Difference between revisions of "Team:Edinburgh/Notebook/HeroinPurity"

Heroin Purity

Week 1
01/06

Order MorA (morphine dehydrogenase). Sequence from Dr Chris French.


Week 4
23/06

Digest the MorA (from IDT) as well as the LacI expression vector (BBa_K314103) and pSB1C3 (BBa_J04450). For MorA into LacI use XbaI/PstI, for LacI use SpeI/PstI, MorA into pSB1C3 use EcoRI HF/PstI, and pSB1C3 use EcoRI HF/PstI.

PCR purification for MorA. Gel purification for pSB1C3 and LacI.


24/06

Ligate LacI to MorA (XbaI) and pSB1C3 and MorA (EcoRI/PstI), control ligations of backbones only.

Transform 3µL ligation reaction into Dh5α.


25/06

No growth on the control plates. Definite colonies on ligation experiments. Inoculate colonies.


26/06

No growth in culture. Streak 8 colonies from each plate onto new plates to grow at room temperature (18ºC) over the weekend.


Week 5
29/06

No growth on streaked plates.
Digest MorA for LacI (XbaI/PstI) and pSB1C3 (EcoRI HF/PstI) as well as re-digesting LacI (SpeI/PstI) and pSB1C3 (EcoRI HF/PstI).
PCR purify LacI and MorA (both digestions).
Gel purify pSB1C3.

Ligate MorA into LacI and pSB1C3 again for an overnight reaction at 16ºC.


30/06

Transform ligation results.


01/07

Transformations all worked except for MorA in pSB1C3.

Try different restriction sites to see if that makes a difference. Digest MorA with EcoRI HF/SpeI and XbaI/PstI. Digest pSB1C3 with EcoRI HF/SpeI and XbaI/PstI.
Gel purify the pSB1C3 backbones. PCR purify the MorA inserts.

Ligate pSB1C3 (EcoRI HF/SpeI) to MorA (EcoRI HF/SpeI) and pSB1C3 (XbaI/PstI) to MorA (XbaI/PstI).


02/07

Digest NADPH-FMN oxoreductase with EcoRI HF/SpeI and XbaI/PstI and LacI with SpeI/PstI. PCR purify.
Ligate pSB1C3 (EcoRI HF/SpeI) to NADPH-FMN oxoreductase (EcoRI HF/SpeI) and pSB1C3 (XbaI/PstI) to NADPH-FMN oxoreductase (XbaI/PstI). Transform the ligations.

Sequence LacI+MorA1 and LacI+MorA2. Diagnostic digest LacI+MorA1 and LacI+MorA2.


03/07

It appears NADPH-FMN oxoreductase went into LacI.
Try putting MorA and NADPH -FMN oxoreductase into pSB1C3 using the linearized backbone from the distribution kit. Digest, ligate, transform.


04/07

Growth appeared on all of the ligated plates with no growth on the controls.


05/07

Inoculate colonies from the plate.


Week 6
06/07

Miniprep cultures.
Diagnostic digest.

Inoculate LacI+NADPH-FMN and pSB1C3+NADPH-FMN.


07/07

Sent pSB1C3+MorA1, pSB1C3+MorA2, LacI+MorA1 and LacI+MorA2 off for sequencing.

Miniprep cultures.
Just realized NADPH-FMN oxoreductase gblock has PstI site in it so re-order without that site as it is necessary.


08/07

Digest CBDs (5.18A, 5.16O, BBa_K863111 and BBa_K863101) with AgeI/SpeI for N terminal fusions and XbaI/NgoMIV for C terminal fusions. Digest MorA with XbaI/AgeI for C terminal fusions and NgoMIV/SpeI for N terminal fusions.
Treat CBDs with Antarctic Phosphatase.
PCR purify CBDs. (Note: could not gel purify MorA because it was not fully cut, so not much of a band to purify).

Transform LacI+MorA into BL21 for expression.
Digest pSB1C3+MorA overnight for a C (XbaI/AgeI) and N (NgoMIV/SpeI) terminal fusion.


09/07

LacI+MorA seems like it went into BL21.

Gel purify the overnight digest.

Make a batch culture of LacI+MorA in BL21.


10/07

Ligate all of the CBDs to MorA with both N and C terminal fusions.
Transform the ligations.


11/07

Some ligations appeared to have potentially worked. Place the plates in 4ºC. Miniprep and make glycerols of cultures.


12/07

Make a batch culture of LacI+MorA in BL21.


10/07

Ligate all of the CBDs to MorA with both N and C terminal fusions.
Transform the ligations.


11/07

Some ligations appeared to have potentially worked. Place the plates in 4ºC. Miniprep and make glycerols of cultures.


12/07

Inoculate transformants that appeared to have worked.


Week 7
13/07

Make glycerols of cultures and mini prep. Diagnostic digest.

Digest MorA again for both N (NgoMIV/SpeI) and C (XbaI/AgeI) terminal fusions. Digest the four CBDs (BBa_K863111, BBa_K863101, 5.16O, 5.18A) for N (AgeI/SpeI) and C (XbaI/NgoMIV) terminal fusions.

Ligate MorA to CBDs.


14/07

Transform ligations.

Make two batch cultures of LacI+MorA in BL21. Each has 500 µL. Induce with IPTG.


15/07

It appears that all of the transformations worked. Inoculate transformations.


16/07

Inoculations all grew except 5.16O+MorAC (1). Miniprep and make inoculations that grew. Diagnostic digest.


Week 8
20/07

Sequence 5.18A MorA C 2, 5.18A MorA N 3, BBa_K863101 MorA C 1, BBa_K863111 MorA C 2, BBa_K863111 MorA N 2, BBa_K863101 MorA N 3 and 5.16 O MorA N4.

Transform heroin esterase.


21/07

Inoculate heroin esterase.


22/07

Nanodrop heroin esterase 1 and 2.

Try to put heroin esterase into pSB1C3 and into LacI using EcoRI/PstI and XbaI/PstI respectively.

Treat backbones with Antarctic Phosphatase. Ligate.


23/07

Digest heroin esterase (XbaI/PstI) and LacI (SpeI/PstI). Ligate. Transform.


24/07

Nanodrop LacI + heroin esterase 1, 2 and pSB1C3 + heroin esterase 1 and 2.
Diagnostic digest. Diagnostic digest was inconclusive due to bands bleaching out.


26/07

Inoculate everything that did not show up on the diagnostic digest.


Week 9
27/07

Make glycerols of all of the cultures.
Miniprep cultures.
Nanodrop.
Diagnostic digest.


28/07

Fuse heroin esterase to 5.16 O, 5.18 A, BBa_K863101 and BBa_K863111 on both the N and C terminals. The heroin esterase is digested with XbaI/AgeI for C terminal and NgoMIV/SpeI for N terminal. The CBDs are digested with XbaI/NgoMIV for C terminal and AgeI/SpeI for N terminal fusions.
Send of pSB1C3 + heroin esterase 3 for sequencing.
Try to put MorA fusions (CBDs+MorA N and C terminal) in pSB1A3 + LacI using XbaI/PstI for the fusions and SpeI/PstI for the LacI backbone. Treat backbone with Antarctic Phosphatase and then ligate everything together.


29/07

Digest CBD fusions and pSB1C3 + heroin esterase for fusions into LacI.
Inoculate transformants from 28/07.


30/07

Miniprep cultures and nanodrop.
Diagnostic digest. No insert visible on gel.
Inoculate LacI + 5.18 A + MorA C 1 and LacI + 5.18A + MorA C 2.


31/07

Fuse heroin esterase to 5.16 O, 5.18 A, BBa_K863101 and BBa_K863111 on the C terminal. The heroin esterase for C terminal is digested with EcoR1/Age1. The CBDs are digested with EcoR1/NgoMIV.
Digest CBDs + MorA and pSB1C3 + heroin esterase for fusion into LacI. CBD + MorA fusions and pSB1C3 heroin esterase were digested with Xba1/Pst1. LacI was digested with Spe1/Pst1.
Antarctic phosphatase treat the CBDs and LacI.
Ligate with appropriate controls and transform.
Make glycerol stocks, miniprep and nanodrop cultures.
Diagnostic digest. Indicates the presence of insert.


02/08

Inoculate successful transformants from 31/08


Week 10
03/08

Make glycerols, miniprep and nanodrop cultures.
Diagnostic digest.
Send LacI + 5.18A MorA C 1 and LacI + 5.18A MorA C 2 for sequencing.
Transform LacI + MorA into E.coli strain BL21.


04/08

Sequence 5.18A + heroin esterase C 1, 5.18A + heroin esterase C 2, BBa_K863101 + heroin esterase C 2 and BBa_K863111 + heroin esterase C 2.
Inoculate LacI + MorA in BL21.


05/08

Digest heroin esterase gBlock with Xba1/Age1 for fusion to the C terminal of CBD 5.16 O. CBD was digested with Xba1/Spe1.
Antarctic phosphatase CBD, ligate and transform.


07/08

Fuse LacI promoter into MorA + CBD fusions. Digest LacI insert with EcoR1/Spe1. Digest CBD + MorA fusions with EcoR1/Xba1.
No phosphatase treatment.
Ligate and transform.


09/08

Inoculate successful transformants.


Week 11
10/08

Make glycerols, then miniprep and nanodrop cultures.
Diagnostic digest.


11/08

Sequence 5.16O + heroin esterase, LacI + BBa_K863101 + MorA C 2, LacI + BBa_K863111 + MorA C 2, LacI + 5.18A + MorA N 1 and LacI + BBa_K863101 +MorA N 1.


13/08

Fuse heroin esterase and MorA to CBD CipA at both N and C terminals. Digest heroin esterase and MorA with NgoMIV/Pst1 for N terminal fusion and with EcoR1/Age1 for C terminal fusion. Digest CBD CipA with Age1/Pst1 for N terminal fusions and EcoR1/NgoMIV for C terminal fusions.
Fuse LacI promoter into heroin esterase. Digest heroin esterase with EcoR1/Xba1, and digest the LacI insert with EcoR1/Spe1. Phosphatase treat the digestions
Ligate and transform.


14/08

Retry fusing LacI to heroin esterase using the same enzymes. Phosphatase treat, ligate and transform.


16/08

Inoculate successful transformants from 13/08 and 14/08.


Week 12
17/08

Fuse LacI into CBD MorA fusions, namely 5.18A MorA C 2, BBa_K863111 MorA N 2, 5.16O MorA N 4 and 5.16O MorA C 2. Digest LacI with EcoR1/Spe1 and digest the CBD MorA fusions with EcoR1/Xba1.
Antarctic phosphatase, ligate and transform into Top10s.
Make glycerols and miniprep cultures from 16/08.
Nanodrop and diagnostic digest.


18/08

Sequence LacI + heroin esterase, CBD CipA + heroin esterase C and CBD CipA + MorA C.
Retry failed heroin esterase to CBD fusions, namely 5.16O + heroin esterase N, 5.18A + heroin esterase N, BBa_K863101 + heroin esterase N, BBa_K863111 + heroin esterase N and 5.16O + heroin esterase C. Digest CBDs with Age1/Pst1 for N terminal fusions and EcoR1/NgoMIV for C terminal fusions. Digest heroin esterase with NgoMIV/Pst1 for N terminal fusions and with EcoR1/Age1 for C terminal fusions. Phosphatase treat, ligate and transform into Top10s.
Amplify heroin esterase by PCR, PCR purify and nanodrop. Diagnostic digest showed PCR amplified the correct sequence.
Inoculate successful transformants from 17/08


19/08

Make glycerols, miniprep and nanodrop cultures.Diagnostic Digest indicated the presence of insert in all but BBa_K863111 fusions.
Amplify MorA by PCR, PCR purify and nanodrop. Diagnostic digest showed PCR amplified the correct sequence.
Inoculate successful transformants.


20/08

Sequence LacI + 5.16O + MorA N 1, LacI + 5.16O + MorA N 2, LacI + 5.16O + MorA C 1, LacI + 5.16O + MorA C 2, LacI + 5.18A + MorA C 1 and LacI + 5.18A + MorA C 2.
Fuse LacI promoter into MorA and heroin esterase CBD fusions. Digest LacI with EcoR1/Spe1. Digest MorA and heroin esterase with EcoR1/Xba1. Phosphatase treat, ligate and transform into Top10s.
Make glycerols, miniprep and nanodrop cultures. Diagnostic digest indicated no insert was present.


21/08

Retry fusion of heroin esterase and MorA to CBDs. Digest CBDs with Age1/Pst1 for N terminal fusions and with EcoR1/NgoMIV for C terminal fusions. Digest inserts with NgoMIV/Pst1 for N terminal fusions and with EcoR1/Age1 for C terminal fusions. Phosphatase treat, ligate and transform into Top10s.
Transform LacI + 5.18A + MorA N1, LacI + BBa_K863101 + MorA N1, LacI + BBa_K863101 MorA C2, LacI + BBa_K863111 MorA C2 and LacI + heroin esterase into BL21s.


3/08

Inoculate successful transformants from 20/08 and 21/08.
Make starter cultures for BL21 transformants.


Week 13
24/08

Retry failed fusions for heroin esterase to CBDs, and MorA to CBDCipA. Digest CBDs with Age1/Pst1 for N terminal fusions and with EcoR1/NgoMIV for C terminal fusions. Digest inserts with NgoMIV/Pst1 for N terminal fusions and with EcoR1/Age1 for C terminal fusions. Treat with antarctic phosphatase for 1 hour. Ligate and transform into Top10s.
Transform LacI + 5.16O + MorA N1, LacI + 5.16O + MorA C2 and LacI + 5.18A + MorA C1 into BL21s.
Make glycerols and miniprep transformants from 20/08. Nanodrop and diagnostic digest. Digest indicated the presence of inserts in some of the samples.


25/08

Sequence LacI + 5.18A + heroin esterase C1, LacI + BBa_K863101 + heroin esterase C1 and LacI + BBa_K863111 + heroin esterase C1.
Fuse LacI into CBDCipA + MorA C2 and BBa_K863111 + MorA N2. Digest LacI with EcoR1/Spe1, and digest the backbone with EcoR1/Xba1. Phosphatase treat and ligate. Transform into Top10s.