Pili Expression Team

This team includes Marc Singer, Kevin Joseph, and Alena Wu.

June/July

6/28/2010

Made a 500 mL batch of LB broth

• When pouring in distilled water, pour a few mL at a time to avoid clumping.
• It takes 20g of powder to make 1 L of broth

Sterilized broth using the autoclave

• The temperature setting on the autoclave is off by a little bit
• Set dial 2 notches below 134°C.

6/29/2010

Started growing E. coli K12 cultures

• Poured 2 mL of LB broth into a Falcon tube
• Used strain of K12 from Dr. Lin's freezer
• Placed in incubator shaker for 24 hrs.

Added and inventoried supplies from Dr. Pinto's lab.

• Regular trash can be thrown out by going down one floor, then going outside to the trash bins.
• Chemical wastes must be cleaned by calling OSEH.

6/30/2010

Cryopreserved stock of K12

• 1 mL located in the iGEM box in the Lin lab.
• 1 mL located in the lab freezer.

7/1/2010

Cryopreserved DH5α according to protocol procedure on 6/30/2010

• Put 1 stock in -20°C fridge in 1239
• Put the other stock in the -80°C fridge in the Lin lab

7/7/2010

Obtain genomic DNA of CFT073 E. coli strain from Dr. Mobley's Lab

• stored in -20°C fridge on ice

7/21/2010

Kevin, Marc, Alena

Met in Dude to determine sequence of fim operon. Arranged meeting with Dr. Mobley's group next Tuesday to learn more about hyperpiliation and the cloning process.

7/27/2010

Kevin, Marc, Alena

Met with Chris Alteri from Dr. Harry Mobley's research group to discuss the best route to hyperproduce the pili. Chris recommended that we create a plasmid by cloning FimB into pBAD, and then inserting that plasmid in MG1655. In theory, that should activate flocculation in the E. coli, inducible by arabinose. Chris was able to give us the procedures for creating a plasmid with FimB, as well as the procedures for knocking out a gene.

In order to test how effectively the pili flocculate, we are planning to create an E. coli strain with fimE knocked out.

8/7/2010

Kevin, Marc, Alena

PCR #1 Used a gradient from 40C to 60C for the first 3 cycles to find the optimum anneling temperature.

All of the annealing temperatures gave a good result according to the gel.

8/7/10 notes

8/9/2010

Kevin, Marc, Alena

Used a 57C degree annealing temperature to get enough DNA for the digest and ligation.

4 out of the 5 PCR reactions worked well according to the gel.

The 5th well could have been a loading problem or there wasn't enough DNA.

8/9/10 notes

8/11/2010

Kevin, Marc

Met with Chris, received advice for updating digest and ligation protocols.

8/12/2010

Kevin, Marc, Alena

Met and discussed protocols for digestion and ligation of FIMB into pBAD.

1. Added 5 mL of LB broth each to 2 50 mL falcon tubes in the ERB lab using sterile technique.

2. Added 5 microliters of Kanamycin to each of the 50 mL tubes in step 1.

Went to the budget committee meeting for 1 hour with the tubes.

3. Obtained the cryostock of pBAD from the Lin -80C freezer (iGEM box cell #73)

4. Stabbed cryostock using a sterile 200 microliter pipette tip and pipetted into media from step 2.

5. At 8:05PM placed the two falcon tubes from step 4 into the incubator/shaker at 30C.

8/14/2010

Kevin, Marc, Alena

Miniprep pBAD plasmid

1. inoculate 5.0mL LB in 50mL conical tubes w/ 100ug/mL of ampilicin
   1. the cultures (2 of them) grew for ~12hrs (8am to 8pm)
2. centrifuge (using the 50mL conical tubes) for 5000rpm for 10min at 4C
3. carefully discard the supernatant and resuspend the pellet with 250uL of P1 buffer (kept in the 4C fridge)
4. transfer into a labelled 1.5mL eppendorf tube (set pipetman to 500uL just in case)
5. add 250uL P2 Buffer--> invert 4-6times (should turn blue)--> add 350uL N3 buffer--> invert 4-6times (should become vicious or clumpy)
   1. centrifuge for 13,000rpm for 10mins
6. pipet out supernatant into a QIA spin column--> centrifuge for 60s--> discard flow through
   1. did not pipet out all of the supernatant
7. add 500uL PB buffer --> centrifuge 60s--> discard flow through
8. add 750uL PE buffer--> centrifuge 60s--> discard--> centrifuge 60s again
9. transfer into a labeled 1.5mL eppendorf tube--> add 50uL EB (elution buffer)
   1. allow it to sit for 1 min (it helps to release the DNA from the column)

fimB PCR product Purification

1. Used sample A and B of PCR product (save C and D for later)
   1. total volume of PCR product = 81 uL (40.5uL separately)
2. add 5 volumes of PB buffer (202.5uL) to 1 volume of PCR product (40.5uL); invert
3. transfer into a QIAquick spin column (provided in the Qiagen kit)
   1. set pipetman to 260uL to be sure to get all of the mixture
4. centrifuge spin column at 13,000rpm for 60s
5. discard the flow through (in the collection tube) and add 750uL PE buffer (to wash the DNA)

repeat step 4 again

1. discard flow through and centrifuge again to get the remain buffers out
2. place the column into a labeled 1.5mL eppendorf tube
3. add 50uL EB buffer (to elute out the DNA) directly to the white inner circle of the column (avoid touching the pipet tip to the column)
   1. allow the mix to sit in the column for 1 min, then centrifuge for 1 min (13,000rpm)
   2. remember to point the cap of the eppendorf tube in the opposite direction of the centrifuge machine

fimB Digest

1. pre-programmed PCR machine to Digest (DIG1)
2. use 5 PCR reaction tubes for each; 5 for fimB and 5 for pBAD
3. add the following amounts in that order (total volume of 20uL)

• 16 uL of the DNA (fimB and pBAD to their respective tubes)
• 2 uL of NEB 2 buffer
• 1 uL NcoI
• 1uL HindIII

1. incubate for 37C overnight (12hrs)
   1. place into 4C fridge the next day

8/16/2010

Kevin, Marc, Alena

Alena order/picks up NEB Ligase (T4 DNA Ligase, 20,000U/mL) from MSRB II enzyme store

Attempted experiment:

• Added 1uL of CIP (Calf Intestine Phosphatase) to plasmid reactions only (pBAD)
  • CIP will cut off the 3' phosphate to prevent the plasmid from folding back on itself
• incubate CIP-pBAD at 37C for 1 hr and then heat-shock (65C for 15 min)

We made a huge human error when setting up our thermal cycle program on the PCR machine. We did not realize we only set the thermal cycle for our digest on 8/14/2010>> [1] for 12 minutes (12:00) rather than the intended time period of 12 hours (12:00:00). This explains the condensation present in the PCR machine (it stayed at 4C for too long). We discovered our mistake after running the incubation of CIP+pBAD for 37C for (1:00) 1 minute then heat-shock at 65C for 15 minute. It is VERY IMPORTANT to triple check one another when entering in any program.

Rest of the day:

• headed over to the ERB and made two cultures of 5mL LB-Amp (in 50mL conical tubes)--> placed in the Lin Lab 4C fridge

8/17/2010

Kevin, Marc, Alena

Marc inoculated the two cultures made last night (8/16/2010) with pBAD and grew in 37C shaker (~9am to ~6p, 9hrs)

NOTE REGARDING LB CULTURE MADE ON 8/16/2010

• Marc noticed the 400mL LB media made in the ERB to be contaminated.
• Our two cultures could have been contaminated but pBAD grew pretty well (saturated) so we're not too worried

fimE and fimB knock out (K.O.) strains came in

• Marc adds 50uL of kanamycin onto each LB-agar plate and spread evenly with sterile glass beads
  • LB-agar plate ~25mL
  • Jeremy Minty's advice: more Kan is better than too little --> Kan 100 instead of Kan 50
  • allow the plate to absorb the kanamycin for 2 hours before applying the strains
• Alena follows Jeremy Minty's example of carefully taking out the small filter circle out of the foil with sterile techniques onto a LB-agar plate (previously labeled)
  • add ~75uL of LB onto the filter circle
  • streak the filter paper w/ inoculating streakers (used 3 of them)
• incubate plates w/ filter paper (still on the agar) upside (agar side up) at 37C overnight

Followed 8/14/2010 protocol (above) for:

• Miniprep for pBAD plasmid
  • Last time we used the incorrect spin column (QIAquick spin column; purple; used for PCR purification). This time we used QIAprep spin column (blue; had no cap on the column)
• PCR purification for fimB
  • used PCR products labeled C and D tubes
• Digest fimB and pBAD
  • This time, made sure the time on the program was set to 12:00:00
  • 5 rxns for each fimB and pBAD (10 total tubes, 20uL total volume in each tube)

8/18/2010

Kevin, Marc, Alena

Met with Chris to discuss about yeast agglutination

• Chris sent us a possible paper with a decent protocol to look at for the assay Media:YeastAgglutinationPaper.pdf

NOTE ABOUT K.O. STRAINS

• fimE K.O. did not grow out (which contradicts the prediction that fimE K.O grows faster than fimB K.O.)
• try letting the plate grow at 37C for a longer period of time (one colony observed)
• remove the filter circle paper and put onto a new LB-agar plate
• suspect too much kanamycin on the plate

Lab work:

1. add CIP (calf intestine phosphatase= cuts off the 3' phosphate to prevent plasmid from closing back on itself) to plasmid (pBAD) reaction tubes only--> incubate at 37C for 1 hour (using PCR machine)
2. heat/inactivate pBAD and fimB reaction tubes for 15 min at 65C (again using PCR machine)
3. perform DNA purification on the digests (follow 8/16/2010 procedure)
   1. using 210uL and 200uL of PB buffer for pBAD and fimB respectively
4. ran a gel on the digest

1. ran nanodrop3.0.1 on the digest
   1. use EB buffer as the blank
   2. ALWAYS clean twice when done using the machine

8/22/2010

Kevin, Marc

Ran Ligation of FimB and pBAD for 16 hrs O/N

Prepared culture of DH5a for electroporation tomorrow.

8/23/2010

Kevin, Marc

Growing competent cells for electroporation.

Colony started growing at 2:00.

Measured ODs

Precipitate Ligation product w/butanol

Add to eppendorf tube:

1. 50 ul ultrapure H2O
2. Ligation product
3. 500 ul butanol

Spin at 4C, 13000 rpm for 20 min

Made ampicillin plates

• Add 25 ul Amp100 to LB plates

6 plates:

• Cells only
• Plasmid only
• 2x pBAD+FimB undiluted
• 2x pBAD+FimB 1:100

9/2/2010

Kevin

Electroporation of pBAD+FimB into K12 and ΔFimB::kan cells.

15 plates
1x K12 control (amp)
1x Plasmid control (amp + kan)
1x ΔFimB control (amp + kan)
6x ΔFimB (amp + kan)
6x K12 (amp)

9/7/2010

Kevin

Started overnight cultures of 1:1000 dilutions of K12 and ΔFim::kan, both with the pBAD+FimB plasmid.

9/8/2010

Kevin

Created frozen stocks of K12 and ΔFim::kan w/the plasmid from overnight cultures. Stored them in box 1 in the ERB -20C freezer.

Ran miniprep of K12 and ΔFim::kan, using 5 ml of each culture. Note: Centrifuge in ERB will only go up to 5000 rpm w/50 ml tubes, therefore we transferred the culture to several eppendorf tubes. This was probably not a good idea, because we were left with a lot of leftover supernatant and that could have diluted the buffers. In the future, we should centrifuge the culture 1 ml at a time, and add 1 ml after each cycle.

Stored the product from miniprep in box 2 in the ERB -20C freezer.

9/9/2010

Kevin

Cryopreserved frozen stocks of K12 and ΔFimB::kan w/the plasmid.

Ran digest to determine whether the plasmid and FimB were actually in the cell. Used protocol from previous digest on 8/14.

9/10/2010

Kevin

Ran gel of previous day's digest, unfortunately the gel was inconclusive. There is an undetermined error with the gel electrophoresis machine.

10/2/2010

Kevin

Prepared tubes with K12 and ΔFim (Both w/insert) for an agglutination assay. We added algae and arabinose at an OD600 of 0.8 and then let the tubes grow

10/3/2010

Kevin

We were able to observe pellets in the tubes containing ΔFimB w/insert. This is exciting, but there are several things to note:

• By looking at the pellets under a microscope, we were able to see many flocs, some of which were a combination of E coli and algae, and some of which were pure E coli. This is a promising result.
• Both induced and uninduced tubes of ΔFimB formed flocs, however the arabinose induced tube had a much larger floc. This is possible evidence of leaky expression.
• None of the K12 tubes formed flocs.

We are planning to run a more quantitative assay later this week.

10/7/2010

Kevin

Ran flocculation assay.

1. Prepared O/N cultures of K12 and ΔFimB, both with the FimB plasmid.
2. Created 5 tubes: LB, LB+ΔFimB (both uninduced and induced), LB+K12 (both uninduced and induced)
   • Used 5 ml of cell culture due to low algae culture available
3. Added 100 ul arabinose during exponential phase
4. Added 5 ml algae to every tube
5. Shake for 10 min
6. Take 200 ul samples and measure OD600.

This graph illustrates the results. We can see a decline in the OD600 for all species, but there is no clear distinction between our control tube (LB+Algae) and the other tubes containing E. coli.

Suggested Modifications:

• This procedure was originally developed to measure a fast-acting chemical flocculant. Clearly, the time-scale will have to be changed to adjust for the slower E. coli.
• In between measurements, the tubes were allowed to sit at room temperature. It may be more beneficial to put the tubes in the incubator shaker, and take measurements less frequently.
• If we wait 2-3 hrs for the arabinose to fully induce and then start taking measurements, we should get better results.

Marc ran another qualitative assay, with positive results.

10/13/2010

Kevin

Ran PCR of FimB in preparation of making our final iGEM BioBrick standard part.

10/15/2010

Kevin

Ran gel of PCR products from 10/13. It appears that all of our wells contain our intended product, however 3 wells appear to contain unexpected products as well.

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10/18/2010

Kevin

PCR purified the FimB PCR product, using the PCR purification kit in 1230 ERB. Started o/n cultures of ΔFimB and K12 in preparation of another OD assay.

In the Lab