Difference between revisions of "Team:ETH Zurich/Notebook"
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<h3>Test 1A: Construction of pNorV and norR plasmids</h3> | <h3>Test 1A: Construction of pNorV and norR plasmids</h3> | ||
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| − | + | We performed a Gibson assembly to create a norR plasmid with J23118 promoter and T0 terminator. | |
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| − | + | ||
| − | + | ||
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</p> | </p> | ||
<h3>Test 3: Switch based on recombinases</h3> | <h3>Test 3: Switch based on recombinases</h3> | ||
<p> | <p> | ||
| − | < | + | <ul> |
| − | + | <li>We did PCR to create fragments with overhangs for Gibson assembly for parts containing recombinases phiC3, bxb1 and tp901, with attB site in front of recombinase and attP site after terminator. </li> | |
| − | < | + | <li>We did transformations of sfGFP + ssRa and mNectarine + ssRa plasmids created a week before. Adding ssRa tag to sfGFP was not successful.</li> |
| − | < | + | </ul> |
| − | </ | + | |
| − | + | ||
| − | + | ||
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</p> | </p> | ||
<h3>Directed evolution</h3> | <h3>Directed evolution</h3> | ||
<p> | <p> | ||
| + | We did PCR of fragments for construction of hsvTK-GFP with overlaps to esaboxes. | ||
</p> | </p> | ||
<h3>General</h3> | <h3>General</h3> | ||
<p> | <p> | ||
| + | <ul> | ||
| + | <li> We repeted all previously failed digestions to exchange resistances on empty backbone plasmids.</li> | ||
| + | <li> After transformations of plasmids created with GA or site directed mutagenesis, colonies were picked in the evening for overnight culture and miniprep was done the next day in the morning.</li> | ||
| + | </ul> | ||
</p> | </p> | ||
Revision as of 17:58, 18 October 2016
NOTEBOOK
JULY
WEEK 1 (27.6. – 5.7.)
Test 1A: Construction of pNorV and norR plasmids
We ordered gBlocks for:
- norR without forbidden restriction sites
- two versions of pNorV: one with the native spacer after transcription start site and one without
Test 1B: Construction of promoters with esaboxes and esaR plasmid
We ordered gBlocks for promoters with esaboxes. E. coli colonies with plasmid with esaR from addgene arrived.
Test 3: Switch based on recombinases
We ordered gBlocks for 3 different codon optimized recombinases without forbidden restriction sites:
- bxb1
- phiC31 and
- tp901
General
- We ordered first oligos
- We prepared first TFB1 and TFB2 buffers for competent cells. The next day we prepared the first batch of competent TOP10 cells (80 transformations).
- We did first transformations:
- interlab study plasmids
- pSEVA backbone plasmids
- plasmids from distribution kit to get J23118 promoter, terminator, prefix and suffix
- Transformation of plasmids with fluorescent proteins we might use: sfGFP, mCherry, mNectarine, mTurqouise.
- Followed were first overnight cultures of transformations and first minipreps of the plasmids from transformations and addgene colonies.
- We prepared first batch of ingredients for M9 media.
- We poured first LB-agar plates with single resistances for carbenicillin, kanamycin and chloramphenicol.
WEEK 2 (6.7. – 12.7.)
Test 1A: Construction of pNorV and norR plasmids
We used two approaches to get norR and pNorV fragments:
- PCR to extract genomic copy of norR and pNorV
- PCR to multiply norR and pNorV from gblocks.
Test 1B: Construction of promoters with esaboxes and esaR plasmid
We did PCR to get a fragment with esaR from addgene plasmid and added overhangs to it.
Test 3: switch based on recombinases
We did site directed mutagenesis (PCR) to add ssRa tag to mNectarine and sfGFP.
Directed evolution: make EsaR specific to AHL present in the gut
- We prepared electro-competent DH5alpha -uptr cells.
- We transformed respective cells with plasmid with uracil phosphoribosyltransferase (upp or uptr).
- - We performed Tecan plate reader experiment to test the response of cells with and without the plasmid towards 5-fluorouracil. Upp/gfp plasmid is under control of dmpR/phenol.
- 5-FU had an inhibitory effect on growth with all concentrations. There was no growth difference between un-induced and induced state
- We attempted to construct CAT-UPTR fusion protein:
- We did PCR to get fragments with CAT (chloramphenicol resitance) and UPTR.
- We did Gibson asesembly of CAT and UPTR fragments to create fusion protein.
- We attempted to construct hsvTK (herpes simplex virus thimidine kinase)-APH-Stop-GFP operon:
- We did PCR to create APH and hsvTK fragments and performed Gibson assembly. Colony PCR did not show any results. However, since the cells grew, they were Amp resistant.
General
- We repeated failed transformations.
- We transformed additional pSEVA empty backbone plasmids.
- We did PCR to linearize pSEVA backbones and to get fragments with prefix + J23118 and terminator + suffix for Gibson assemblies.
- We did digestion to exchange oris on two plasmid backbones.
- Following all successful transformations we did overnight culture and minipreps.
WEEK 3 (13.7. – 19.7.)
Test 1A: Construction of pNorV and norR plasmids
We performed a Gibson assembly to create a norR plasmid with J23118 promoter and T0 terminator.
Test 3: Switch based on recombinases
- We did PCR to create fragments with overhangs for Gibson assembly for parts containing recombinases phiC3, bxb1 and tp901, with attB site in front of recombinase and attP site after terminator.
- We did transformations of sfGFP + ssRa and mNectarine + ssRa plasmids created a week before. Adding ssRa tag to sfGFP was not successful.
Directed evolution
We did PCR of fragments for construction of hsvTK-GFP with overlaps to esaboxes.
General
- We repeted all previously failed digestions to exchange resistances on empty backbone plasmids.
- After transformations of plasmids created with GA or site directed mutagenesis, colonies were picked in the evening for overnight culture and miniprep was done the next day in the morning.
WEEK 4 (20.7. – 26.7.)
Test 1A: Construction of pNorV and norR plasmids
Test 1B: Construction of promoters with esaboxes and esaR plasmids
Test 2: AND gate promoter pNorV+esabox
Test 3: Switch based on recombinases
Test 4: AND gate promoter pNorV+LldO
Test 5: switch based on Cpf1 system
Directed evolution
General
WEEK 5 (27.7. – 2.8.)
Test 1A: Construction of pNorV and norR plasmids
Test 1B: Construction of promoters with esaboxes and esaR plasmids
Test 2: AND gate promoter pNorV+esabox
Test 3: Switch based on recombinases
Test 4: AND gate promoter pNorV+LldO
Test 5: switch based on Cpf1 system
Directed evolution
General
AUGUST
WEEK 6 (3.8. – 9.8.)
Test 1A: Construction of pNorV and norR plasmids
Test 1B: Construction of promoters with esaboxes and esaR plasmids
Test 2: AND gate promoter pNorV+esabox
Test 3: Switch based on recombinases
Test 4: AND gate promoter pNorV+LldO
Test 5: switch based on Cpf1 system
Directed evolution
General
WEEK 7 (10.8. – 16.8.)
Test 1A: Construction of pNorV and norR plasmids
Test 1B: Construction of promoters with esaboxes and esaR plasmids
Test 2: AND gate promoter pNorV+esabox
Test 3: Switch based on recombinases
Test 4: AND gate promoter pNorV+LldO
Test 5: switch based on Cpf1 system
Directed evolution
General
WEEK 8 (17.8. – 23.8.)
Test 1A: Construction of pNorV and norR plasmids
Test 1B: Construction of promoters with esaboxes and esaR plasmids
Test 2: AND gate promoter pNorV+esabox
Test 3: Switch based on recombinases
Test 4: AND gate promoter pNorV+LldO
Test 5: switch based on Cpf1 system
Directed evolution
General
WEEK 9 (24.8. – 30.8.)
Test 1A: Construction of pNorV and norR plasmids
Test 1B: Construction of promoters with esaboxes and esaR plasmids
Test 2: AND gate promoter pNorV+esabox
Test 3: Switch based on recombinases
Test 4: AND gate promoter pNorV+LldO
Test 5: switch based on Cpf1 system
Directed evolution
General
SEPTEMBER
WEEK 10 (31.8. – 6.9.)
Test 1A: Construction of pNorV and norR plasmids
Test 1B: Construction of promoters with esaboxes and esaR plasmids
Test 2: AND gate promoter pNorV+esabox
Test 3: Switch based on recombinases
Test 4: AND gate promoter pNorV+LldO
Test 5: switch based on Cpf1 system
Directed evolution
General
WEEK 11 (7.9. – 13.9.)
Test 1A: Construction of pNorV and norR plasmids
Test 1B: Construction of promoters with esaboxes and esaR plasmids
Test 2: AND gate promoter pNorV+esabox
Test 3: Switch based on recombinases
Test 4: AND gate promoter pNorV+LldO
Test 5: switch based on Cpf1 system
Directed evolution
General
WEEK 12 (14.9. – 20.9.)
Test 1A: Construction of pNorV and norR plasmids
Test 1B: Construction of promoters with esaboxes and esaR plasmids
Test 2: AND gate promoter pNorV+esabox
Test 3: Switch based on recombinases
Test 4: AND gate promoter pNorV+LldO
Test 5: switch based on Cpf1 system
Directed evolution
General
WEEK 13 (21.9. – 29.9.)
Test 1A: Construction of pNorV and norR plasmids
Test 1B: Construction of promoters with esaboxes and esaR plasmids
Test 2: AND gate promoter pNorV+esabox
Test 3: Switch based on recombinases
Test 4: AND gate promoter pNorV+LldO
Test 5: switch based on Cpf1 system
Directed evolution
General
OCTOBER
WEEK 14 (30.9. – 6.10.)
Test 1A: Construction of pNorV and norR plasmids
Test 1B: Construction of promoters with esaboxes and esaR plasmids
Test 2: AND gate promoter pNorV+esabox
Test 3: Switch based on recombinases
Test 4: AND gate promoter pNorV+LldO
Test 5: switch based on Cpf1 system
Directed evolution
General
WEEK 15 (7.10. – 13.10.)
Test 1A: Construction of pNorV and norR plasmids
Test 1B: Construction of promoters with esaboxes and esaR plasmids
Test 2: AND gate promoter pNorV+esabox
Test 3: Switch based on recombinases
Test 4: AND gate promoter pNorV+LldO
Test 5: switch based on Cpf1 system
Directed evolution
General
WEEK 16 (14.10. – 19.10.)
Test 1A: Construction of pNorV and norR plasmids
Test 1B: Construction of promoters with esaboxes and esaR plasmids
Test 2: AND gate promoter pNorV+esabox
Test 3: Switch based on recombinases
Test 4: AND gate promoter pNorV+LldO
Test 5: switch based on Cpf1 system
Directed evolution
General
Thanks to the sponsors that supported our project:
