme Digest: Inserts cut with E & X, vectors cut with E & X
- SeaPlaque 0.8% for 1 hour at 60V
- Nusieve 4% for 2 hours at 40V
- Transformation of VhB, SOD, Arabinose with HAO and VhB, SOD, Arabinose with CYT into E. coli
- Created precultures from plated TAT sequences
- Observed plates from the previous day
- Miniprep of N. europaea TAT GFP + RFP and Pc. denitrificans TAT GFP + RFP
- Top row: HAO + Sod, Cyt + Vhb, Cyt + arab, HAO ligation control
- Bottom row: Cyt + Sod, HAO + Vhb, HAO + arab, Cyt ligation control
- Miniprep of Promoters + CYT/HAO
- Preculture of SOD/HAO biobrick
- Biobrick Formation via 3A Assembly into pSB1k3: arab/HAO + arab/CYT; VhB/HAO + VhB/CYT
- Restriction Enzyme Digest
- 300 ng of insert 1 (digested with 0.5uL of E and S), 300 ng of insert 2 (digested with 0.5 uL of X and P), 100 ng of vector (digested with 0.5 uL of E, P & DpnI)
- 2 uL Cutsmart, 20uL total rxn volume for master mix
- Incubated at 37 degrees C for 30 min, heat kill at 80 degrees C for 20 min, ligation overnight
- Redoing digests from 7/21 and running a new gel
- Received sgRNA from biobasic, prepared and stored the sgRNA at -20°C
- Digested CRISPR RNA insert and Cas9 vector
- Performed digests with BSA1 on the CRISPR vector and insert
- Performed a ligation reaction between the vector and insert
- Realized that the primers used to PCR the LeuS gene were incorrect - Therefore, our T1 and T2 biobricks did not contain the correct DNA sequences
- Continued the PrG uptake test - purification of the cell lysate
- Performed a miniprep for the T1 and T2 terminators (for biobricking)
- Transformed and plated KanR gene from kit
- Streaked pS1M27 cells from gel stab to Tet plate
- Re-transformed and plated KanR gene on CAM plates
- Re-streaked pS1M27 plate
Lab work:
- Miniprep performed for pS1M27
- Re-transformed KanR DNA
- Performed a PCR reaction to obtain LeuS gene from genomic DNA
- Confirmatory digest performed on PCR product (LeuS gene)
- Restriction digest for biobrick #1
- Performed ligation reactions between the LeuS gene and the T1 and T2 genes
- Performed a ligation reaction between CRISPR Cas9 genes and sgRNA sequence
- Minipreped ZeoR, KanR, T1, and T2 plasmids
- Confirmatory digests performed for T1 and T2 plasmids
- Performed a ligation reaction with T1 and T2 genes, transformed products
- PCR performed to confirm CRISPR/Cas9 and sgRNA gene ligation
- Digests performed to redo LeuS and terminator ligation
- Ligation reaction performed
- Received IDT DNA: Penicillin G Acylase pieces 1 and 2
- Sequencing of Cas9 plasmid
- Digested PCRed LeuS product
- Transformed cells with T1 LeuS lig and T2 LeuS lig
- Began preparing competent JW strain E. coli cells
- Restriction digest of pac1, pac2, kan, and zeo resistance genes
- Restriction digest of terminators followed by cipping
- Terminator digest Ex + Sip gel
- LeuS PCR purification
- Performed transformations using the following genes:
- Promoter from iGEM distribution kit
- RFP for cell competency check
- T1-LeuS ligation reaction products
- T2-LeuS ligation reaction products
- Restriction digests on pac1 and pac2
- Ligation reaction for pac1-pac2-terminator
- Miniprepped 9 terminator-LeuS ligation plasmids
- Transformed pac-terminator ligation
- Precultured promoter into pac vector
- Minipreped Pro1 and Pro2
- Performed PCR on CRISPR plasmid
- Performed a restriction digest to confirm LeuS-terminator plasmid
- Performed a PCR purification
- Performed restriction digests on terminator 1, terminator 2, LeuS from PCR, Cas9 plasmid, and on sgRNA insert
- Performed sequencing on CRISPR insert
- Miniprepped 5 pac-term
- PCR of LeuS gene
- Completed PCR purification
- Performed ligation on zeo and piece E
- Performed a transformation of zeo-E-tet
- Restriction digest of LeuS
- Redoing competent JW cells
- Took nanodrop concentrations of minipreppred ligation reaction
- Restriction digests
- Made primer solutions
- SDM of Pst 1
- Transformation and sequencing
- Performed digests of T1
- Digest PCR LeuS
- Ligated T1 and LeuS
- Transformed cells
- Preculture of term-leuS lig
- 3 colonies of 6:1 ligation and 1 colony of 8:1 ligation
- Miniprep of leuS term ligation
- Ran a gel
- Performed PCR
- Performed transformation
Lab work:
- Preculture of Pst sdm
- Performed miniprep
- Ran confirmatory digest
- Performed restriction digests
- Performed ligations
- Preculture for transformations
- 3A assembly of zeo-E-Kan backbone
- Sequencing
- Performed restriction digest
- Performed ligation
- Performed restriction digest
- Performed ligation
- Nanodrop used to determine concentrations
- Nanodrop to determine concentrations and sequencing performed
- Ran term and enzyme gels
- Digest of term performed
- Miniprep of term and pro
- Performed digest
- Digest of terminator
- Success! Got term in lane 5
- Ligation performed
- PCR of mutant LeuS
Lab work:
- PCR purifed mut LeuS
- Digest performed
- Ligations performed
- Miniprep x 27
- Digest MT 7.1 → CIP
- Digest piece A and controls
- Ligations of mut term and piece A performed
- Miniprep of HA and mut and term
- Restriction digest of enz-term and promoter
- Preculture JW cells
- Competent cell procedure for JW
- Confirmation test followed by plating
- Restriction digest
- JW5807 competent cell protocol
- Miniprepped promoter-enzyme-terminator plasmid
- Confirmatory digest of promoter-enzyme-terminator plasmid miniprepped on 10/9
- Sequencing of promoter-enzyme-terminator plasmid miniprepped on 10/9
- Made minimal medium plates with nothing, 3mM leucine, or 3mM CBZ-leu
- digest, gel, ligation of parts into CAM backbones
May | ||||||
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S | M | T | W | T | F | S |
1 | 2 | 3 | 4 | 5 | 6 | |
7 | 8 | 9 | 10 | 11 | 12 | 13 |
14 | 15 | 16 | 17 | 18 | 19 | 20 |
21 | 22 | 23 | 24 | 25 | 26 | 27 |
28 | 29 | 30 | 31 |
June | ||||||
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S | M | T | W | T | F | S |
1 | 2 | 3 | ||||
4 | 5 | 6 | 7 | 8 | 9 | 10 |
11 | 12 | 13 | 14 | 15 | 16 | 17 |
18 | 19 | 20 | 21 | 22 | 23 | 24 |
25 | 26 | 27 | 28 | 29 | 30 |
July | ||||||
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S | M | T | W | T | F | S |
1 | ||||||
2 | 3 | 4 | 5 | 6 | 7 | 8 |
9 | 10 | 11 | 12 | 13 | 14 | 15 |
16 | 17 | 18 | 19 | 20 | 21 | 22 |
23 | 24 | 25 | 26 | 27 | 28 | 29 |
30 | 31 |
August | ||||||
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S | M | T | W | T | F | S |
1 | 2 | 3 | 4 | 5 | ||
6 | 7 | 8 | 9 | 10 | 11 | 12 |
13 | 14 | 15 | 16 | 17 | 18 | 19 |
20 | 21 | 22 | 23 | 24 | 25 | 26 |
27 | 28 | 29 | 30 | 31 |
September | ||||||
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S | M | T | W | T | F | S |
1 | 2 | |||||
3 | 4 | 5 | 6 | 7 | 8 | 9 |
10 | 11 | 12 | 13 | 14 | 15 | 16 |
17 | 18 | 19 | 20 | 21 | 22 | 23 |
24 | 25 | 26 | 27 | 28 | 29 | 30 |
October | ||||||
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S | M | T | W | T | F | S |
1 | 2 | 3 | 4 | 5 | 6 | 7 |
8 | 9 | 10 | 11 | 12 | 13 | 14 |
15 | 16 | 17 | 18 | 19 | 20 | 21 |
22 | 23 | 24 | 25 | 26 | 27 | 28 |
29 | 30 | 31 |