Used regular expressions to find the concensus sequence of A8 and A9 motifs in the adenylation domain

To read: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2773127/

Explorations of catalytic domains in non-ribosomal peptide synthetase enzymology - Read Up Fam

This will start when the DNA from IDT arrives.

Purify with nickle affinity chromatography

Use the nanodrop on the pET-16b vector received from the Allingham lab. the vector had a concentration of 34.7 ng/ul. Due to the low concentration it was recommended we transform and propagate the vector.

The pET-16b plasmid was used to transform a culture of XL1 Blue cells. 5 ul of DNA was used for the transformation. Plates were made with Amp resistance.

1/10 and 1/100 dilutions were prepared.

Two colonies were found on the 1/10 dilution plate.

Another transformation was attempted with DH10B cells using the pET-16b vector. 5 ul of pET-16b vector was used and the transformation was plated on AmpR plates.

An overnight culture with the two colonies found on the 1/10 dilution plate was started. 4 ul of Amp was added to the culture tube.

The over night cultures were mini prepped using a Nucleospin Miniprep Kit. Multiple colonies on transformation plate, none on control.

The DNA concentration of each mini prep was found to be (1) 126.45 ng/ul and (2) 76.5 ng/ul

A 0.8% gel was ran using the two miniprep samples after they were digested with EcoRV. The gel was streaky including the ladder, but two bands were visible in accordance to a EcoRV digest. Possible troubleshooting: properly heat inactivate the enzyme. Pour deeper gel wells since they felt shallow when pipetting.

An over night culture was prepared using two colonies from the pET-16b DH10B cells plate. 4 ul of Amp was added to the LB of the ON.

Mini prepped the two over night cultures. DNA was clumpy instead of wispy for one of the mini prep tubes. Resulted in low concentration of DNA and impure yield.

pET-16b_DH10B #1 : 93.85 ng/ul of DNA. pET-16b_DH10B #2 : 39.60 ng/ul of DNA.

EcoRV digest of pET-16b_XL1B #1, pET-16b_XL1B #2, pET-16b_DH10B #1. Ran a 1% gel.

Insert Gel Here. Two bands present around 4.2 and 1.5 kb which corresponds to pET-16b vector cuts. Air bubble in gel may have caused lane shit upwards.

The next step since pET-16b was confirmed is to amplify the oxidaiton domain ordered from IDT with the set of Gibson primers and assemble it in the pET-16b vector using Gibson assembly.

PCR Amplification of Codon Optimized Oxidation Domain (CO Ox Domain) with DDGibsFwd and DDGibsRev2. 50 ul PCR reaction was prepared.

Insert Gel

The PCR product was cleaned up using a NucleoSpin PCR cleanup Kit (Insert Protocols)

The pET-16b Vetor was linearized using a double digest. It was digested using NdeI and BamHI. 5 ul of vector was used in order to meet the requirements of Gibson assembly after dilution by the digestion.

Break Civic Holiday

2ul of the Gibson Assembly product was used to transform DH5α NEB Cells.

Mixed up the control and the colony plates. Good colony growth was present on the plate.

8 colonies were picked and over night cultures were prepared. 4ml of LB was poured in each tube and 4ul of Amp was used.

The ON cultures were cloudy in the morning signifying bacterial growth. All of the cultures were mini prepped.

The mini prep was digested with EcoRV in order to verify the insert of the BpsA oxidation domain. The domain contains a EcoRV site so 3 bands were expected on the gel compared to the 2 seen for the regular pET-16b vector.

Insert EcoRV digest gel.

No protein expression was induced, new parts were designed on Benchling and ordered through IDT.

BL21 (DE3) cells were transformed using the mini prep product from miniprep #2 . a 1/10 dilution was also done

3 over night cultures were prepared using colonies form the 1/10 diluted transformation plate. Each on had a volume of 3.5 ml and 3.5 ul of Amp was added

The ON cultures were mixed and 0.5 ml was used to inoculate 25ml of LB in a 125ml flask.

The OD was checked periodically and 0.5 mM and 0.2 mM of IPTG were used to induce protein production when the cultures were at an OD600 level of 0.8. The cultures were left at 37 oC and 30oC over night (12 h)

The cultures were sonnicated and protein levels were examined using an SDS-PAGE Gel.

The Gel was left to destain over night

No significant band was present at 52 KDa which would have been the mass of the oxidation domain and linker.

Future Steps:

Use a higher level of IPTG for induction 1mM - 1.5 mM in order to stimulate protein production

Take colony samples at 2, 4, and 6 hours after IPTG induction.

Large band was present around 30 KDa. Using Phyre2 the conserved oxidation domain region was constructed through homology modeling. The oxidation domain itself would be 34.2 KDa. There might be some form of protease activity.

Test will be re run again but using a larger 250/500 ml flask.

The purpose of creating a PadE like module with an interrupted adenylation domain containing the Oxidation domain from BpsA is to demonstrate that the nature readily uses the hypervariable loop between A8 and A9 motifs in the adenylation domain as a place for inserting tailoring domains.

The module will be fused with the oxidation domain through Gibson assembly and expressed in BAP1 cells

Prepared 3 ON cultures using BL21 cells . Use 100 mg/ml of Amp for the resistance .

added 1 ml of ON culture to 50ml of LB with 100mg/ml Ampicillin. Cells contained pET-16b vector with oxdation domain gene

OD600 of 0.704 was reached in 1h 25 min and 1 mM of IPTG was ued to induce protein expression. flasks were swirled after IPTG addition and incubated at 30C

10ml sample of uninduced bacteria was taken. The sample was centrifuged at 4000 rpm for 15 min and the supernatent was discarded. Pellet was stored at -80 C

10ml samples were taken 2h, 4h, 6h after induction The samples were centrifuged at 4000 rpm for 15 min and the supernatent was discarded. Pellets were stored at -80C

ON sample was taken and centrifuged at 4000 rpm for 15 min and the supernatent was discarded. Pellet was stored at -80 C

Cells were resuspended in 1ml of lysis buffer and sonnicated at amplitude 4 for 2 min using 20 second pulses or until the samples became a a clear opal colour. (all samples)

Total cell lysate samples were taken. 50ul samples of the TCL were spun down and the supernatent was collected. The pellet was re suspended in 50ul of dH2O.

All samples were denatured along with SDS loading dye

The denatured samples were ran on a SDS-PAGE and imaged.

Insert GEL HERE !

a small fraction of the protein was visible in the supernatant while the majority of the protein was in icclusion bodies and insoluble.

Protein in the supernatant peaked at the 4h mark. None of it was present in the supernatent in the ON culture.

Linor prepared a ON culture using the BL21 Cells containing the pET-16b oxidation domain construct.

2 5ml ON tubes contained LB and 100mg/ml of Amp

The 2 5ml ON samples were mixed and 1 ml was used to innoculate 50 ml of LB in a 250 ml Erlenmeyer flask.

OD 600 = 0.65 was reached 2h after the initial inoculation. The cells were induced with 1mM IPTG and grown at 30C for 4h.

50 ml of Cell samples were spun down and frozen at -80C

Small Scale Protein Purification

1 ml of lysis buffer was added to the cell pellet

the pellet was resuspended and sonnicated for 1 min using 10 seocnd intervals until the lysate was clear

Protease inhibitor was added to the cell lysate 400 ul

the cell lysate was centrifuged at 14.5k for 10 min

1000ul of Nm was taken from the product

the pellet was resuspended in 1 ml of lysis buffer

Ni resin was prepared: 150 ul of the resin slurry was added to a spin column. It was centrifuged at 13.3k for 1 min

it was washed with 10 volumes of dH2O

The SN was added to the column and rocked for 30 min @ 4C

The column was washed once with lysis buffer

it was washed 4 times with 500 ul of elution buffer

the column was eluted with 100ul of elution buffer

10ul samples of each were taken and mixed with 10 ul of SDS loading dye.

the samples were denatured at 97 C for 10 min

A 12% SDS-PAGE was ran using the columns. 90V for 15 min stack and then 120v until the elution line reached the bottom of the gel

INSERT GEL PIC HERE

Goal: Create and express the thioesterase domain of BpsA in order to determine if it is responsible for the cyclization of glutamine SNACs

Goal: Create and express the thioesterase domain of BpsA in order to determine if it is responsible for the cyclization of glutamine SNACs

Rehydrate primers as well as BpsA domain. Linearized pET16b vector has a concentration of 23.46 ng/ul after digest

To rehydrate primers add 10x ul of H2O where x is primer amount in uM. Concentration is now 100 uM, Make a 1/10 dilution in order to get a concentration of 10 uM.

Rehydrate gene in 100ul of H20. Prepare a 1/10 dilution and use 1ul for PCR.

PCR Amplification of TE domain with gibson primers

Prepare a Gel, run the product on a gel and perform a gel extraction

Nanodrop PCR product to determine the concentration

Prepare Gibson using pET16b backbone vector.

Transform into DH5a cells

Rehydrate primers as well as BpsA domain. Linearized pET16b vector has a concentration of 23.46 ng/ul after digest

To rehydrate primers add 10x ul of H2O where x is primer amount in uM. Concentration is now 100 uM, Make a 1/10 dilution in order to get a concentration of 10 uM.

Rehydrate gene in 100ul of H20. Prepare a 1/10 dilution and use 1ul for PCR.

PCR Amplification of TE domain with gibson primers

Prepare a Gel, run the product on a gel and perform a gel extraction

Nanodrop PCR product to determine the concentration

Prepare Gibson using pET16b backbone vector.

Transform into DH5a cells

Large amount of colonies observed. Run ON at the end of the day

Prepared 5 ON cultures, 5ml of LB with 5ul of 100mg/ul of Amp

Large amount of colonies observed. Run ON at the end of the day

Prepared 5 ON cultures, 5ml of LB with 5ul of 100mg/ul of Amp

Prepared an ON culture with 50 ml of LB broth in a 250ml flask. Added 50ul of 100mg/ul of Amp to a total concentration of 100 ug/ul

A pipette tip was used to pick a BL21 colony containing the pET-16b and oxidation domain construct

Mini Prepped the ON cultures using Quiagen Miniprep Kit

Checked the concentration of all 5 samples : 1. 2. 3. 4. 5.

Digested the peET -16b BpsA TE DRE assembled vector with EcoRV. Expected to see 3 bands based off of virtual digest.

Ran 20ul of the digested product on a 1% Agarose Gel. Mixed

Gel Pic

Mini Prepped the ON cultures using Quiagen Miniprep Kit

Checked the concentration of all 5 samples : 1. 2. 3. 4. 5.

Digested the peET -16b BpsA TE DRE assembled vector with EcoRV. Expected to see 3 bands based off of virtual digest.

Ran 20ul of the digested product on a 1% Agarose Gel. Mixed

Gel Pic

Protein purification with 1L of LB.

measured out 1L of water and 20g of LB into large flask. Mixed with magnetic stir bar and autoclaved.

Inoculated with 10 ml of ON culture and 500ul of Amp in a sterile environemnet

Incubated until OD reached 0.7

Induced expression with 1 mM of IPTG

Incubated at 20C over ngiht

Used peET -16b BpsA TE DRE Sample # 3 in order to transform BL21 (DE3 ) Cells

Used peET -16b BpsA TE DRE Sample # 3 in order to transform BL21 (DE3 ) Cells

Spun down 1L of culture with large centrifuge 4000 RPM for 30 min

Re suspended in a small amount of supernatent and discarded the rest

Added 30 mL of Lysis Buffer w/ Lysozyme

ADD BUFFER RECIPE

incubated on ice for 30 min

Sonicated for 50 seconds 2 min rest. Amplitude 7 for 5 rounds

Spun down cell lysis for 16k RPM @ 4C for 35 min

Filled an equilibrated nickle column with Ni resin. Filled with the supernatent from centrifugation,

Rocked back and forth for 1h at $C

Washed 4 times with wash Buffer

Eluted with 50,100,...,30 mM of imidazole

Elutions 2 and 3 were yellow possibly flavin co-purifying with the oxidation domain

Elution 2 and 3 placed in a dialysis tube. SDS-PAGE showed the majority of the protein was in fraction 4 and 5.

SDS-PAGE HERE

Used half plating method there were small colonies on the control side.

Redoing the transformation next week

Used half plating method there were small colonies on the control side.

Redoing the transformation next week

Concentrated fractions 4,5, and 6.

Fractions were placed in dialysis for 6 hours with a buffer exchange at 3 hours

Protein concentrated, total concentration of 5.7 mg/mL

Colony growth on the plate was adeuate, No colonies present on the control plate

Colony growth on the plate was adeuate, No colonies present on the con

Beginning of enzymatic assay

We believe that the oxidation domain is responsible for oxidizing the cyclized glutamine, or pyroglutamine

Pyro-Gln was ordered from Astatech. It was resuspended in dH₂O. It had a blue colour when suspended in solution. Auto oxidation of Pyro-Gln might occur creating indigoidine.

5 mg was suspended in deuterated ethanol. The compound was insoluble in DMSO.

H NMR was performed in order to verify compound.

INSERT NMR HERE

Protein Assay

Pyro-Gln has a peak of absorption at 595nm due to the blue colour. Indigoidine also has the same OD in H₂O.

Protein in phosphate buffer was used as a reference

800 ul Protein and 200 ul of Pyro-Gln was the reaction

Pyro-Gln with phosphate buffer used as a secondary reaction

incubated at 30C checked back every 20 min,

No apparent change in OD