Philip, Julien

gBlocks PCR amplification: IDT-OMPHT,NSP4-tyrosinase,Mefp-5

PCR reaction: Prepare reaction mix on ice. Primers for IDT-OMPHT are 100-F/R, for NSP4 tyrosinase are 113/114-F/R, for Mefp-5 are 105-F/R. Concentrations: IDT-OMPHT is 11.0 ng/μl, NSP4-Tyrosinase is 16.7 ng/μl, Mefp-5 is 18.2 ng/μl.

PCR setting: Tm for IDT-OMPHT is 68°C, for NSP4 tyrosinase is 62°C, for Mefp-5 is 65°C.

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052019 gBlocks PCR fragments checking via gel running

Repeat gBlocks PCR amplification: IDT-OMPHT,NSP4 tyrosinase,Mefp-5

052191 gBlocks PCR fragments checking via gel running

Gel running: Make two different size gels. The small one is 25ml TAE with 0.25g agarose and 1.25ml SYBR safe dye. This gel is for checking the 052019 PCR fragments. The big one is 50ml TAE with 0.5g agarose and 2.5ml SYBR safe dye. This gel is for checking the 052119 PCR fragments. Mix 5μl PCR product and 1μl 6X loading dye. Run at 130V for 30 min and then check the image.

PCR reaction and setting: The reaction mix is the same as 052019. Only difference we made is that we changed the Tm into three sets in order to figure out whether Tm led to the incorrect bands for image052019. Tm for Set A is 65°C, Tm for set B is 57°C, and Tm for Set C is 53°C.

After rechecking, we found that we mistakenly ordered the primers for NSP4 tyrosinase. Actually the fragment we ordered has already contained the homologous regions that we wanted.

Restriction digestion of plasmid vectors

Recheck gBlocks NSP4 tyrosinase

Digestion reaction:

Incubate at 37°C overnight

Gel running: Run the PCR products of IDT-OMPHT and Mefp-5, and directly run the gBlocks NSP4 tyrosinase at 130V for 30 min and then check the image.

We got the correct band for NSP4 tyrosinase even though it was very weak. We would try to amplify after ligation.

Tyrosinase secretion system construction

Gel running: 50 μl digested sample + 10 μl loading dye. The order of loading is shown below. Then separate 60μl into two wells on the gel because the well will be overflowing if we put all 60 μl into one well.

Run at 120V for 23 min in order to separate the digestion products and obtain the target vector (pBAD24, 4516 bp) from unused fragments (26 bp).

Gel purification: We’re using protocol from Illustra GFX PCR DNA and Gel Band Purification Kit Gel. Gel slices weight are 258 mg (used 258 μl CBT3) and 280 mg (used 280 μl CBT3)*CBT3 = capture buffer type 3

Sample capture → sample binding → wash & dry → elution 

We used 1 Microspin column and 1 collection tube for purification of 2 gels. Then Elution with 20μl elution buffer type 6 

Gibson Assembly reagents calculation:

Volume used (Vector : Insert = 1 : 4)

Gibson assembly reaction:

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Tyrosinase secretion system construction

Gibson assembly reaction as negative control for 052419:

Incubated at 50°C for 15 minutes.

Transformation of NSP4-Tyrosinase-pBAD24 into competent cells:

a. 50 μl competent cells + 5 μl Gibson assembly mix made on 052419

b. 50 μl competent cells + 5 μl negative control made on 052519

c. Spread on two plates with Ampicillin and incubate at 37°C overnight

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Tyrosinase secretion system construction

Colony picking and incubation:

Prepare the LB Broth with Ampicillin antibiotic and tubes with 1 mL – 5 mL LB Broth with Ampicillin antibiotic. 

Pick the only one single colony from the pBAF24-NSP4 tyrosinase transformation plate.

And randomly pick 5 colonies from the negative control transformation plate. Incubate at 37°C overnight.

The negative control has much more colonies than our pBAD24-NSP4 tyrosinase. So we guessed we might mistakenly record the two plates. The results would show us whether our assumption was true after the plasmid extraction and gel running.

Tyrosinase secretion system construction

Plasmid extraction:

a. Collect 3 mL of overnight suspension culture from the five negative control tubes.

b. Elute with 50μl elution buffer. Did the elution steps three times with the same 50μl elution buffer. Incubate 4min between each centrifuge.

c. Then use Nanodrop to test the extracted plasmid concentration. 

Restriction enzyme digestion reaction: Total 50μl

Incubate at 37°C overnight

DNA plasmid extraction: They are 12.81ng/μl, 18.38ng/μl, 31.41ng/μl, 27.2ng/μl and 28.44ng/μl.

Tyrosinase secretion system construction

Plasmid identity checking for 052619: Run the digested plasmid from five randomly picked colonies on 052619. Loading order for the gel: Left to right 1 kb ladder -> Colony 1 -> Colony 2 -> Colony 3 -> Colony 4 -> Colony 5

Repeat transformation of bacteria: 5 μl Gibson assembly mix made on 052419 and 5 μl negative control made on 052519. Spread on two plates with Ampicillin and incubate at 37°C overnight.

Besides the result of colony 3 that we don’t know how to explain it. Its band is neither the vector nor the NSP4 tyrosinase which was 1022 bp, but this band is around 1500 bp. All the other bands are our vectors which is pBAD24. However, they do not have the insert so they are not the proper plasmid.

Tyrosinase secretion system construction

Colony picking and incubation

Pick the two colonies from the pBAF24-NSP4 tyrosinase transformation plate.

And pick the 3 colonies from the negative control transformation plate. Incubate at 37°C overnight.

Tyrosinase secretion system construction

Plasmid extraction:

a. Collect 3 ml of overnight suspension culture from the two pBAD24-NSP4 tyrosinase tubes.

b. Elute with 50μl elution buffer. Did the elution steps three times with the same 50μl elution buffer. Incubate 4min between each centrifuge.

c. Then use Nanodrop to test the extracted plasmid concentration. They are 46ng/μl and 28ng/μl.

Restriction enzyme digestion reaction: Total 25μl

Incubate at 37°C overnight

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Tyrosinase secretion system construction

Plasmid identity checking for 053019: Run the digested plasmid from two picked colonies from pBAD24-NSP4 tyrosinase plate on 053019 at 120V for 25 min. Loading order is 1 kb ladder, sample1, sample2, 1 kb ladder. Load 25μl digestion product with 5μl loading dye.

No correct colonies. Through these two transformation experiments, we thought our competent cells have some problem. So we would change to use a new one.

Vector identities checking via gel running

Expected band size for each plasmid backbone:

pBBR1MCS2 = 5148 bp and pBAD24 = 4542 bp

I: First gel running around 10 am by Hugh

Samples loaded :

10μl pBBR1MCS-2 + 2μl loading dye (x6)

10μl pBAD24 + 2μl loading dye (x6)

Ran for 120 V for 25 minutes. Initial observation was that the samples were floating and not settling down even after the addition of the loading dye. Hugh did not run this gel and prepared a new one instead because the TA said maybe the gel preparation is not good.

II: Second gel running around 11 am by Hugh

Samples loaded:

10μl pBBR1MCS-2 + 2μl loading dye (x6)

10μl pBAD24 + 2μl loading dye (x6)

Ran for 120 V for 25 minutes. Gel image is figure 1.

III: Third gel running around 5 pm by Philip

Samples loaded:

24μl pBBR1MCS-2 + 20μl loading dye (x6)

24μl pBAD24 + 20μl loading dye (x6)

Ran for 120 V for 25 minutes. Initial observation: Even after mixing the large volume of loading dye. When loading the sample it was still floating away. We now suspected that there might be something wrong with the sample. We think the sample contains ethanol. Gel image is figure 2.

IV: Fourth gel running around 6 pm by Philip

Samples loaded:

10μl pBBR1MCS-2 + 2μl loading dye (x6)

10μl pBAD24 + 2μl loading dye (x6)

Ran for 120 V for 25 minutes. Initial observation: Same thing as the previous gel

No correct band at all, I think it’s due to the loading dye making the fragments too heavy and thus it did not run fast in the gel, therefore our Xiao Xiong (One of our TAs)  recommended to prepare a new gel and use a smaller well size. After this result, Xiao Xiong has put the stock plasmid backbone tubes onto a heating block; she opened the cap of the tubes and heated the tubes at 55 C, I reclaimed the stuff after maybe 30 minutes; this was to evaporate possible ethanol contaminants.

Sample might have floated away and none were actually run in the gel.

No correct band at all, I think it’s due to the loading dye making the fragments too heavy and thus it did not run fast in the gel, therefore our Xiao Xiong (One of our TAs) recommended to prepare a new gel and use a smaller well size. After this result, Xiao Xiong has put the stock plasmid backbone tubes onto a heating block; she opened the cap of the tubes and heated the tubes at 55 C, I reclaimed the stuff after maybe 30 minutes; this was to evaporate possible ethanol contaminants.

It seems that the band is still not at the correct place. There is not even a trace of the correct band for pBAD24 it seems like the band is located more than 10kb. For pBBR1MCS-2, it seems like the DNA sample all floated away and what we saw that settled were probably just the loading dye. Next time we should mix the loading dye and the DNA sample better.

Maybe there is something wrong with the plasmid? After consultation with Professor Zheng Jun’s PhD. Student, apparently we should first digest the plasmid vector with at least one enzyme to linearize it. Only then should we run it in the gel and check the size. Apparently if we do not linearize the plasmid before running, due to its circular form it would run slower along the gel causing it to show a non-true band.

Repeat vector identities checking via gel running

Restriction enzyme digestion reaction for vectors: Total 10μl A1 is pBBR1MCS-2 041719, A2 is pBBR1MCS-2 050519, B1 is pBAD24 041719, B2 is pBAD24 050519.

Incubate at 37℃ for 1h. Gel running: 120V for 25 min. 10μl digestion product + 2μl loading dye. Gel arrangement is 1kb ladder, A1, B1, B2, A2, 1 kb ladder. Restriction enzyme digestion reaction for insert: Total 50μl. Incubate overnight.

After digestion with EcoRI-HF for 1 hr at 37 C. We ran the plasmid stock that has kept (041719) along with the stock plasmid solution that has prepared (050519). The 041719 are at the expected size: pBBR1MCS2 = 5148 bp and pBAD24 = 4542 bp.

Vector and insert checking via gel running

Sample:55μl digested insert + 10μl loading dye

Order: 1 kb ladder(μl), sample(30μl), sample(30μl), 1 kb ladder(2μl)

Run at 120V for 30 min.

Our expected size is 4515bp for vector pBAD24 and 1004bp for NSP4+tyrosinase

The left one is for vector pBAD24 and the other one is for insert NSP4+tyrosinase. The right one was the supposedly digested insert but we did not see any band there.

pBAD24, pBBR1MCS2 plasmid extraction for sequencing

Pellet 4 ml of the 5 ml overnight culture. Pipette 1 ml into microcentrifuge tube and centrifuge for 3 min. Discard supernatant and add next 1 ml into same tube. Repeat until you’ve pelleted 4 ml worth of bacteria culture. (12 min of centrifugation)

Follow protocol in QIAGEN (miniprep) plasmid extraction kit.

 *Miniprep is for 5 ml bacteria culture, yield is low because starting vol. is low. 

    Mediprep is for 50 ml, maxi for 100, and giga for 1L. 

P1 is resuspension buffer. 

P2 is lysis buffer. (Alkaline, with many salts)

N3 is neutralization buffer. (Balance alkalinity)

You’d see some white precipitate after adding N3. The centrifuge step in protocol is to spin down most of that ppt. for you to collect supernatant. This prevents clogging (blocking) of the spin column for downstream procedure. 

Nanodrop at Prof. Xu’s Lab. Clean with water and blank, then measure. 

pBAD24: 66.4ng/μl (acceptable)

pBBR1MCS2: 37.6ng/μl (still pretty low)

Afternoon: Philip sent them for plasmid sequencing. 

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Resuspension of gBlock fragments

Centrifuged the tube at 4500 x g to make sure the dried pellet is at the bottom of the tube. Added 100μl TE buffer to all of the fragments (I was expecting to get 10ng/μl). All products that were delivered were 1000 ng. Vortexed briefly. Incubated at 50 C for 20 minutes. Vortexed then centrifuged. Got the concentration using Nanodrop, we used TE buffer as blank. After the last step, I aliquoted 50μl to another tube so that we would avoid exposing the main stock to many freeze/thaw cycles which could damage the DNA too. So there is about 50μl fragment volume left on the main IDT tubes that we have received.

Resulting concentration:

 Pmol calculations through https://nebiocalculator.neb.com/#!/dsdnaamt.

Restriction digestion of pBBR1MCS2

Restriction digestion reaction: (pBBR1MCS2 concentration:75.5ng/μl)

Put in thermocycler for 1h. Settings are as following: 37℃ for 1h, 4℃ for infinite

Gel running: Loading samples (50μl+10 loading dye). Put original plasmid to compare with digested one. Run at 120V for 20 min.

Gel purification: After excise bands, we did gel purification using protocol from Illustra GFX PCR DNA & Gel Band Purification Kit Gel. Final elution volume is 40μl. Then using Nanodrop to check its concentration which is 7.278ng/μl. It is not a good concentration result. So we repeated the experiment again.

Repeat restriction digestion: Prepare 4 tubes, 50μl each, total 200μl. The reaction is the same as previous. Overnight digestion at 37℃.

Both are the digested products, we just separated them into two wells like usual because one well might overflow and we would lose DNA. Both of them pBBR1MCS2 are at the expected size.

Repeat gel purification

Gel running: This time we put all digested products into one big well. (200μl sample+40μl loading dye). Order is 1kb ladder, sample, and 1 kb ladder. Then Still run at 120V for 20 min.

Gel purification: After excising the gel and following the same protocol, we eluted it with 20μl elution buffer.

pBBR1MCS2+OPHTIII construction

Gibson assembly:

Volume used (Vector : Insert = 1 : 3)

Gibson assembly reaction:

Incubate at 50℃ for 1h.

Transformation of bacteria: The only changes that we usually make are the following:

1. Incubation times for both thawing and incubating with DNA. Varies from 25-30 mins
2. The amount of LB broth to put for the outgrowth step varies from 500-900 ul (This case Hugh used 500 ul)
3. Incubation time for the outgrowth step varies from 45 mins - 1 hr (This time we used 1 hr)
4. Centrifuge 3000 x g for 2 minutes after outgrowth step to pellet the bacteria then remove some supernatant to leave 250 ul LB solution then resuspend the pellet with the leftover 250 ul LB solution then plate all the bacteria on the plate.
5. Dry the plates for 10 minutes before inverting and putting into the 37 C incubator

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pBBR1MCS2+OPHTIII construction

Colony picking and incubation

1. Picked 6 colonies from the plate
2. Put 8 mL LB broth total and added 8 uL Ampicillin
3. Put in 37 C shaking incubator overnight for 12-16 hrs

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pBBR1MCS2+OPHTIII construction

Plasmid extraction: using QIAprep Spin Miniprep Kit.

6 tubes containing 8ml transformed bacteria cultured with LB broth. Pellet 6ml bacterial overnight culture from each tube. Add 250μl buffer D1, then 250μl buffer D2, 350μl buffer N3. Centrifuge at 13000 rpm for 10 min. Transform the 800μl supernatant into spin column. Centrifuge at 13000 rpm for 60s and discard flow through. Add 500μl buffer PB with ethanol. Then centrifuge again at 13000 rpm for 60s and discard flow through. Add 750μl buffer PE, centrifuge at 13000 rpm for 60s and repeat twice. Put the spin columns into new tubes and add 35μl buffer EB. Stand for 5min then centrifuge again. Using Nanodrop to measure their concentration.

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pBBR1MCS2+OPHTII Construction

Gibson assembly

Transformation of bacteria

Failed: subsequent days were used as diagnostic and review

pBBR1MCS2+OPHTII Construction

Colony picking and overnight culture

None

Repeat pBBR1MCS2+OPHTII Construction

Gibson assembly

Transformation of bacteria

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Repeat pBBR1MCS2+OPHTII Construction

Plasmid Extraction of pBBR1MCS2: using Midi Kit

Transfer the overnight culture into four 50ml tubes.

Nanodrop result is 54.42 ng/ul

Restriction digestion

PCR for 2 hours at 37 degrees, infinite hold at 4 degree

Gel run for 25 mins at 130V

(We didnt save the gel image results), we cut the lower band and did gel purification for the pBBR1MCS2 vector.

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Repeat pBBR1MCS2+OPHTII Construction, Bacteria Transformation

Gibson Assembly of pBBR1MCS2 and OPHT2

Run for 1 hour at 50 degrees, infinite hold at 4 degrees

Transformation:

We thawed the competent cells for only 12 minutes, we should thaw them for a longer time. Then we spread the bacteria on Kan+ agar plates, culture them for 16 hours.

Transformation: It has no colony on positive control plate but few colonies on OPHT2 plate.

Repeat pBBR1MCS2+OPHTII Construction, Start OPHM construction and Transformation of OPHM

Colony picking and bacteria incubation:
Picked 4 colonies from OPHTII agar plate for overnight LB broth culture
LB broth volume 40ml + Kanamycin volume 0.04ml

Gibson assembly for pBBR1MCS2+OPHM construction:

(insert) OPHM concentration = 5.164ng/ul = 0.0047pmol

(vector) digested pBBR1MCS2 = 12.60ng/ul = 0.004pmol

60ng of vector = 60/12.60 = 4.76ul

4.76ul x 0.004pmol = 0.019 pmol

vector:insert=1:2

Insert = 0.019pmol x 2 =0.038pmol

Volume for insert = 0.038/0.0047 = 8.09ul

2. pBBR1MCS2+OPHM (vector:insert=1:2)

2. Positive control

3. Negative control

Incubate at 50℃ for 1h.

Transformation:

a. Heat shock at 42℃ for 45 secs using water bath.

b. Incubate at 37℃ for 60 mins, then plated them into three plates and cultured them for 16 hours.

Transformation: The positive control plate has no colony due to our mistake of plating the bacteria to Kan+ agar plate, it should be plated on Amp+ agar plate.The negative control plate has many colonies which should not happen, we suspect that the plasmid is self-ligated although we transformed with vector without insert. The OPHM agar plate has man colonies which is the expected results.

Repeat pBBR1MCS2+OPHTII Construction
 Plasmid Extraction of pBBR1MCS2-OPHT2

Restriction digestion for plasmid identity checking

Plasmid Extraction: We eluted with 40ul elution buffer for x3 times.

Restriction digestion reaction:

Put in thermocycler for 2hrs. Settings are as following: 37℃ for 2hrs, 4℃ for infinite hold.

Gel running: Load 4 samples (10μl+2 loading dye) and 1kb ladder (2ul) into wells. Put original plasmid to compare with digested one. Run at 120V for 20 min.

Restriction digestion reaction: (pBBR1MCS2 concentration:75.5ng/μl)

We made two tubes of the reaction above. Put in thermocycler for 14hrs. Settings are as following: 37℃ for 14hrs, 4℃ for infinite.

Concentration of OPHT2 plasmid:

colony 1(C1)=82.28ng/ul

colony 2(C2)=87.78ng/ul

colony 3(C3)=85.39ng/ul

colony 4(C4)=88.74ng/ul

Gel run: The expected results should be two bands shown on the gel as the plasmids should be digested by enzyme at the restriction sites. However, there was only a band for each sample, it might due to the plasmid is undigested or not fully digested. Therefore, we have to reconstruct the plasmid backbone.

Repeat pBBR1MCS2+OPHTII Construction

pBBR1MCS2+OPHM Construction

Prepare LB agar plates

Gel purification: Run gel. Order is 1kb ladder, pBBR1MCS2, pBBR1MCS2, none, OPHT II, pBBR1MCS2, pBBR1MCS2. Used 20μl elution buffer type 6. Concentration is 25.42ng/μl.

Gibson assembly reaction:

OPHT II concentration: 7.645ng/μl = 0.0055pmol

pBBR1MCS2 concentration: 25.42ng/μl = 0.008pmol

OPHM concentration: 5.164ng/μl = 0.0047pmol

1.pBBR1MCS2+OPHTII (insert: vector=3:1)

2. pBBR1MCS2+OPHM (insert: vector=2:1)

3. Negative control

Incubate at 50℃ for 1h.

Transformation of OPHT2, OPHM and negative control:

a. Heat shock at 42℃ for 45secs using water bath.

b. Incubate at 37℃ for 45 min then plated them into three plates.

c. Culture them for 12 hrs.

Prepare LB agar plates: 200ml LB agar= 8g powder+ 200ml MilliQ water.

The ratio for antibiotics and solution is 1:1000. So 200μl Amp/Kan to each kind of plate.

We made totally 18 plates with Kan+ and 21 plates with Amp+.

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Colony picking for OPHT2, OPHM
Transformation of OPHT1, OPHT3 and NSP4-Tyrosinase

Amplify the plasmid

Plasmid Extraction of OPHT2, OPHM

Restriction digestion to check plasmid identity

Colony picking: We picked 6 colonies from each transformed plasmids and incubate them in 5mL LB with antibiotic for 12-14 hrs.

Transformation: Transform OPHT2(#2),  OPHT3(#2), NSP4(#6) into Dh5 strain E.coli and incubate at 37 degrees.

Amplify the plasmid by streaking bacteria glycerol stock and culture in 5mL LB broth with antibiotic.

Plasmid Extraction: Save overnight culture before extraction. Used 30ul elution buffer and eluted 3 times, each time incubated 3 minutes. Concentration of DNA is measured by using Nanodrop.

Restriction digestion reaction:

Put in thermocycler for 4hrs. Settings are as following: 37℃ for 4hrs, 4℃ for infinite hold.

Run the samples on the agarose gel and observe the DNA bands under UV. The estimated correct band size of plasmid colonies are sent for RNA sequencing.

Concentration of DNA:

OPHM                                 OPHT2

#1=95.65ng/ul                   #1=117.1ng/ul

#2=94.81ng/ul                   #2=124.3ng/ul

#3=94.47ng/ul                   #3=97.22ng/ul

#4=204.6ng/ul                   #4=109.4ng/ul

#5=98.68ng/ul                   #5=108.2ng/ul

#6=91.51ng/ul                   #6=102.3ng/ul

Gel run and observation:

OPHT2 (#2 ans #5)

OPHM(#2 and #6)

Philip, Julien

1.Preparation of 20% Arabinose

2.SDS-Dye preparation

3.Protein samples preparations

1: Prepared 5 mL 20% arabinose

Mixed 1051.9 mg Arabinose + 5 mL milliQ water

2: 4x SDS sdye preparation

3: Protein samples preparation

 OPHT, OPHTIIII and NSP4 Pellets

-Resuspended them all with 75 ul Milli-Q water + 25 ul 4 x SDS Page dye. Stored in -20C fridge

Stored 1 mL of the supernatant of the induced culture tube for later protein expression test.

Prepared loading sample for supernatant. 75 ul NSP4 supernatant + 25 ul 4x SDS page dye

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Renee

pBBR1MCS2+OPHTII(sequenced) Construction

pBBR1MCS2+OPHM(sequenced) Construction

Transformation:

2.5ul OPHTII #5 with 50ul competent cell

2ul OPHM #2 with 50ul competent cell

Plated at 16:15 and incubated at 37 degree overnight.

None

Philip

SDS-Page gel running and Coomassie blue staining

Gel percentage used 12.5%

Protein samples that were ran:

NSP4 = 37.17 kDA

OPHTI = 52.18 kDA

OPHTIII = 52.83 kDA

10x Prepared running buffer:

Gel running arrangement:

NSP4 supernatant -> NSP4 pellet -> OPHT3 -> OPHT -> Ladder -> NSP4 supernatant -> NSP4 pellet -> OPHT3 -> OPHT -> Ladder

Running time: 60V for 1hr then 120 V for 1 hr

Coomasie blue staining:

1. Washed the SDS away from the gel
2. Soaked the gel with the gel stain for 1 hr
3. Wash the excess gel stain with dH2O.
4. Put water after 3 times washing, boil for 5 minutes in the microwave then let it shake overnight. Start time 10:27 pm
5. Remove the water and take picture

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Philip, Renee

Make glycerol stock of pBBR1MCS2+OPHTII(sequenced) and pBBR1MCS2+OPHM (sequenced)

200ul 5% glycerol + 800ul bacteria culture

Separate 10 tubes SDS dye

None

Renee

Test adhesion system in three cell lines

Transformation: 

TOP10           OPHT#1(1ul)     OPHT3 #3(1ul)     07/05 vector (3.5ul)

BL-21            OPHT#1(1ul)     OPHT3 #3(1ul)     07/05 vector (3.5ul)

MG1655        OPHT#1 (1ul)     OPHT3 #3(1ul)     07/05 vector (3.5ul)

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Renee

Test adhesion system in three cell lines

Pick colony: three colonies per plate for TOP10 and BL-21 with OPHT, OPHT3 and vector, totally 18 tubes, culture at 37 degree shaker overnight

Transformation:

TOP10          OPHT2       OPHM

BL-21           OPHT2       OPHM

MG1655       OPHT2       OPHM

No colony grew on all MG1655 plates. Repeat the plating with all the remainder MG1655 culture.

Renee

Test adhesion system in three cell lines

Pick colony: three colonies per plate for TOP10 and BL-21 with OPHT2 and OPHM, totally 12 tubes, culture at 37 degree shaker overnight

Still no colony grew on all MG1655 plates. MG1655 was not suitable for our expression system.

Renee, Philip

pet11a+OPHT/OPHT2/OPHT3/OPHM/OPHA1/OPHA2 Construction

1. Gel making
2. Gel running: 50ul PCR product + 10ul loading dye, load 30ul per well for pet11a+OPHT/OPHT2/OPHT3/OPHM/OPHA1/OPHA2; 3x50ul PCR product + 30ul loading dye, load 90ul per well for pet11a vector. Run at 120V 15mins

Loading order: Gel1-ladder/OPHT1/OPHT1/OPHT1/OPHT2/OPHT2

                           Gel2-blank/ladder/OPHT3/OPHT3/OPHM/OPHM

                           Gel3-ladder/OPHA1/OPHA1/blank/OPHA2/OPHA2

                           Gel4-ladder/vector/vector

Gel cut weight: OPHT1     0.36g           Buffer type 3: 360ul

                            OPHT2     0.27g                                     270ul

                            OPHT3     0.31g                                     310ul

                            OPHM      0.31g                                     310ul

                            OPHA1     0.16g                                     160ul

                            Vector1    0.57g                                     570ul

                             Vector2   0.43g                                     430ul

Concentration: OPHT1   41ng/ul

                            OPHT2   38.36ng/ul 

                            OPHT3   18.95ng/ul

                            OPHM   21.67ng/ul

                            OPHA1  10.02ng/ul

                            Vector1+ Vector2  54.12ng/ul

No band for OPHA2

Philip, Julien, Renee

Repeat pet11a-OPHA2 Construction

Continue pet11a-OPHT/OPHT2/OPHT3/OPHM/OPHA1 Construction

1. OPHA2 PCR followed by running gel

2. Gibson assembly:

3. Transformation: 2ul DNA+50ul competent cell Stbl3
Transformed OPHT1, OPHT2, OPHT3, OPHM, OPHA1 and positive ctrl; plated 300ul transformed bacteria on agar plates

Still no band showed on the gel for OPHA2

Philip, Julien

Plasmid Extraction

Used 30ul elution buffer for elution

DNA Concentration (ng/ul):

Philip, Julien

Tyrosinase Activity Assay

Sample 1: Induced NSP4 pellet
Sample 2: Induced NSP4 supernatant

Sample 3: Uninduced NSP4 pellet

Sample 4: Uninduced NSP4 supernatant

Homogenize cells with 500ul ice-cold Tyrosinase Assay buffer to perform lysis and keep on ice for 10 minutes followed by centrifugation at 10000xg for 15 minutes at 4 degree. Collect the supernatant and estimate protein concentration by using BCA protein assay kit (protein concentration should range between 1 to 2.5 ug/ul)

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Philip, Julien, Renee

Prepare NSP4 for Negative control

NSP4 activity test preparation

1. Took one tip of cells from glycerol stock, incubate with 5ml LB broth + 5ul Ampicillin at 37 degree shaker at 17:05
2. Pull down assay for NSP4 induced supernatant.
3. Sonication for NSP4 bacteria culture

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Philip, Julien

Nickel pull-down assay

Tyrosinase activity test

1. Prepare nickel resin: Pipette 30ul of nickel resin. Wash resin in 1ul H2O two times followed by washing with 1ml binding buffer two times.
2. Pull-down assay: Centrifuge the induced BL-21 (NSP4 transformed colony 3) as well as the uninduced one and take the supernatant as pull-down assay sample. Pipette 30ul resuspend resin into supernatant. Incubate and rotate it at 4 degree for  45mins. Spin at 3900rpm for 10mins, remove the supernatant which will be stored just in case, then leave 1ml fraction in  resuspend resin and transfer resin to  1.5ml tube. Spin down at 4000g 2min to get resin. Discard supernatant. Wash resin with 750ul binding buffer three times (we washed the resin with Tyrosinase Assay buffer in the third wash). Measure protein concentrationProceed to Tyrosinase Activity Assay.
3. Tyrosinase activity test: Homogenize cells with 500ul ice-cold Tyrosinase Assay buffer to perform lysis and keep on ice for 10 minutes followed by centrifugation at 10000xg for 15 minutes at 4 degree. Collect the supernatant and estimate protein concentration by using BCA protein assay kit (protein concentration should range between 1 to 2.5 ug/ul)

As the induced supernatant concentration is negative, there is literally no protein in the supernatant, therefore we did not continue proceed the Tyrosinase Activity test.

Julien

Make an overnight culture of BL-21 (NSP4 transformed colony 3) from glycerol stock

Make a 20mL culture of BL-21 glycerol stock (NSP4 transformed colony 3) with Ampicillin antibody (1:1000 dilution) and start incubation in 37 degree shaking incubator at 6.25pm

None

Julien, Renee

pBAD24 NSP4 #3 transformation with BL-21

Induce overnight BL-21 (NSP4 transformed colony 3)

1. Transformation: 1.5ul pBAD24 NSP4 #3 + 50ul BL-21 competent cell
2. Induction: separate 5ml out of 20ml overnight BL-21 as uninduced BL-21 stock and put into -80 degree, for the rest of 15ml, add 150ul 20% arabinose for induction, then culture overnight in 37 degree shaker

None

Philip, Julien, Renee

Preparation for NSP4 activity assay

Colony picking

1. Picked a colony from the transformation yesterday. Prepared a 20 mL culture with amp+ and incubated in 37 C at 6:49 am
2. Centrifuge the overnight induced culture of NSP4 Colony #3 BL-21, separate pellet and supernatant, then store in -80degree. Centrifuged 5mL uninduced sample NSP4_plasmid#3 then stored in -80C. Induced the rest 15 mL (OD=1.1227 13-14 hr culture) with 150 ul 20% arabinose at 20:08 and incubated in shaking incubator.

None

Philip, Julien, Renee

NSP4 activity assay

1. Nickel pull down both induced and uninduced Nsp4 SUPERNATANT (new and old stock), total 4 samples
2. Scrapped OPHT2 bacteria (three stocks) from glycerol stock 082719 and cultured in ~5mL volume of Lb broth with amp+ and then put in 37C shaking incubator at 16:45. Glycerol stock for all OPHT2 colonies were thawed completely by accident watch out for the quality of this stock for next scrapping.
3. Homogenize 4 samples with tyrosinase assay buffer and test protein concentration with BCA protein assay (30mins incubation)

None

Philip

OPHT2 induction

NSP4 activity assay

Material stock preparation

1. Induced 5mL cultures of pet11a_OPHT2 from yesterday with 1mM IPTG (10 ul of 500mM IPTG stock) and incubated at 37 C shaking incubator at 4:45. OD600 =0.7144
2. Tyrosinase activity assay started plate OD monitoring (510nm) at 8:02 am in the plate reader in 37 C temperature for 1 hr. Stored all proteins back in the “Protein sample box” in -80C
3. Made two 500mL LB broth and one 110 mL LB agar for making 10 amp+ plates. Made a stock of 50 mL LB Broth amp+ and put in 4 degree fridge.
4. Diluted induced pet11a_OPHT2 #1 from early this morning. 1:1000 dilution ratio by putting 10 ul of that induced culture into 10 mL LB Broth amp+ then stored in shaking incubator 37 C overnight.

Result of tyrosinase activity test today

Philip

BL-21 OPHT2 #1 EGFP expression

eGFP expression is still very high even after ~28 hrs after induction for our bacteria Bl-21 OPHT2 colony #1

Philip, Julien

Material stock preparation

BL-21 OPHT2 #1 EGFP expression

Adhesion system reconstruction

1. Made 11 Amp+ agar plates by adding 100ul (1:1000) to 100ml agar solution.
2. eGFP expression is still very high even after ~40 hrs after induction for our bacteria Bl-21 OPHT2 colony #1
3. PCR reaction for the amplification of adhesion system for ligation to pet11a for 1 hr and 26 mins. Started at 15:34

Philip, Julien, Renee, Jia Ying

Adhesion system reconstruction

Preparation digested pet11a vector stock

1. Incubated a new BL_21 OPHT2 colony #1, 5 mL volume, at 37 C

2. Run the gel with opht1,2,3 and opha1 samples (each well 30ul, each sample 2 wells) at 120V for 20 mins. Then gel purification.

3. Gibson assembly to ligate Pet11a vector with Opht1,2,3 and Opha1 inserts. Calculation as below:

75ng of vector=75/54.12=1.39ul

1.39ulx0.015pmol=0.02pmol

vector:insert=1:3

Insert=0.06pmol

4.Restriction digestion for pet11a vector. We did it for 100ul, incubate for 14hrs at 37 degree, infinite hold at 4 degree. Calculation as below:

pET11a concentration: 49.1ng/ul

5. Transformation stock OPHT1, OPHT2, OPHT3, OPHM, and OPHA using BL-21; stock pBBR1MCS2_OPHT2 using BL-21; stock pet11a_OPHT1, OPHT2 using Rosetta. Put into the incubator at 21:00

Transformation:

Julien, Renee, Jia Ying

Colony picking for transformed bacteria

Bacteria transformation

Ammonia iron (III) citrate solution preparation

Induction of IPTG and Ammonia iron (III) citrate in OPHT2 bacteria

Gel run and gel purification

Colony picking: Picked 1 colony for OPHT1,2,3,M and OPHA1 (BL-21), OPHT1,2 (Rosetta). Picked 3 colonies for pBBR1-Opht2 (BL-21). Incubate the picked colonies with 5ml Amp+ (for pET11a plasmid) or Kan+ (for pBBR1MCS2 plasmid) LB broth, put in 37degree shaking incubator at 4.30pm.

Transformation with new constructed pet11a_OPHT, OPHT2, OPHT3 and OPHA using the Stbl3 strain E.coli. Put them into 37 degree incubator at 18:40.

Prepared the Ammonia iron (III) citrate solution at concentration:100mM with 26.3g iron(III) powder in 263mL water at 18:00.

We added 10ul IPTG and 1ml ammonium iron(iii) citrate into BL-21 OPHT2 at 6pm. We run the gel for digested pET11a vector, the expected band size is 5.6kb. Then we purify the excised gel (band1 weight 0.371g and band2 weight 0.372g) with 30ul elution buffer type 4 three times, each time incubated 5minutes.

Gel run results:

The concentration for band1 is 2.24ng/ul and band 2 is 2.49ng/ul.

Philip, Julien

Glycerol stock making

Colony picking and bacteria overnight culture

Picked up adhesion system plasmids transformed plates from 37C, parafilmed and put it in 4C. All constructs have colonies. Picked three colonies for each transformed plasmids in Stbl3 with 5ml LB, incubated in 37 shaking incubator at 6.50pm.

Collected the overnight culture of picked colonies at 1.30pm [amplified adhesion system-old stock: OPHT1,2,3,M and OPHA1 (BL-21), OPHT1,2 (Rosetta) and pBBR1-Opht2 #1,2,3 (BL-21)]. Made 1ml glycerol stock of these with 500ul 50% glycerol and 500ul bacteria culture, stored in -80 degree fridge’s Glycerol stock box.

Checked EGFP expression of pbbr1-opht2 with fluorescence microscope:

Julien

Sequencing samples preparation

Glycerol stock making

Collected overnight culture of picked colonies (pet11a-stbl3) and prepared sequencing samples(1ml bacteria, 120ul 106FOR, 30ul 102 FOR), sent for sequencing.

Made 1ml glycerol stock with 500ul 50% glycerol stock and 500ul bacteria culture, stored in -80 degree fridge’s glycerol stock box.

None

Julien

Protein extraction

Protein extraction for overnight bacteria culture (pet11a adhesion system-BL21, Rosetta) with 500ul lysis buffer, boiled at 98degree for 10minutes. Measured protein concentration after 30minutes BCA incubation. Run western blot at 1.40pm.

The order and volume loaded shown as below:

Ladder - OPHT1 (Rosetta) - OPHT2(Rosetta) - OPHT1 (BL-21) - OPHT2 (BL-21) - OPHT3 - OPHM - OPHA1

(5ul for ladder, 20ul for the rest protein samples)

Incubated with antibody and shaking in 4 degrees fridge at 5.20pm

None

Philip, Julien

Continue western blot

Washed the membrane with about 3 times.

Incubated 10 mL OPHT2 Colony #1 in LB amp+ at 37 C shaking incubator at 23:30.

Magnetization of E.coli incubated in Ferric ammonium citrate(3 days incubation at 37 C) for 3 hrs with magnet. Made a video from the fluorescence microscope

Western Blot results:

Philip

Incubation of bacteria culture, adhesion system functional test

Incubated 15 mL culture of all the new pET11a adhesion system glycerol stock at 37 C shaking incubator at 14:01. Will extract after 12 hrs.

Induced the overnight BL-21 with 1mM IPTG (50ul in 10 mL) and put in 37C shaking incubator. Inducing for 3 hrs.

Added 2 ml of 100mM ferric ammonium citrate into the 3 hr IPTG induced BL-21 OPHT2 colony #1(Checked egfp signal and it’s positive) and put it in 37C shaking incubator at 17:08. Incubating for 24 hrs.

Made 5 mL overnight culture of BL-21 for adhesion system functional test. Stored in 37 C shaking incubator at 20:50 . Used all colony #1 from the old pet11a glycerol stock. Incubating for 12 hrs.

None

Philip

Plasmid extraction

Induction of adhesion system

Magnetization test set up

Extracted plasmids of the 15 mL new pet11a adhesion system and sent samples for sequencing.

Stored new pet11a plasmids in -20C in the new construct box.

Induced 5 mL overnight for functional test of adhesion system using 1mM IPTG(10 ul 500 mM stock -> 5 mL culture) for 3 hrs in 30C shaking incubator in Prof.Xu’s lab started at 16:30.

Started the magnetization test setups at 00:05. Prepared three dishes. Two that was incubated with fe3+ and one negative ctrl. One of the two dishes has been put in the fluorescence microscope and picture is being taken at 30 sec intervals for 6 hrs.

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Philip

Incubation of bacteria culture

Started the incubation of overnight culture of Tyrosinase (500 U/ml) 2 mL + 2 mL of our adhesion system bacteria that was resuspended with 10 uM Phosphate buffer (Which prepared at 092419). Incubated in Professor Xu's lab's shaking incubator temperature 25-27 C, shaking at 75 rpm.

Picked up our overnight culture E.coli with Tyrosinase at 19:45 (Total 17 hrs incubation).

Started the incubation of our "Activated E.coli with Tyrosinase" at 20:30. Mixed 150 ul of the nanoparticle PFODTBT (30 ppm) with the 4 mL overnight culture. Put the solution in Professor Xu's shaking incubator at 25C 70 rpm for 1 hr.

Put the activated bacteria incubated with the nanoparticle in the 4 Degree of PRofessor Xu's lab.

Julien

Checking of nanoparticle binding with bacteria

Centrifuged the nanoparticles incubated bacteria culture and resuspended the pellet with 1mL phosphate buffer. Get 10ul on glass slide and observed under a fluorescence microscope. We have negative ctrl(without tyrosinase), nanoparticles only and 5 bacteria samples.

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Julien

Bacteria culture

Restriction digestion

Prepare 5mL of pet11a adhesion system BL-21 bacteria culture with Amp+, shaking in 37 incubator of Prof. Henry’s lab.

Restriction digestion for pBAD4 vector and pBAD4-tyrosine vector with XbaI enzyme. Put in thermocycler 37 degree for 14 hours and 4 degree for infinite hold.

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Philip, Julien, Renee

DNA purification

Induction of samples

Gibson assembly

Transformation

Separated 1ml culture as uninduced sample for each. Induced with 10uL IPTG for each samples except negative ctrl and incubated in Prof’s Xu lab 30 degree shaking incubator at around 11.40pm.

Purified DNA of digested pBAD24 and pBAD24_NSP4 from agarose gels. Concentration of pBAD24 is 125.1ng/ul and pBAD24_NSP4 is 61.31ng/ul.

Resuspend ftnA fragment with 25ul TE buffer followed by 50degree incubation for 20min. Gibson assembly of ftnA+pBAD24 and ftnA+pBAD24_NSP4.

Gibson assembly for pBAD vector+ftnA and pBAD NSP4 +ftnA. Put in thermocycler for 1hr at 50degree. Calculation as below:

pBAD NSP4 conc=61.31ng/ul = 0.017pmol

pBAD conc=125.1ng/ul = 0.042pmol

ftnA conc=20ng/ul = 0.052pmol

100ng vector (pBAD NSP4)=1.63ul

(vector)1.63 x 0.017=0.028pmol

vector:insert=1:3

(insert)=0.083pmol

ftnA volume=0.083/0.052=1.6ul

100ng vector(pBAD)=0.8ul

(vector)0.8 x 0.042=0.034pmol

vector:insert=1:3

(insert)=0.102pmol

ftnA volume=0.102/0.052=1.96ul

Incubated our 1 mL bacteria with tyrosinase 500U/mL in Professor Xu’s shaking incubator at 25 C 90 rpm for 24 hrs.

Transformation of ftnA+pBAD24 and ftnA+pBAD24_NSP4 into stbl3 strain. Start incubation at 22:17.

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Philip

Colony picking

Picked three colonies for FtnA+pBAD24 and made 25 mL bacteria solution with amp+ at 17:30. Unfortunately there were no colonies for the pBAD24_NSP4+FtnA. Will repeat this transformation later.

None

Philip, Julien

Plasmid extraction

Plasmid extraction for three overnight culture of pbad24-ftna. Used 30ul EB, repeated 3times and each time incubated 5 mins. Extracted concentration listed in "Results"

Incubated 100 ul of activated adhesion system + bacteria from 092819 with 10 ul (1 mg/mL concentration) of 1.0%w/v FP-00552-2 Yellow fluoresscent particle, 0.04-0.09 um size in 25C and mixing at 300 rpm for 1 hr. Started at 23:40 (Samples were covered with aluminum foil to prevent exposure to light).

Concentration of extracted plasmid:

pBAD24-ftnA #1: 169.3ng/ul

pBAD24-ftnA #2: 200.9ng/ul

pBAD24-ftnA #3: 137.8ng/ul

Philip

Adhesion functon test

Adhesion test fluorescence checking using 4th floor plate reader machine. Result: Failed will elaborate later.

Third functional test using 092819 1000ul activated bacteria. Put 5 ul of the yellow nanoparticle from Prof Xuanjun 30ppm into the 1000ul bacteria. Incubated at 25C for 1hr with shaking for 400 rpm. Started at 19:00.

Incubated new adhesion system bacteria for functional test. Picked all Colony #1. Prepared 5 mL overnight culture amp+, will subculture tomorrow to 20 mL culture (Need more bacteria concentration for the tests). Started the incubation at 22:30 at shaking incubator 37 C.

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Philip

Incubation of bacteria culture

Induction of adhesion system

Subcultured 5 mL overnight cultures into 20 mL LB amp+ and incubated at 37 C for 3 hrs. Started incubation at 13:00. Saved the 5 mL overnight culture as uninduced samples. Will measure OD of both later, need OD=2 for activation and functional tests.

Induced Subcultured 20 mL adhesion system bacteria culture with 1 mM IPTG each (OPHT, OPHT2, OPHT3, OPHM, & OPHA). Incubated at 30 C for 4 hrs in Professor Xu's shaking incubator.

Update on the magnetization effect after incubation with ammonium ferric citrate of the BL-21 strain of E.coli. They can still be attracted to magnet until now since 092419.

Update on adhesion 20 mL culture, I will induce it overnight instead of 4 hrs. Will harvest them tomorrow morning instead.

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Philip

Checked sequences of newly constructed plasmid

Checked the sequences of the newly constructed pet11a adhesion system plasmids. Conclusions are as follows：

OPHT1 colony # 3 = Should be correct (Has a single point mutation but it's peak looks like a sequencing error)

OPHT2 colony #1 = Correct and usable if needed

OPHT3 = no correct or intact colony

OPHA Colony #1 and #3 = Correct and usable if needed

None

Philip

Sent sample for sequencing

Send ftna samples that Julien extracted from 093019 to Tech dragon sequencing.

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Philip, Renee

Functional test of the combination of tyrosinase and adhesion system

Incubated 100ul bacteria with 50ul 1500U/ml tyrosinase and 1ul of 1%w/v yellow nanoparticle together for 4 hrs and 8hrs. Will check the fluorescence reading after these two time periods.

Cultured 20 mL bacteria using glycerol stock of colony number 1 for OPHT, OPHT2,OPHT3, OPHM, OPHA1 with Amp+ and one for BL21 only without Amp.

Current conclusions for adhesion system assay parameters: bacteria amount (Need OD=2), time of incubation with tyrosinase (4hrs, is the best I think), amount of tyrosinase (U/mL) (500U/ml or less), amount of nanoparticle (10ml/L or 10ppm is good for our experiment), centrifugation speed(3000xg 3 mins no nanoparticle pelleting), learning the plate reader machine(440 nm first filter and 480nm second filter), choosing the nanoparticle(1% w/v Yellow fluorescent particle from spherotech and red nanoparticle of Prof Xuan Jun), assay format in 96 well plate (As shown in the picture just now), Amount of bacteria and nanoparticle total volume (150 ul) so we can plate in 96 well.

ligated pBAD24_NSP4 with fntA, and a negative control only has pBAD_NSP4 without insert then transformed them into DH5 alpha. Started incubation at 20:23.

Cultured 18 tubes 20ml of bacteria from glycerol stock NSP4-Tyrosinase colony 3.

Current conclusion based on this fluorescence plate reader result is that after 4 hrs of incubation with tyrosinase there seems to be a significant decrease of supernatant fluorescence signal compared to our inactivated supernatant samples. Most notable OPHM.

Philip, Julien, Renee

Induction of NSP4 tyrosinanse

Induced with 5 concentrations of arabinose in 15 tubes of nsp4 tyrosinase bacteria overnight culture. Another 3 tubes are not induced with arabinose act as negative ctrl. The volume of arabinose added shown as below:

Induced overnight OPHT1, OPHT2, OPHT3, OPHM and OPHA with 1 mM 40 ul of (500mM IPTG) per 20 mL culture and incubated in Professor Xu's shaking incubator 30 C for 4 hrs. Started ~approximately 12:08.

Collected 4 hr incubation with 5 concentrations arabinose’s bacteria culture.

Nickel pull down for nsp4 4 hr arabinose induced (supernatant only), stored the pulled down protein in -80c protein samples box.

Collected 8hr samples and centrifuging at 3000xg for 10 mins. Will separate supernatant into another tube.

Incubated adhesion 100219 and 100519 bacteria with yellow nanoparticle started incubation at approx~ 21:00.

Checked the construction of three extracted plasmids pbad24-fntA on 093019 by PCR and gel running.

Nickel pull down for nsp4 8 hr arabinose induced (supernatant only), stored the pulled down protein in -80 protein samples box.

Collected 12hr samples and centrifuging at 3000xg for 10 mins. Stored separated-supernatant into another tube and stored in -80c.

Nanoparticle Fluorescent signal of inactivated and activated supernatant of OPHT, OPHT2, OPHT3, OPHM, OPHA, Negative control, and Positive control.

Philip

Incubation of bacteria containing FtnA plasmid

Functional test of adhesion system

Tyrosinase activity assay

Incubated transformed bl-21 with FtnA plasmids. Diluted outgrowth solution 1:100 and plated 350 ul of the bacteria. Put it in Prof Xu’s 37 degree incubator at 1:16 am.

Incubated BL-21 with pBAD24_FtnA at 14:20 in Professor Xu's 37 C incubator. Only plated pBAD24_FtnA#1 and pBAD24_FtnA#2.

Tyrosinase activity assay for 4,8,12 hours incubation with arabinose at 5 different concentrations. We cultured them at 30 degree accidentally.

Tyrosinase activity assay reagents set-up:

Sample Background Control (SBC) mix: add this into SBC wells

Reaction mix: add this into assay background control, samples and positive control wells

Positive control well: 2ul positive control + 48ul assay buffer

Assay background control: 50ul Assay buffer

Plate:

Results for the adhesion system functional test. Conclusion: The results are quite consistent. Plate was read three times with the same machine and averaged all the reads as shown in the picture data set. Then I averaged each the three repeats for each samples and obtained final reads for each 100219 and 100519 samples and made the graph based on them. We can see a clear difference with the activated and inactivated one. Negative control (Wild type, just BL-21) is too close to our inactivated samples, not sure if the activated readings would be considered significant, but this negative control sample has been prepared along with the 100519 sample so it actually cannot act as a negative control for the 100219 samples but I just put it anyway. We need a third biological repeat for all these to have a final conclusion. Anyways, the data is very consistent, activated sample's readings are always lower than the inactivated ones for these first two biological repeats.

The results of plates after 1 hour measurement in the plate reader (30seconds interval, 37 degree incubation, OD 510nm) is saved.

Julien

Nickel pull-down for NSP4 tyrosinase protein

Incubated 15 tubes of 5ml nsp4 bacteria with 5 different arabinose concentration (2%, 0.2%, 0.02%, 0.002% and 0.0002%) at 12.30pm.

Collected nsp4 tyrosinase bacteria after 4hr incubation (at 4.30pm), centrifuged and keep the supernatant. Nickel pull down for 4hr incubation batch nsp4 bacteria’s protein and stored in -80 fridge’s bacteria box. (Because no space in protein sample box). Collected 8 hr incubation of different concentrations arabinose in nsp4 at 8.30pm. nickel pull down for 8hr incubation batch nsp4 bacteria’s protein and stored in -80 fridge’s bacteria box. (Cuz no space in protein sample box). Collected 12 hr arabinose incubation at 5 different concentrations of nsp4 bacteria at 12.30am . Centrifuged and kept the supernatant in -80 with a green holder.

None

Philip, Julien

Nickel pull down

Nickel pull down

Diluted protein samples by 10x and measure the protein concentration using BCA assay.

Protein concentration:

Philip, Julien, Pinto

Tyrosinase activity assay

Protein extraction

Western Blot

Tyrosinase activity assay for 4,8,12 hours incubation with arabinose at 5 different concentrations. We cultured them at 37 degree this time.

Tyrosinase activity assay reagents set-up:

Sample Background Control (SBC) mix: We use only one SBC for each time point incubation of protein samples as we were running out of reagents.

Reaction mix: add this into assay background control, samples and positive control wells

Positive control well: 2ul positive control + 48ul assay buffer

Assay background control: 50ul Assay buffer

Plates:

SDS-page gel loading sequence:

First gel (OPHT-adhesion system):

Ladder- Ladder- Control- OPHT1 - OPHT2 - OPHT3 - OPHM - OPHA1

Second gel (ftNA):

Ladder - Control - ftnA1.1 - ftnA1.2 - ftnA1.3 - ftnA2.1 - ftnA2.2 - ftnA2.3

In the functional test results page

Philip, Renee

ftnA functional test

Centrifuged the second bench ftnA test samples and resuspend in LB broth. Measure the OD value. Begin magnet attraction test at 9:30 and will check their states every hour.

Transformation of pBAD24-FtnA #2 into seven strains which are Stbl3, DH5 alpha, BL-21 DE3, BL-21 Star, BL-21 Rosetta, BL-21 pLys, TOP10. Started incubation at 13:00.

Reset pBAD24-FtnA 1.1, 1.2, 2.1, 2.2 attraction test with only one magnet under it at 14:50.

Induced 5 tubes 5ml bacteria pBAD24-FtnA 1.1 with 1ml 100mM ferric ammonium citrate at 16:20.

101219: Incubated 20 mL BL-21 NSP4 tyrosinase on 37 degree for adhesion system functional test with our own enzyme.

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Philip

Adhesion system functional test

Started adhesion system functional test. Incubated adhesion system bacteria with yellow nanoparticle. Would collect the samples at 4 different time points, 1 hr, 2 hr, 3 hr and 4 hr and compare the difference in the relative fluorescence.

None

Philip, Julien

Bacteria culture, Incubate with antibody for immunoblotting membrane

Incubated 5ml culture of FtnA-Stbl3,Dh5alpha,BL-21(De3),BL-21(star), BL-21(plys), TOP10, BL-21(Rosetta), And Bl21(WT) at 37 degree shaking incubator started 14:39.

Pinto made SDS gel and store in Buffer. Measured protein concentration of FtnA protein samples and stored in -80C.

Autoclaved some tips blue and yellow tips and 1.5 mL centrifuge tubes.

Incubated FtnA samples and RpoB internal control with anti-flag and anti-rabbit primary antibody. Put 5 mL and 3 mL respectively of the antibodies. Put in 4 degree in Henry’s fridge at 20:20.

Incubated 6 20mL culture of OPHT, OPHT2,OPHT3, OPHM, OPHA and WT bl-21 all picked from glycerol stock colony #1 in 37 degree shaking incubator overnight(start 21:00) Has white tape mark, will induce it with 1mM IPTG for 4 hrs after 12 hrs (9:00). For time dependent functional test with yellow fluorescent nanoparticle and Professor Xuan Jun’s nanoparticle.

Incubated 5x 20 mL culture of NSP4_BL21 subcultured from yesterday’s overnight culture(1:100 ratio) at 37C started at (21:00) . Will induce later with 0.2% arabinose after 3hrs (00:00). And let it produce tyrosinase into the supernatant for overnight(12-16 hrs) Has green tape mark. This is for testing our own secreted tyrosinase whether it would be able to activate our capture system bacteria.

Incubated 6x 5mL tubes of OPHT,OPHT2,OPHT3,OPHM, OPHA and negative ctrl at 37 C has yellow tape mark started (21:00). Picked from glycerol stock colony #1. Will incubate overnight and induce tomorrow after 16 hrs with IPTG and incubate in 30C for 4 hrs (12:00).

Incubated 7x 5mL tubes for each bacteria strains we had cultured overnight FtnA-Stbl3,Dh5alpha,BL-21(De3),BL-21(star), BL-21(plys), TOP10, BL-21(Rosetta), And Bl21(WT) at 37 degree shaking incubator started 21:00. Has red tape. Subcultured 1:100 ratio will induce with 0.2% arabinose later after 3 hrs (00:00) and incubate for 12-16 hrs. These samples are for protein concentration check for FtnA based on different strains. Will extract proteins tomorrow and measure protein concentration along with the preparation of protein samples for western blot running on Wednesday.

Induced earlier log picked strains incubated this morning from 2:39 5ml cultures of FtnA-Stbl3,Dh5alpha,BL-21(De3),BL-21(star), BL-21(plys), TOP10, BL-21(Rosetta), And Bl21(WT) with 0.2% arabinose (50 ul of 20% arabinose) and stored in 37 degree shaking incubator has purple tape. Started at 21:42. Will incubate overnight for 12 hrs. Then would put with ~20 mM ammonium ferric Citrate 1 mL each and incubate for 24 hrs for magnetization test on Wednesday morning 9:00 am.

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Philip, Julien

Western blot

Induced red and green taped samples from previous logs with 0.2% arabinose at 00:44 and put in the 37 C shaking incubator. Plan to incubate for 12-16 hrs (Probably 16 hrs).

Induced yellow tape marked tubes and white marked tape tubes with 1mM IPTG at 12:00, put them in 30C shaking incubator of Professor Xu's lab.

Washed the membrane with tbst 3 times and incubated with anti-rabbit 2nd antibody at 12.40pm

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Philip, Julien

Western blot

Incubated purple taped samples from 101419 with ~20 mM ferric ammonium citrate. At 37 C starting 00:49.

Run SDS-Page gel. Loading order as below:

First gel

Ladder-Ladder-Control-2%-0.2%-0.02%-0.002%-0.0002%

Second gel

Ladder-BL-21WT-Rosetta-BL-21(star)-TOP10-BL-21(plys)-Dh5alpha-BL-21(De3)

Incubated membranes with primary antibody for overnight. (Anti-flag for 19kda ftna, Anti-rpob for 150kda internal ctrl).

None

Philip

Immunofluorescence

Incubated immunofluorescence slide samples with histidine primary antibody for 12-16 hrs. Put in 4 degree in Henry’s fridge.

None

Philip, Renee

Cell viability testing

Started incubation of bacteria cell viability with ZnO and AgNP at 04:30. Will test the following time points (Hrs) or OD600 (2, 4,6,8,10,12,14,16). 6:30, 8:30, 10:30, 12:30, 14:30, 16:30, 18:30 are the time points. I would need help getting the absorbance values for the 10:30, 12:30, 14:30 timepoints.

Incubated 20 mL overnight culture OPHt,OpHT2, OPHT3, OPHM, OPHA, -ctrl in 37 C shaking incubator started 17:00.

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