NOTEBOOK
- Shelf organization: Acids, Proteins, Buffers, Gels, Agarose, Salts, Carbs
- Competent Cell Formation (K12)
- See comp. cell prep protocol in VA iGEM 2016 Lab Protocols
Agenda:
- PrG Determination (1-4)
- Orthogonality Research
- Competent Cell Formation Cont. (K12)
- Growing K12 cells and make buffer (sterilized transformation buffer)
- Plans to grow another batch with XL1 Blue tomorrow
Modeling:
- Research docking software and test
Competent Cells:
- Making Sterilized Transformation Buffer (500ml)
- Took out cells at 9:30am
- Transferred to 20°C at 11:00 PM, pour one of XL1-Blue and K12
Competent cells:
- Cultures left 18°C
- 11pm tonight: into LB + back into 18°C
- Sterilized filtration buffer
Protecting group:
- 2?:Phenyl acetyl protecting group, cleaved by penicillin g acylase
- Truncated from 5 PGs to 3
- 1: Dipeptide (maybe leu-leu), with a D aa on N-terminus
- A protease w/ cleavage activity acting on D aa is being researched for increased specificity
Modeling:
- AD4 returned zero errors w/ synthetase and leucine as ligand
- Still unsuccessful AG4 and AD4 returned
- Researching alternatives to direct another approach
- Unpacked Rosetta ligand modeling
- IGEM dock successful modeling; concerns about synthetase model
CRISPR
- Found biobrick to insert plasmid
- An alternative approach to silencing RNAs and transformation
Funding
- Anders finished half application for grant
Removing competent cells from 18°C
After 17 hrs:
- XL1 blue at an OD of 0.012
- K12 at an OD of 0.16
Put XL1 blue back into 18°C incubator, put K12 into 4°C fridge
Competent cells:
- Removed XL1 blue at a OD of 0.13, centrifuged with buffer, added DMSO, froze with liquid nitrogen
Lab work:
- Made CAM plates
- Made SOC media
- Competency test
Decided on 4 different PrGs:
- Cbz leu, pro leu, phenyl acetyl leu, N-methoxy leu
Modeling:
- Corrected enzyme mistake, now using correct configuration of enzyme
Lab work:
- Competency test control plated at 4:50pm
Lab work:
- No growth on control plates
Lab work:
- JW cells subcultured on LB agar plate in 37°C incubator
Lab work:
- JW cells grown in overnight liquid culture at 37°C shaking
Lab work:
- JW cells frozen in glycerol stock at -80°C (5 tubes)
Lab work:
- Preculture of JW in 20 ml LB
- Made 50 ml of sterile LB and 0.1g of pro-leu
Lab work:
- Began pr-leu uptake test
- Take OD: 0.07 for each preculture, incubate
- Test for lawn growth or individual colonies on a spread plate (for CRISPR)
Lab work:
- Uptake test OD measurements
- AM measurements: 1.137 for LB, 1.422 for LB + pro-leu
- 7 pm measurements:1.104 for LB, 1.994 for LB + pro-leu
- CRISPR plate - lawn produced
Pr-leu uptake test:
- Test failed, bacteria were fixed by ethanol overnight and could not be lysed
- Restarted test, XL1-blue innoculated in LB at 5:20pm
Policies and Practices:
- Meeting with Rivanna river waste treatment plant
- Meeting with open bio labs
Tested spectrophotometer in the PLSB lab - Not consistent with results in Prof. Kozminski's lab
Pr-leu uptake test:
- Failed again, cells did not lyse
- Started a new pr-leu uptake procedure
Wiki team meeting - wiki design
Created standards for LC-MS
Lab work:
- Transformed XL1-blue with Bba-K1218011 CRISPR Cas9 plasmid
- Transformed XL1-blue with BBa-B0010 forward terminator plasmid
- Transformed XL1-blue with RFP control plasmid
Lab work:
- Growth on CRISPR, terminator, and RFP plates, no growth on controls
- Cultures prepared for glycerol storage of transformed bacteria
Lab work:
- Optical densities taken for second round of pr-leu uptake and enzyme tests
Lab work:
- Optical densities taken again for second round of pr-leu uptake and enzyme tests
Lab work:
- Sample taken from enzyme reaction and frozen in -20° freezer
Lab work:
- Second sample taken from enzyme reaction and frozen in -20° freezer
Lab work:
- Redoing growth uptake test with aerated growth conditions
Lab work:
- OD measurements taken for growth test
- Extracted and purified E. coli genomic DNA via a minikit
- Began PCR on genomic DNA to obtain the leuS gene DNA
Lab work:
- Purified the PCR product
- Ran a gel to confirm product - bands ran together
- Took OD measurements for growth test
Lab work:
- Extractions performed for enzyme test
- Redo growth uptake test again
| Growth Uptake Test Results |
| Supplement | Growth at 10:05am | Growth at 4:00pm | Growth at 9:08pm |
| 1 None | None | None | None |
| 1 None | None | None | None |
| 3 None | None | None | None |
| 1 Leu | None | Yes | Yes |
| 2 Leu | None | Yes | Yes |
| 3 Leu | None | Yes | Yes |
| 1 Z 3 | None | None | None |
| 2 Z 3 | None | None | None |
| 3 Z 3 | None | None | None |
| 1 Z 8 | None | None | None |
| 2 Z 8 | None | None | None |
| 3 Z 8 | None | None | None |
Lab work:
- PCR performed on LeuS gene and product was purified
Lab work:
- Confirmatory digest performed on LeuS PCR product
- XL1-blue cells transformed with LeuS-ampR plasmid - plated and incubated at 10:45pm
Lab work:
- Inoculated precultures of LeuS and terminator transformed cells for miniprep and sequencing
Lab work:
- Redoing enzyme test (again) with a new protocol
- Performed a miniprep to obtain LeuS and terminator plasmids
Lab work:
- Sent off minipreped LeuS and terminator plasmid DNA for sequencing
- Performed confirmatory digests on LeuS and terminator plasmid DNA
Lab work:
- Performed PCR and PCR purification
Lab work:
- Talked with Professor Kozminski
- Set up another PCR reaction and PCR purification
Lab work:
- No colonies grew on the terminator transformation plate
- Restarted enzyme efficacy test
Lab work:
- 9:50am: Removed and stored a sample of the enzyme test
- Observed growth on the RFP, T1, and T2 plates, no growth on control
- Started precultures with colonies from the terminator 1 and 2 plates
Lab work:
- Performed gel electrophoresis, obtained wrong product
- Extracted DNA from gel
- Performed minipreps
Lab work:
- Confirmatory digest of minipreps from 7/15
- Performed a ligation reaction for T1 and T2
Lab work:
- Transformed XL1-blue cells with T1, T2, and an RFP control
- Extracted digested products from agarose gel
- Plated again using transformed cells from 12am at 1:30pm
- Transforme and plated new cells at 3:30pm
- Ligation reaction performed for T1 and T2 using DNA extracted from gel
Lab work:
- Transformed XL1-blue cells with the 2 ligation products (T1 with PCR, T2 with PCR)
- Began a preculture of XL1-blue cas9 cells in LB+cam at 5:45pm
Lab work:
- Performed a restriction digest
- Gel electrophoresis at 1:45am
- Gel weights: T2-173g, T1-163g, PCR1-192g, PCR2-202g
- Nanodrop densities: T2-13.5ng/µl, T1-17.4ng/µl, PCR1-13.5ng/µl, PCR2-17.2ng/µl
- Cells transformed and incubated at 37°C at 9:45am
- T1, T2, RFP, and control removed and plated at 11:15am
- Started uptake and enzyme activity tests with N-methoxy-leu
Lab work:
- Continued performing uptake test for N-methoxy-leu
Lab work:
- Continued performing uptake test for N-methoxy-leu - lysed cells and stored cell lysate
- Performed minipreps and confirmatory digests - No result from digests because no EtBr was in the gel
Lab work:
- 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
Lab work:
- Performed digests with BSA1 on the CRISPR vector and insert
- Performed a ligation reaction between the vector and insert
Lab work:
- 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
Lab work:
- Continued the PrG uptake test - purification of the cell lysate
- Performed a miniprep for the T1 and T2 terminators (for biobricking)
Lab work:
- Transformed and plated KanR gene from kit plate
- Streaked pS1M27 cells from gel stab to Tet plate
Lab work:
- 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
Lab work:
- 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
Lab work:
- 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
Lab work:
- PCR performed to confirm CRISPR/Cas9 and sgRNA gene ligation
Lab work:
- Digests performed to redo LeuS and terminator ligation
| May |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
|
|
|
|
|
