| Dates | Results | Description & Notes | Figures |
|---|---|---|---|
| 7/7 | • Lysogeny broth (LB) made • Agar Plates made |
• Preparations for future experiments | N/A |
| 7/12 | • Concentration test | • Test to confirm whether old constructs can be used | N/A |
| 7/15 | • Transformed DH5a with PSB1C3 containing plasmid | • Diluted pSB1C3-pSTE12-18, pSB1C3-RFP, and pSB1C3-CmR according to the concentration tested on 7/12 for 1ng/ul transformation [1-1] • Transformed and plate with DH5𝛼 (for plating we grow bacteria overnight 12~16 hrs) [1-2~1-5] |
7/15 |
| 7/16 | • Colonies grown in 7/15 were moved into LB | • SB1C3-CmR didn’t grow • Selected colonies for the other two and grew them in liquid LB (30℃) |
7/16 |
| 7/17 | • Verified that pSB1C3 was contained | • Did digestion tests with different restriction sites to verify and extracted pSB1C3 (used RFP construct) [2] • Extracted backbone (pSB1C3) and measured its concentration [1] |
7/17 |
| 7/29 | • First cloning cycle | • Resuspended ordered constructs (ccdA, ccdB, mleR-first half, chitinase-first half) • Digested backbone on E-S restriction sites and ligated with each of our constructs individually • Transformed and plate the ligated products into DH5𝛼, wait for colonies to grow overnight [1~4] |
7/29 |
| 8/3 | • Verification (first cloning cycle failed) | • Did colony PCR with primers • All the bands are way too largely |
8/3 |
| 8/6 | • Second try for cloning cycle • Redo cloning cycles for ccdA and ccdB |
Improvements and discussion made • 1ul Digestion Enzyme • Verification of backbone and RFP band • Reminder: heat shock before transformation • Digested vector (pSB1C3-RFP) and extracted backbone • Re-attempt cloning cycle (same steps as 7/29) • Refilled LB |
N/A |
| 8/7 | • Verified pSB1C3+chitinase & pSB1C3+mleR (failed) • Redo cloning cycle for all constructs (third re-try) |
• Colony PCR results didn’t look good [1] • Re-attempt cloning cycle • Digested and extracted vector and fragments [2] • Overnight ligation |
8/7 |
| 8/8 | • Grow DH5𝛼 with our ligated products | • Transformed and plated the four ligated constructs with DH5𝛼 (thanks for Bryan’s assistance) | 8/8 |
| 8/12 | • Verifying for ligated construct (failed) | *50x TAE buffer was accidently added • decided to redo Colony PCR the next day (lost a few samples while loading) |
N/A |
| 8/13 | • Chitinase-1st half success | • Redo Colony PCR • One chitinase colony had the correct band • Unfortunately other constructs where wrong |
8/13 |
| 8/14 | • Success colony grown in LB | • Moved successfully ligated chitinase colony into LB • Grew 4 tubes of chitinase 1st half and one tube of vector with DH5𝛼 |
N/A |
| 8/15 | • Vector and Chitinase first-half extracted | • Miniprep pSB1C3-RFP & pSB1C3-Chitinase first-half (colonies 1~4) | 8/15 |
| 8/18 | • Vector digested | • Prepare backbone for future cloning cycle [2] • Gel was cutted and preserved in -20℃ fridge • concentration was tested [1] |
8/18 |
| 8/19 | synthesize designed chitinase full construct | 3rd round cloning cycle • Chitinase 2nd-half resuspension and digestion • Gel electrophoresis & extraction • Ligate Chitinase 2nd-half with pSB1c3-Chitinase 1st-half • Transform and plate chitinase-full construct |
N/A |
| 8/20 | Verify chitinase full construct (failed) | Colony PCR, bands didn’t match | 8/20 |
| 8/21 | • Verify chitinase full construct (succeed) • 3 plates of DH5𝛼, each contains one construct: pSB1C3-ccdA, pSB1C3-ccdB, pSB1C3-mleR-1st half | • Tried Colony PCR with the same plate again, but chose 1x more colonies this time to increase chance of obtaining our construct [1] 4th round colony cycle: • Redo cloning cycle for the other constructs: psb1c3-ccdA, psb1c3-ccdB, psb1c3-mleR-1st half • Digest fragments and vectors [2]-> concentration test TE buffer misused for vector • Did second vector digestion [3]-> gel extraction ligate backbone with fragments transform and plate with DH5𝛼 |
8/21 |
| 8/26 | • Same as 8/21 | • colony PCR-> gel electrophoresis [1] * Retransformation and plate previous ligation products but with recovering [2] |
8/26 |
| 8/27 | • Cloning Cycle (Success) • Grew bacterias that contain our constructs in LB |
• Colony PCR— ALL BANDS CORRECT • Grow correct colonies into LB |
8/27 |
| 8/28 | • Constructs finally collected: • pSB1C3-ccdA • pSB1C3-ccdB • pSB1C3-mleR-1st half |
• Miniprep and concentration test | N/A |
| 8/29 | • Cloning cycle (success) for mleR-2nd half+pSB1C3-mleR 1st half (got the full mleR construct | 5th round Cloning cycle (for mleR full construct) • Vector and fragment digestion [1] • Run gel-electrophoresis and extracted backbone • Leaved ligation overnight For psB1C3-ccdA-His/ccdB-Myc -tag addition • Q5 mutagenesis (PCR) • Run gel for verification [2] • KLD reaction-> transformed and plated with DH5𝛼 |
8/29 |
| 8/30 | • DH5𝛼 grown with (supposedly) mleR-full construct • Starting for chitinase expression • Grow colonies that contain tag-added constructs |
For mleR-full construct: • transform and plate yesterday’s ligation product For Chitinase protein production: • transformed and plated chitinase-full plasmid into BL21 |
N/A |
| 9/1 | • Verification on the mleR full construct (success) • Verification on whether if chitinase is successfully transformed (failed) • Verification on tag-added constructs (success) |
For mleR full construct • Colony PCR bacteria grown yesterday [1] • run gel electrophoresis [2] • Grow colony that has been successfully transformed into LB For Chitinase protein production: • Colony PCR For psB1C3-ccdA-His/ccdB-Myc -tag addition • Colony PCR Gel electrophoresis and grow correctly transformed colonies into LB |
9/1 |
| 9/2 | • Successfully collected mleR-full construct • Verification on whether if chitinase is successfully transformed (success) |
For mleR full construct • Miniprep For chitinase protein production: • Retry Colony PCR with same plate • Grow successfully transformed colony into LB |
N/A |
| 9/3 | • Expressed and collected chitinase (waiting for verification) | *Did concentration test for yesterday’s mleR miniprep For chitinase protein production: • Move small amount of colonies into starter culture • OD measurement (check colony concentration if reached log phase)-> protein expression and extraction/collection • Start Chitinase protein production • 19:20 begin induction, OD 0.354 25C, 250rpm => sample collection (0h, 1h, 2h, 3h) • 20:20 begin induction 2, OD 0.348 => sample collection (0h, 1h, 2h, 3h) |
9/4 |
| 9/4 | • Grow bacteria containing CcdB and mleR (individually) |
For protein expression CcdB & mleR (two are separated) • Transform and plate CcdB & mleR (BL21) [2] |
9/4 |
| 9/5 | • Collected tag added constructs (success) |
For psB1C3-ccdA-His/ccdB-Myc -tag addition • Miniprep and test concentration with spectrophotometer |
9/5 |
| 9/7 | • CcdA-6xHis E-S digestion quantification • Got bacteria transformed with pSB1C3-mleR-ccdA-His |
For psB1C3-ccdA-His/ccdB-Myc -tag construct building
Cloning Cycle (6th) • Digested pSB1C3-mleR-full construct gel electrophoresis-> extraction-> Miniprep-> concentration test • Ligate mleR-full with CcdA-6xHis Transform and plate |
N/A |
| 9/8 | • Transformed separated and combined constructs • Started starter culture-> ready for protein expression |
For ccdA, ccdB, mleR and other combined constructs • Cloning cycle & CPCR & plating: CcdA-mleR, Ccda-6xHis, CcdB-Myc tag • Starter LB mleR, CcdB BL21 |
N/A |
To view the specific procedures for our experiments, please download the file attached below.