iGEM 2021 — Team Michigan

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# Team:Michigan/Notebook

Notebook | iGEM Michigan

[iGEM Michigan](https://2021.igem.org/Team%3AMichigan)

* Team

  [Team](https://2021.igem.org/Team%3AMichigan/Team)[Attributions](https://2021.igem.org/Team%3AMichigan/Attributions)[Collaborations](https://2021.igem.org/Team%3AMichigan/Collaborations)
* Project

  [Contribution](https://2021.igem.org/Team%3AMichigan/Contribution)[Description](https://2021.igem.org/Team%3AMichigan/Description)[Model](https://2021.igem.org/Team%3AMichigan/Model)[Experiments](https://2021.igem.org/Team%3AMichigan/Experiments)[Engineering](https://2021.igem.org/Team%3AMichigan/Engineering)[Notebook](https://2021.igem.org/Team%3AMichigan/Notebook)[Results](https://2021.igem.org/Team%3AMichigan/Results)[Implementation](https://2021.igem.org/Team%3AMichigan/Implementation)
* Parts

  [Parts](https://2021.igem.org/Team%3AMichigan/Parts)
* [Human Practices](https://2021.igem.org/Team%3AMichigan/Human_Practices)

[ ]

# Notebook

Timeline of Project

---

##### Contents

Wednesday June 9 2021

* Team Members: Amogh, Bonnie, Elizabeth
* Protocols Followed: (Non-His Tag) Alpha Factor + mNeon Green PCR with DpnI Digestion and Gel Electrophoresis
* Purpose: Incorporate alpha factors, which will aid endocytosis into yeast, into the existing mNeon plasmid using primers.
* Results:

![PCR Gel](https://static.igem.org/mediawiki/2021/3/3a/T--Michigan--img--n1.png)

* Lane 1: 10 kb DNA ladder, Lane 2 and 3: PCR product
* Gel was not run long enough to confirm the product was 7.5kB as expected, as the ladder was too compacted.

Monday June 14, 2021

* Team Members: Bonnie, Elizabeth
* Protocols Followed: Pouring Plates, Making Overnight Cultures
* Purpose: To prepare for future experiments
* Results:
* Autoclaved/Made LB, water, and poured plain, no abx agar plates
* Plates are parafilmed and in USB fridge
* LB and water are on the counter in main USB room

Wednesday June 16, 2021

* Team Members:
* Bonnie and Elizabeth did the first half of making chemically competent cells with BL21 plain overnights
* Steven, Amogh, and Carolyn finished this up
* Protocols Followed: Making Competent Cells, Making Overnight Cultures, Making Glycerol Stocks
* Purpose: To prepare competent cells to be used for transformation
* Results:
* Made and autoclaved 80% glycerol
* Ran PCR with new MA76 plasmid using GoTaq
* PCR failed, no bands
* Known errors:
* Possible extension time error, adjusted for day 2
* TBE buffer was added after sampled were added to wells, could have caused them to wash out

Thursday June 17, 2021

* Team Members: Bonnie, Elizabeth, Amogh, Alec
* Protocols Followed: PureLink MiniPrep Kit
* Purpose: To purify out encapsulin plasmid for sequencing for confirmation of alpha factor addition
* Results:
* Completed plasmid mini-preps of MA76 and TG27 strains from Cassie using the PureLink quick plasmid miniprep kit
* Samples were taken to nanodrop in Anthony's lab
* Samples are stored in USB lab freezer (-20 C)
* Reran same PCR with GoTaq, made extension time longer according to online protocol (from 4 min to 7.5 min)
* Holding at 4 C until tomorrow
* Cells were transported back to USB freezer

Friday June 18, 2021

* Team Members: Elizabeth, Bonnie, Adam, Alec
* Protocols Followed: His Tagged PCR: Alpha Factor + mNeon Green
* Purpose: Digest PCR Product, Make a New Batch of PCR Product
* Results:
* Attempted to dpnI digest Thursday's 06/17/21 PCR product, however contents of a mislabeled tube were added instead of dpnI.
* Reran new PCR with GoTaq to replace the one that was incorrectly digested. Holding at 4C until Monday 06/21/21.
* Filled and autoclaved 4 boxes of 0.1-10ul and 3 boxes of 1-20ul pipette tips

Wednesday June 23, 2021

* Team Members: Elizabeth, Bonnie, Anya
* Protocols Followed: Pouring Plates Protocol, Chemical Transformation Protocol
* Purpose: Purify Plasmid for Transformation to reproduce plasmid
* Results:
* Made 4 LB/Agar/Ampicillin Plates for transformation (Followed Pouring Plates Protocol, adding 100ul amp for 10ml LB/Amp after autoclaving)
* Performed Gel Extraction on the first PCR product gel from June 9th (Platinum Taq PCR product)
* Completed transformation (following Chemical Transformation Protocol) with two eppendorf tubes, one labeled BL21 and another labeled BL21 control (no DNA added)
* Plates with ampicillin were streaked with transformed cells and are in 37 C incubator (not the shaking one)

Thursday June 24, 2021

* Team Members: Amogh, Elizabeth, Bonnie
* Protocols Followed: Chemical transformation protocol, mNeon+His+SpyTag plasmid Protocol
* Purpose: To test for why transformation was unsuccessful
* Results:
* Transformation plates from yesterday have no growth.
* Today, I did a transformation of the comp cells with pUC19 to "test" if the comp cells are alright to use. Will check the plate tomorrow with growth (sitting at 37 in the incubator).
* Used chemical transformation protocol
* Made a couple more ampicillin LB plates which are in the fridge.
* Made glycerol stocks from the three plates in the fridge. Those are now in the -80 and should be used for overnights/plates in the future.
* There's also another restreaked BL21 plate in the incubator for no particular reason other than to have an extra on hand especially if we need to make more comp cells.
* Amogh did a DpnI digest on a green sample from the freezer thinking that it was the PCR product for the mNeon+His+SpyTag plasmid, but it wasn't actually a PCR product (gel was run the next day with no bands).

Friday June 25, 2021

* Team Members: Bonnie, Elizabeth
* Protocols Followed: Gel Electrophoresis
* Purpose: To confirm whether or not the PCR was successful
* Results:
* Moved newly streaked BL21 plate and transformation plate from yesterday (Thursday June 24, 2021) into the fridge.
* Made AMP plates (~50). Labeled and parafilmed.
* Cleaned freezer of old primers.
* Made and ran gel of PCR product from yesterday (Thursday June 24, 2021). The gel showed no bands in the PCR product column, and on further investigation, we found the PCR tube that should have been digested on Thursday still in the freezer. This means that the PCR digest with dpnI performed on Friday was not performed on the correct PCR product.
* The correct PCR product was digested with dpnI. A new gel was made and run.
* When visualized, no bands showed.
* Correct PCR Product/GoTaq WITH Purple Loading Gel (Couldn't see bands under UV light):
* Correct PCR Product/ GoTaq WITHOUT Purple Loading Gel (Still couldn't see bands under UV light):

![PCR Results](https://static.igem.org/mediawiki/2021/2/20/T--Michigan--img--n2.png)

Figure 2: PCR Results

* We now have one gel's worth of gelred left. May need to get a stain if gelred does not come in soon.
* Streaked the two plates from Cassie (MA76 and TG27). These will be left out on the counter until Monday (June 28th, 2021).

Monday June 28, 2021

* Team Members: Bonnie, Elizabeth, Amogh
* Protocols Followed: mNeon+Histag+Spytag plasmid using High Fidelity Platinum Taq DNA polymerase, Gel Electrophoresis
* Purpose: Attempt to confirm whether or not PCR was successful in adding alpha factor to the mNeon plasmid
* Results:
* Made overnights for comp cells for BL21 cells

![PCR Results](https://static.igem.org/mediawiki/2021/6/6d/T--Michigan--img--n3.png)

Figure 3: PCR Results

* Did PCR on the mNeon+Histag+Spytag plasmid using High Fidelity Platinum Taq DNA polymerase
* Performed a DpnI digest on completed PCR product
* Used PCR product to run gel electrophoresis

Tuesday June 29, 2021

* Team Members: Bonnie, Elizabeth, Amogh
* Protocols Followed: Making Competent Cells, Chemical Transformation
* Purpose: To attempt to transform plasmid into competent cells to amplify plasmid quantity
* Results:
* Made competent cells using the BL21 overnight cultures from yesterday (6/28).
* Transformed the competent cells using the PCR product from yesterday (6/28) and from the gel extraction from (6/09), along with. a positive and negative control. Plated on Amp+ plates.

Wednesday June 30, 2021

* Team Members: Bonnie, Elizabeth, Amogh, Carolyn
* Protocols Followed: Chemical Transformation
* Purpose: Reattempting successful transformation of modified T4 encapsulin plasmid
* Results:

![New Plates](https://static.igem.org/mediawiki/2021/6/6a/T--Michigan--img--n4.png)

Figure 4: New Plates

* Redo Transformation from yesterday 6/29 with the following plate:
* pUC19 (Carolyn's BL21)
* pUC19 (MSBT BL21)
* PCR Product From 6/28 (Carolyn's BL21)
* PCR Product From 6/28 (MSBT BL21)
* PCR Product From 6/28 (MC1091)
* Negative Control (MSBT BL21)

Friday July 2, 2021

* Team Members: Bonnie, Elizabeth, Amogh
* Protocols Followed: Invitrogen Miniprep Kit Protocol
* Purpose: Miniprep plasmids from transformation for sequencing to confirm presence of alpha factor
* Results:
* Mini Prepped overnight cultures from yesterday, using invitrogen miniprep kit (protocol in box)
* Some cultures did not grow very well, so we only mini prepped cultures B, C, D, E, H
* Used Anthony's nanodrop to measure concentrations of plasmid
* B: 52.244 ng/ml
* C: 62.679 ng/ml
* D: 35.689 ng/ml; gave us error message
* E: 39.187 ng/ml
* H: 62.921 ng/ml
* Concentrations of D and E were too low and probably would not give good results, so we did not send them in to sequencing
* Sent samples B, C, and H in for sequencing through Eurofins

Monday July 12, 2021

* Team Members: Bonnie, Elizabeth, Amogh
* Protocols Followed: Gel Electrophoresis
* Purpose: Gel extract the PCR product to remove extraneous DNA
* Results:
* Made and ran a gel with the PCR product from 06/28/21 then performed gel extraction in order to clean up the pcr and send it in for sequencing. Epindorf tube of this is in the freezer labelled "Purified PCR Prod. for Seq." and contains 12ul of solution
* Made re-streaked and made overnight from the transformed plate.
* Results as of Tuesday 07/13/21 at 10am. Streak towards the bottom of the plates labeled A-H is the streak done on 07/12/21. The streak towards the top of those same plates is from 07/01.

![New Plates](https://static.igem.org/mediawiki/2021/7/7e/T--Michigan--img--n5.png)

Figure 5: New Plates

Tuesday July 13, 2021

* Team Members: Bonnie, Elizabeth, Amogh, Alec
* Protocols Followed: Invitrogen Miniprep Kit Protocol, Making Overnights Protocol
* Purpose: Collect plasmids from overnights to send for sequencing
* Results:
* More Eppendorf tubes were autoclaved.
* OD of a randomly selected overnight was 2.148. All of the overnights appear to be about the same turbidity. The corresponding streak plates all showed growth as well.
* 4 of the overnight cultures were selected and mini-prepped. These are in the freezer labelled A-D with the date (7-13) labelled on the side of the tube. Do not confuse it with the previous minipreps form 07/01 or 07/02, which also have tubes labelled B, C, and D.
* The other 4 overnights were left in the incubator to continue growing along with the overnight of the old Cassie plate (not the re-streak of the Cassie plate which should be miniprepped tomorrow).
* The 4 overnights that were in the shaking incubator were centrifuged for 5 mins and then the LB was decanted. The pellets were then stored in the freezer.
* The old Cassie plate was used to make an overnight dish which is in the shaking incubator. This should be mini prepped tomorrow.

Wednesday July 14, 2021

* Team Members: Bonnie, Elizabeth, Amogh, Alec
* Protocols Followed: Making Overnights
* Purpose: Assess minipreps for ability to sequence and increase plasmid concentration in our minipreps
* Results:
* Made overnights with amp for expression, purification, and isolation protocol.
* Nano-Drop of Minipreps of plasmid from yesterday (7/13):
* A 40.209 ng/uL
* B 27.69
* C 30.678
* D 46.937
* We threw these plasmids away because the concentrations were too low to send in for sequencing. We remade overnight to rerun mini prep tomorrow.
* Anthony's suggestion: Use around 30 uL of elution buffer while doing mini prep to increase plasmid concentration

Thursday July 15, 2021

* Team Members: Bonnie, Elizabeth, Amogh
* Protocols Followed: Making Overnights
* Purpose: Redo overnights for plasmid purification because previous overnights were contaminated
* Results:
* The LB was contaminated so a new LB was made and autoclaved.
* The overnights made yesterday did not have AMP in them and so they were thrown away. New overnights were made using the new LB.
* The plasmids will need to be mini prepped and sent in for sequencing tomorrow afternoon.

Friday July 16, 2021

* Team Members: Bonnie, Elizabeth, Amogh, Anya
* Protocols Followed: Invitrogen MiniPrep Kit Protocol
* Purpose: Collect plasmids from overnights to send for sequencing
* Results:
* ****MAKE SURE TO ELUTE THE MINIPREPS IN ONLY 30ul OF ELUTION BUFFER****
* Anya, Bonnie, and Elizabeth mini-prepped 6 (A-F) of the 8 overnights. All of the overnights grew about the same amount and are turbid.
* The mini prepped samples are labelled and in the -20 freezer in the main room.

Tuesday July 20, 2021

* Team Members: Bonnie, Elizabeth, Amogh, John
* Protocols Followed: Expression, Isolation, and Purification of Encapsulins
* Purpose: Prepare for Expression of the Encapsulin Protein
* Results:
* The cassie cell overnight grew to be turbid, the transformed cells did not
* Sent in samples for sequencing
* Continued expression per the protocol
* Made a small (500 uL) aliquot of 1 M IPTG to use for expression
* Made an overnight of MC1061 for both comp cells and a glycerol stock tomorrow
* Restreaked transformed cell plate to determine if cells are completely dead
* Preinduction Sample had OD of 0.837

Wednesday July 21, 2021

* Team Members: Bonnie, Elizabeth, Amogh, Alec
* Protocols Followed: Making Glycerol Stocks, Expression, Isolation, and Purification of Encapsulins
* Purpose: Begin protein expression process
* Results:
* Made new LB media since the one we had looked contaminated. Made 3 100mL aliquots of LB, using the instructions in the Making Overnights protocol.
* I labeled two of the jars "Not Used" to indicate that they are still 100% sterile from the autoclave. Try to keep using the one that is not labeled to minimize how much of the LB gets contaminated.
* Made glycerol stock of MC1061 cells as described in benchling protocol and stored in -80 freezer.
* May want to redo this if LB was contaminated?
* Expression:
* OD of induced sample: 0.579 OD, 5.79 OD/mL
* Dilution: (0.837 OD \* 3 mL)/5.79 OD/mL --> 0.434\*1000 --> 434 uL to PBS
* Finished the expression protocol, except for the SDS PAGE gel. The uninduced and induced tubes are both in the -20.
* Made comp cells with the MC1061 plate from Anthony

Friday July 23, 2021

* Team Members: Elizabeth, Amogh, Alec
* Protocols Followed: MA76+Alpha Factor 2-Step PCR
* Purpose: Attempt adding alpha factor using PCR in a two step process, as opposed to a single step process as before
* Results:
* qRun A PCR:
* One with primers A, B, and C as usual
* We then need to clean with a gel and run another PCR on this with just primers B and C to confirm the presence of alpha factor
* Put the PCR product in the -80C so that the dpnI doesn't do anything weird
* Ran the PCR with MA76 (T4+His+Spy)
* Ligated with quick ligase and t4 ligase buffer. We did three times the amounts listed in the protocol
* Dpn1 digest was started and put in the thermocycler to hold at 4C until monday

Monday July 26, 2021

* Team Members: Bonnie, Elizabeth, Adam
* Protocols Followed: Gel Electrophoresis
* Purpose: To confirm if new PCR process worked
* Results:
* Ran a gel from the ligated, digested PCR product from Friday
* No product :(
* Rerun same PCR and resume tomorrow

![PCR Results](https://static.igem.org/mediawiki/2021/d/da/T--Michigan--img--n6.png)

Figure 6: PCR Results

Tuesday July 27, 2021

* Team Members: Bonnie, Elizabeth, Amogh, Alec
* Protocols Followed:MA76+Alpha Factor 2-Step PCR, Ligation with T4 Ligase
* Purpose: Attempt adding alpha factor using PCR in a two step process, as opposed to a single step process as before
* Results:
* Ligated 20ul with Quick Ligase and t4 ligase buffer, following the Ligation with t4 ligase protocol.
* Possible Issues We can Already Foresee:
* Ligase expired in 2019
* Many of ligase buffers were expired but we did find one that was not
* Digested the product with dpnI.

Wednesday July 28, 2021

* Team Members: Bonnie, Elizabeth, Amogh
* Protocols Followed: Gel Electrophoresis
* Purpose: COnfirm the addition of alpha factor to our plasmid via two step PCR
* Results:
* Ligated 10ul of the PCR product from 06/28/21 following the "Ligation with T4 Ligase Protocol" and holding it in the thermocycler for 30 minutes at room temperature.
* Made a gel electrophoresis gel and stored it in a buffer on the counter.
* Started two new PCR reactions at 12pm.
* One with MA76 and undiluted primers following the "mNeon+Alpha Factor PCR" protocol
* One with ligated 06/28 PCR product and undiluted primers also following the "mNeon+Alpha Factor PCR" protocol
* Ran both PCRs in a gel.
* The ladder is in the bottom/leftmost lane. A new PCR run today with MA76 and primers A, B, and C is in the middle lane. The only PCR product from 06/28 that was ligated this morning and then PCR-ed with only primers B and C is in the top/rightmost lane.

![PCR Results](https://static.igem.org/mediawiki/2021/b/b1/T--Michigan--img--n7.png)

Figure 7: PCR Results

Thursday July 29, 2021

* Team Members: Anya, Bonnie, Elizabeth
* Protocols Followed: Gel Electrophoresis
* Purpose: Determine if ligation of the plasmid occurred
* Ligation Results:

![PCR Results](https://static.igem.org/mediawiki/2021/4/4e/T--Michigan--img--n8.png)

Figure 8: PCR Results

Hold at room temperature for 30 minutes, then heat inactivate as the protocol instructs

* The plasmid that was made yesterday with ABC primers was ligated. There was 55 uL of PCR product, so the amounts in the protocol were multiplied by a factor of 11 (55/5 uL PCR product). I held the ligated mixture for 30 mins rather than 10 mins as the protocol says, but heat inactivated as the protocol says. We ran out of T4 DNA ligase, so I ordered some more.
* A gel was made with the ligated product
* Gel showed positive results, so pcr was started with the ligated pcr product and only primers B and C.

Friday July 30, 2021

* Team Members: Bonnie, Elizabeth, Amogh, Anya
* Protocols Followed: MA76+Alpha Factor 2-Step PCR
* Purpose: Run PCR with only the B and C primers to complete alpha actor addition
* Results:
* Made and ran a gel of the PCR run with only primers B and C

Thursday August 5, 2021

* Team Members: Bonnie, Elizabeth, Amogh
* Protocols Followed: MA76+Alpha Factor 2-Step PCR
* Purpose: Run PCR reagent dilutions to optimize PCR
* Results:
* Started PCR reactions:
* One set of 7 for ABC primers + original plasmid
* One set of 7 for BC primers + pcr product
* Control -- no DNA
* Control -- undiluted DNA
* Dilutions:
* 1:10
* 1:100
* 1:500
* 1:1000
* 1:2000

Tuesday August 10, 2021

* Team Members: Bonnie, Elizabeth, Amogh, John
* Protocols Followed: MA76+Alpha Factor 2-Step PCR
* Purpose: Run PCR at a gradient of annealing temperatures to optimize PCR reaction
* Results:
* PCR of plasmid and alpha factor linker to add overhangs
* Annealing temperature is 56 C
* Reaction to make the insert PCR product (See Inverse Fusion PCR Cloning Paper in Literature Drive).
* Reaction is completed with only the A and B primers and on template DNA MA76 (T4 enc + his + spy).
* Annealing Temperatures: 52.5 - 66 C
* Reaction Quantities
* 0uL Reaction
* 61.6 μL nuclease free water (enough to get to 50 μL total volume per PCR tube)
* `Find in storage room freezer
* 1.6 μL of 10uM (500nM) primer A
* 1.6 μL of 10uM (500nM) primer B
* 1.6 μL mNeon plasmid DNA
* MA76 (make sure it includes his tag)
* 8 μL 10x High fidelity PCR buffer
* 3.2 μL MgSO4
* 1.6 μL dNTP mix
* 0.8 μL Platinum Taq DNA polymerase

Wednesday August 11, 2021

* Team Members: Bonnie, Elizabeth, Amogh
* Protocols Followed: Gel Electrophoresis
* Purpose: Visualize differences annealing temperature made to PCR reaction
* Results:
* Ran gels to confirm PCR results from the PCR reaction with only the A and B primers testing different annealing temperatures and a gel with the vector and plasmid PCR for Gibson assembly.
* Gel for A-G. All not big enough to be 7kB

![PCR Results](https://static.igem.org/mediawiki/2021/c/cf/T--Michigan--img--n9.png)

Figure 9: PCR Results

* A-H are as follows for PCR reactions with the A and B primers
* Annealing Temperatures:
* Gel with H in lane 3, gib fragment in lane 3 and Gibson vector in lane 4.
* H should have been an amplified plasmid (7 kB) and the band is not large enough to be our encapsulin plasmid (This band is in the box below)
* The fragment pcr did not need to be run as the fragment already has overhangs
* The vector pcr should have resulted in amplification of our plasmid (7 kB) and so the band does not indicate that amplification was correct because it is too far down.

![PCR Results](https://static.igem.org/mediawiki/2021/b/b0/T--Michigan--img--n10.png)

Figure 10: PCR Results

Thursday August 12, 2021

* Team Members: Bonnie, Elizabeth, Amogh
* Protocols Followed: Gel Electrophoresis, MA76+Alpha Factor 2-Step PCR
* Purpose: Re-Attempt 2-Step PCR
* Results:
* I had to change our gel electrophoresis power supply (dark blue one) and got a new submarine because the other ones were broken.
* Ran gel on yesterday's PCR product to see if the Gibson assembly plasmid successfully incorporated overhangs.
* Ran PCR on MA76 plasmid with A and B primers with an annealing temp of 55.8 C. Will run gel in the afternoon.

![PCR Results](https://static.igem.org/mediawiki/2021/7/70/T--Michigan--img--n11.png)

Figure 11: PCR Results

* Looking better today! Lane 2 is the plasmid unmodified, lane 3 is primer A, lane 4 is primer B and lane 5 is our pcr product with a and b primers

Friday August 13, 2021

* Team Members: Bonnie, Elizabeth, Amogh
* Protocols Followed: MA76+Alpha Factor 2-Step PCR, Gibson Assembly
* Purpose: Continue re-attempt of 2-step PCR and explore Gibson Assembly as a new addition method for adding alpha factor to our encapsulin
* Results:
* Inverse Fusion PCR Step 2 was started
* PCR was run on the Inverse Fusion PCR product from step one (AB primers only) with only the BC primers.
* 2ul of Gel extracted product for the AB Primer PCR and 0.5ul of the template MA76 were added to the reaction tube since we didn't know the exact concentrations of either
* Annealing temperature was set to 55.8 C, as this was the annealing temperature calculated by a Tm calculator and was the temperature most successful for the AB primer PCR.
* New gel on the top shelf of the fridge for the afternoon group. Please make sure to run control lanes of MA76, The PCR product from the Inverse Fusion with only AB Primers, Primer B, and Primer C.
* There is a label by the thermocycler for the PCR tube that is in it too!
* The results of the gel for today. Both Primers B and C in lanes 4 and 5 can be seen. The Forward Vec and Reverse Vec can also be seen in the last two lanes. The 6th lane contains the PCR product from 8/12 intended to be used for Gibson Assembly. The 3rd lane does not show any bands and holds the PCR product from the Inverse Fusion with only AB Primers. The 1st lane contains the ladder and the second lane contains the MA76 plasmid.

![PCR Results](https://static.igem.org/mediawiki/2021/0/02/T--Michigan--img--n12.png)

Figure 12: PCR Results

* Gibson Assembly was also conducted today. The standard Gibson Assembly Protocol provided by NEB was used. 3 ul in total from the fragments were used, 17 ul of water and 10 ul of the Gibson MasterMix. This was incubated in the thermocycler at 50 C for 15 min and stored at -20 C in the freezer for sequencing. The 3 ul consisted of 0.5 ul from each component in the mix. This included 0.5 ul of the vector, its respective Forward and Reverse fragments, the 1:10 diluted Alpha Linker, and its respective Forward and Reverse fragments. This totaled the 3.0 ul that was added.

Thursday September 9, 2021

* Team Members: Amogh, Adam
* Protocols Followed: Expression, Isolation, and Purification of Encapsulins
* Purpose: Confirm expression was successful in previous test
* Results:
* SDS page gel was run and stained 09/08. Was left in DI water to destine overnight.
* The lane farthest to the left is the ladder, and the labeled band should be 35.8kDa. The second lane is the pre-induced sample, followed by the induced sample. It looks like there is a band in both at about 37kDa which is what we roughly estimated the size of our encapsulin as.

![SDS Page Gel Results](https://static.igem.org/mediawiki/2021/6/6b/T--Michigan--img--n13.png)

Figure 13: SDS Page Gel Results

Monday September 13, 2021

* Team Members: Amogh, Adam, Bonnie, Elizabeth
* Protocols Followed: Gibson Assembly PCR
* Purpose: See if Gibson Assembly could add alpha factor to plasmid
* Results:
* Ran PCR on MA 76 plasmid to add overhangs for Gibson assembly. Added 1 uL of both forward and reverse vector sequences. Annealing temp was 57 C as calculated by NEB Tm calculator (may have been wrong - conflicting Tm is 68 C). Gel electrophoresis was run the next day and nothing showed up.

Tuesday September 14, 2021

* Team Members: Amogh, Adam, George
* Protocols Followed: Gibson Assembly
* Purpose: Continue Gibson Assembly
* Results:
* Ran gel from gibson overhang yesterday. No product seen, and the ladder is very bunched up despite being run for 45 minutes.

Tuesday September 21, 2021

* Team Members: Amogh, John, George
* Protocols Followed: N/A
* Purpose: Prepare for Lab
* Results:
* Made 1X TBE using 100 mL of 10X TBE and adding 900 mL of DI water

Monday September 27, 2021

* Team Members: Elizabeth, Amogh, Adam, George, Alec
* Protocols Followed: Invitrogen Gel Extraction Kit Protocol
* Purpose: Purify DNA and Proteins
* Results:
* Made LB and autoclaved it
* Measured gel extracted plasmid DNA with nanodrop
* 4.44 ng / ul
* high amount of protein / contaminants in DNA sample
* Anthony gave us crystallography columns for protein purification
* Made overnight cell cultures

Tuesday September 28, 2021

* Team Members: Elizabeth, Amogh, Adam
* Protocols Followed: Overnights
* Purpose: Create Overnight Cells
* Results:
* Overnights of cells for minipreps made to produce more plasmid
* MA76 glycerol stock from the -80
* Day 2 of expression protocol

Thursday October 7, 2021

* Team Members: Bonnie, Elizabeth, Amogh, Steven, Adam
* Protocols Followed: Protein Expression
* Purpose: Finish expression
* Results:
* Ran the remainder of the expression protocol up until the point of running the gel and dialysis

Tuesday, October 12, 2021

* Team Members: Adam, Amogh
* Protocols Followed: Gel Electrophoresis
* Purpose: Evaluate Gibson Assembly
* Results: Inconclusive
* Run gel for gibson pcr overhangs

Monday, October 18, 2021

* Team Members: Alec
* Protocols Followed: yeast overnights
* Purpose: created overnight yeast cultures

Wednesday, October 20, 2021

* Team Members: George, Amogh, Alec
* Protocols Followed: endocytosis into yeast
* Purpose: endocytose encapsulin without alpha factor into yeast as negative control
* Results:
* Viewed encapsulin and yeast under fluorescent microscopy
* Believe we saw encapsulin, endocytosis seems unsuccessful as expected although difficult to determine conclusively

![Pure encapsulin (clusters possibly present in white circles)](https://static.igem.org/mediawiki/2021/1/12/T--Michigan--img--n14_2.png)

Figure 14: Pure encapsulin (present in white circles)

![Pure yeast](https://static.igem.org/mediawiki/2021/a/ad/T--Michigan--img--n15.png)

Figure 15: Pure yeast

![Encapsulin + yeast](https://static.igem.org/mediawiki/2021/e/ef/T--Michigan--img--n16.png)

Figure 16: Encapsulin + yeast

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# Team:Michigan/Engineering

Engineering Success | iGEM Michigan

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# Engineering Success

Following the Engineering Design Cycle

---

##### Contents

![Biology Engineering Cycle](https://static.igem.org/mediawiki/2021/3/3e/T--Michigan--img--engin_cycle.png)

# PCR Optimization

While our PCR experiment to add an alpha factor sequence to the encapsulin was not a standout success, its optimization did follow the engineering cycle of designing, building, testing, and learning, and then repeating this process iteratively.

*Design*

We first designed the primers for inverse fusion PCR, considering factors like melting temperature and linker position in designing the sequences.

*Build*

We then used these primer sequences to run inverse fusion PCR.

*Test*

We tested the results by running gel electrophoresis and sequencing the plasmids. While certain gels looked promising, successful sequencing proved elusive.

*Learn*

When the results deviated from our expectations, these discrepancies were analyzed and parameters like primer concentration, annealing temperature, and polymerase type were adjusted before repeating the cycle.

# Encapsulin Protein Purification

Likewise for our purification of the encapsulins without alpha factor, we also followed the principles of the engineering cycle.

*Design*

We first designed a plasmid containing the encapsulin protein carrying an mNeonGreen fluorescent cargo for visualization and a his-tag used for protein purification.

*Build*

Then, we transformed the plasmid into competent BL21 E. Coli cells for the protein to be expressed. Afterward, we performed encapsulin protein purification using the his-tag. In doing so, the encapsulin protein was “built” by bacterial machinery.

*Test*

To evaluate our results, we ran an SDS page gel to confirm the size of the protein matched the expected size of the encapsulin. This assay indicated successful purification. We also viewed the encapsulin under a fluorescent microscope with inconclusive results. The protein concentration was evaluated with a NanoDrop.

![SDS Page Results](https://static.igem.org/mediawiki/2021/6/6b/T--Michigan--img--n13.png)

Figure 2: SDS Page Results

![Fluorescence Microscopy (possible encapsulin clusters circled in white)](https://static.igem.org/mediawiki/2021/1/12/T--Michigan--img--n14_2.png)

Figure 3: Fluorescence Microscopy (possible encapsulin clusters circled in white)

*Learn*

While the assays seemed to indicate successful encapsulin purification, a low protein concentration led us to believe that higher specific activity could be achieved in further iterations of the engineering cycle.

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# Team:Michigan/Experiments

Experiments | iGEM Michigan

[iGEM Michigan](https://2021.igem.org/Team%3AMichigan)

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# Experiments

Overview of Project Experimental Protocols

---

##### Contents

This section displays the experiment protocols used during the cloning, expression, and endocytosis of the protein encapsulin nanocompartment.

# Chemical Transformation

Chemical transformation is a common way to incorporate plasmid DNA into chemically competent bacterial cells. MSBT used this protocol to incorporate the encapsulin plasmid into E. Coli cells. Plates with ampicillin were used to select for the cells that had taken up the plasmid, which contains ampicillin-resistance.

**Materials**

* Comp cells
* Plasmid DNA
* Water bath at 42 C
* SOC media
* Agar plate

**Procedure**

1. Thaw 50 uL aliquot of comp cells in ice bath
2. Gently mix thawed cells ONLY BY TAPPING, do not pipette or vortex
3. Add 2-5 uL of DNA, do not mix
4. Place on ice for 30 minutes
5. Heat shock cells for 45 seconds at 42 C timing is exact here Use the water bath to heat shock
6. Place tubes on ice again for 1-2 minutes
7. Add growth media (SOC or LB) to bring final volume to 500 uL Should be adding about 445 uL if adding 5 uL of DNA in step 3
8. Incubate at shaking icubator at 37 C for one hour 160-225 rpm
9. Spin down cells for 2.5 minutes at 1000 rcf (9000 rpm)
10. Carefully pour off about 80% of supernatant into waste container, leaving 100 uL
11. Resuspend cells in remaining supernatant
12. Spread all cells onto plate and grow at 37 C overnight

# Dpn1 Digestion

The digestion was used following PCR to break down any remaining plasmid template DNA prior to ligation. Dpn1 targets methylated DNA, resulting in cleavage of only the template, not the PCR product.

**Materials**

* PCR product
* 10X CutSmart™ Buffer
* Dpn1

**Protocol**

* Add 50 μL (all) of PCR product with 10 μL 10X CutSmart Buffer and 1 μL DnpI
* Found in main room freezer
* Incubate at 37 C for 1 hour
* Heat-inactivate DpnI by incubating at 80°C for 20 minutes
* After running, place it in the fridge to keep until ready to run a gel

# Expression, Isolation, and Purification of Encapsulins

The expression, isolation, and purification of the encapsulins was performed in two stages. First, a test protocol was performed. This was followed by a large batch general expression and his-tag purification using a nickel column. This protocol resulted in pure encapsulin proteins that can be used in endocytosis.

**Materials**

* 1M solution IPTG
* Ampicillin
* LB Media
* PBS
* Tris (0.0024228 g / 1 mL)
* NaCl (0.008766 g/ 1 mL)
* SDS
* Chicken egg white lysozyme
* Bovine DNAsel

**Procedure**

* Inoculate a few mL overnight LB with ampicillin (1:1000, stock solution = 100 mg) and shaking at 37 C
* Overnight stock: add 5 uL amp to 5 mL of LB + colony
* Next day: inoculate 1 mL of overnight in 50 mL with Amp, place in shaking incubator at 37 C until OD reaches 0.6-0.8
* Take away 3 mL of grown bacteria and freeze in 15 mL falcon tube (note OD before --> this is preinduction sample)
* Add 0.1mM (1:10000 of 1M stock IPTG) to induce protein expression and leave shaking at 18C to express protein overnight
* Use a 1 M solution of IPTG diluted in autoclaved, distilled water
* For a 45 mL culture, add 4.5 uL of IPTG
* Next day: Dilute overnight 1:10 in LB to measure OD and record measurement (example: if you measure 0.43 OD, record 4.3 OD/mL)
* Dilution: (OD preinduced sample\*3mL)/(OD from step 5) = some number S --> Add (S x 1000) uL of induced sample in 3 mL PBS
* Spin both pre-induction and diluted induced sample from step 6 down in 15 mL falcon tube
* Discard supernatant and carefully take away any additional supernatant
* Add 1 mL of 20 mM Tris (pH=7.5) and 150 mM NaCl to supernatant
* Either: a) Sonicate, or b) Freeze-Thaw Method
* Take out lysate, put into 1.5 mL eppendorf, and spin 8500x g for 10 minutes
* Take out all of supernatant, add 1 mL of buffer to the pellet and resuspend pellet
* Run samples on SDS page gel
* Inoculate a few mL overnight LB with ampicillin (1:1000, stock solution = 100 mg) and shaking at 37 C
* Overnight stock: add 5 uL amp to 5 mL of LB + colony
* For a 1 L total make 10 mL of culture
* For 500 mL total use 5 mL of culture
* Next day: inoculate 5 mL of overnight in 500 mL with Amp, place in shaking incubator at 37 C until OD reaches 0.8 OD
* Add 0.1mM (1:10000 of 1M stock IPTG) to induce protein expression and leave shaking at 18C to express protein overnight
* Use a 1 M solution of IPTG diluted in autoclaved, distilled water
* For a 500 mL culture, add 50 uL of IPTG
* Next day: spin down cells, take them out, and put in 50 mL falcon tube Weigh pellet
* Add 5 mL/g of cells of 20 mM Tris (pH=7.5) and 150 mM NaCl buffer tube of 10x solution in cubboard in rack
* Add 0.1 mg of chicken egg white lysozyme and 0.1 mg bovine DNAsel/1mL of buffer
* Resuspend cells (gently until homogenous, will take a hot sec) and then sonicate (or freeze-thaw)
* SPRINT back and forth approx. 7 times
* glass of ethanol in -80
* 42 C for water bath or hot plate with container of water
* 3 minutes per station
* Spin down lysate for 20 minutes at 8500X g
* Add 2 mL of bed volume of Ni-NTA resin to buffer and let bind on rocker for about 2 hours at 4 C slow on side, cold as possible
* Put lysate + resin on a column and collect flowthrough
* Flow 10 Column Volumes (bed volume) of 20 mM Tris (pH=8) over the resin (try to not disturb the resin too much)
* Collect and wash (first wash)
* Flow 10 CV of 20 mM Tris (pH=8) and 300 mM NaCl and 40 mM Imidazole (pH=8)
* Don't disturb resin too much and collect (wash 2)
* Flow 5 CV of 20 mM Tris, pH8 300 mM NaCl, 300 mM Imidazole, pH8 to elute (collect this!!)

# Agarose Gel Electrophoresis

Agarose gel electrophoresis is an important validation step during the DNA assembly process. Running an E-gel on one or more samples of DNA allows any fragments of DNA within each sample to be separated according to molecular weight/DNA length/number of base pairs. MSBT utilized this in many points of experimentation following PCR and Gibson assembly. If the amplicon is of a known size, then an E-gel can be used to confirm the success of the PCR if there is a large presence of the amplicon.

**Materials**

* Agarose Gel
* 50ml of 0.5x TBE buffer
* 0.5g of agarose. (to make a 1% gel)
* 5uL of GelRed
* Digested PCR product
* DNA ladder
* HyLadder 10 kb
* Loading dye
* Gel mold & comb
* Excess 1x TBE buffer

**Procedure**

* Making the Agarose Gel:
* To a 250 mL Erlenmeyer flask add 50 mL of 0.5x TBE buffer.
* Place 0.5 g of Agarose into the flask and microwave on high for 25 s intervals until the agarose has dissolved.
* When the solution has cooled, but not polymerized, add 5 uL of GelRed to the solution and swirl to mix.
* Pour the solution into a casting tray and place the desired combs into position. Running the Electrophoresis
* Allow for approximately 30 mins of polymerization time.
* Fill the buffer chamber with 1x TBE buffer so that the gel is completely submerged.
* In the far left well, add 8 uL of DNA ladder
* From the PCR tube containing the product, remove 6 uL and place in a separate tube
* To the separate tube, add 1 uL of loading dye and mix with pipette
* In a well not containing the ladder, pipette the 7 uL of dyed product making sure to not disturb the integrity of the gel
* Place the casting tray into the gel box in the orientation which allows the negatively charged DNA to run towards the positively charged (red) cathode.
* Run the gel at a constant voltage set to 120V for approximately 30-45 mins or until the dye front has traveled the desired distance, usually about 75% of the way across the gel, checking every so often to ensure the dye does not run too far off the gel.
* Visualize the stained gel using a standard transilluminator (302 or 312 nm) and image the gel using an ethidium bromide filter

# Inverse Fusion PCR

Due to the small size of the alpha factor in comparison to the large plasmid, infusion pcr was used to add the fragment. This is a 2-step PCR process with 3 primers. The best polymerase based on gel electrophoresis results was a high fidelity platinum taq polymerase, as there is a lower error rate. With such a small fragment addition, having a low error rate was crucial to good results.

**Materials**

* FOR THE FIRST PCR REACTION:
* 38.5 μL nuclease free water (enough to get to 50 μL total volume per PCR tube)
* 1 μL of 10uM (500nM) primer A
* 1 μL of 10uM (500nM) primer B
* 1 μL mNeon plasmid DNA
* MA76 (make sure it includes histag)
* 5 μL 10x High fidelity PCR buffer
* 2 μL MgSO4
* 1 μL dNTP mix
* 0.5 μL Platinum Taq DNA polymerase
* FOR THE SECOND PCR REACTION:
* 38.5 μL nuclease free water (enough to get to 50 μL total volume per PCR tube)
* 1ul of 10uM (500nM) primer B
* 1ul of 10uM 500nM primer C
* 1 nM (NOT uL!!!) PCR rxn product from the first PCR
* 10pM original DNA template
* MA76 (make sure it includes histag)
* 5 μL 10x High fidelity PCR buffer
* 2 μL MgSO4
* 1 μL dNTP mix
* 0.5 μL Platinum Taq DNA polymerase

**Thermocycler Settings**

* Thermocycler Settings Reaction 1 exp 004 on thermocycler
* Number of cycles: 1
* Initial denaturation temp: 94 C
* Initial denaturation time: 30 sec
* Number of cycles: 30
* Denaturation temp: 94 C
* Denaturation time: 15 sec
* Annealing temp: 55.8 C
* Annealing time: 30 sec
* Extension temp: 70 C
* Extension time: 7.5 minutes
* Final settings:
* Final extension: 70 C
* Final extension time: 10 minutes
* Hold at 4 C
* Thermocycler Settings Reaction 2
* Number of cycles: 1
* Initial denaturation temp: 94 C
* Initial denaturation time: 30 sec
* Number of cycles: 30
* Denaturation temp: 94 C
* Denaturation time: 15 sec
* Annealing temp: 55.8 C
* Annealing time: 30 sec
* Extension temp: 70 C
* Extension time: 7.5 minutes
* Final settings:
* Final extension: 70 C
* Final extension time: 10 minutes
* Hold at 4 C

**Protocol**

* Get ice, all materials should be kept cold
* Found in storage room, grab a plastic bucket near ice machine to fill
* Program thermocycler
* In an eppendorf (1.5 mL, autoclaved) tube, combine all of the elements in the materials section, leaving TAQ polymerase to the end
* Be sure to place materials back on ice when you are finished
* Wearing gloves, get a PCR tube and place 50 μL of the master mix into the tube
* Place in thermocycler and run
* After running, place it in the fridge to keep until ready to run gel electrophoresis

# Chemically Competent Cells

In order to successfully complete chemical transformation, E. Coli cells have to be made chemically competent. The following protocol describes the process to modify the cells.

**Materials**

* Overnight culture
* LB
* 100 mM CaCl2
* make sure it is cold
* Use 0.5549g CaCl2 per 50mL
* 80% glycerol

**Procedure**

* Create liquid growth culture in 500 uL of overnight culture and 50 mL of LB
* Icubate at 37 C and measure OD until it reaches about 0.5
* Split growth culture into two 50 mL falcon tubes
* Centrifuge cells at 3000 x g for 15 minutes at 4 C
* Make sure centrifuge is balanced and rotor is secure!
* Pour off supernatent
* Resuspend cells in 25 mL of 100 mM CaCl2
* Combine both tubes into one and place on ice for 30 minutes
* Create a balance tube for the centrifuge
* Centrifuge cells at 3000 x g for 15 minutes at 4 C
* Pour off supernatent
* Resuspend cells in 400 uL of cold 100 mM CaCl2 and 75 uL of 80% glycerol
* Freeze 50 uL aliquots at -80 C

# SDS-PAGE Gel Electrophoresis

SDS-PAGE gels are used for protein presence and size evaluation. This was useful in order to identify the presence of encapsulin protein following expression and purification.

**Protocol**

1. Keep samples cold
2. Prepare a gel tank (use the green ones if you made your own gel; use the black ones if using a pre-made gel) a. Insert gel slide (facing outward if pre-made, facing inward if you made it) b. Fill the tank with running buffer i. 10X running buffer is stored on the shelf above the gel tanks ii. Add 100 mL to a large flask and dilute to 1X with ddH 2 O
3. Take a 1.5 mL Eppendorf tube for each of your elution samples
4. Add 8 L of sample to each tube, followed by 2 L of 6X dye (stored at -20 o C)
5. Heat tubes at 95 o C for 5 min.
6. Spin in small centrifuge at 12000 rpm for 5 min.
7. Prepare to load gel plate a. Remove bubbles from each of the wells by taking some of the running buffer and pipetting it into each well until bubbles are gone b. Get protein ladder from -20 freezer
8. Add 1-2 L protein ladder to the far left well, followed by 10 L flow-through sample (well 2)
9. Load 10 L of the remaining samples into the other wells and add another 2 L protein ladder at the end
10. Put lid on top of tank, plug red and black wires into their corresponding outlets
11. Turn on machine, set time for 1 hour and power (?) constant 25 (?)
12. At the end of 1 hour, turn off machine and remove plate from the tank
13. Pry plate open and trim gel to the size that you need
14. Place gel in the bottom of a recycled pipette tip box and pour a thin layer of Coomassie dye over it
15. Let it rock at room temp. for 1 hour
16. After 1 hour, pour off Coomassie dye into the appropriate bottle and add de-staining solution
17. Look for dark band on gel between 25 and 35 kDa; this is your protein
18. Let gel rock with de-staining solution for 2 hours
19. After 2 hours, pour de-staining solution into waste bottle and pour water over gel
20. Cover container and let rock overnight

# Yeast Culture Preparation

Baker's yeast was grown in YPD to be used in future endocytosis experimentation.

**Materials**

* 50g YPD broth powder
* 1L purified H2O

**Protocol**

* Make and autoclave new YPD broth with amounts in materials above
* Incoulate baker's yeast in YPD broth
* Leave shaking at 32 C and 300 rpm

# Endocytosis into Yeast

Yeast was first incubated in YPD growth medium before the introduction of the encapsulin protein. Using a fluorescent microscope for visualization, the encapsulin was added to the yeast. Using mNeon green as a tracker, the location and interactions of the encapsulin protein with the yeast is observed.

**Materials**

* Yeast grown in YPD broth
* Purified encapsulin protein
* Fluorescent microscope

**Protocol**

* On a clean plate, place some of the purified protein on a sample of the yeast culture
* Incubate for 1 hour at 37 C
* Transfer a sample to microscope slide and image using fluorescence microscope
* Look for fluorescence of protein in relation to the yeast cells
* Protein lacking alpha factor should not endocytose

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# Team:Michigan/Results

Results | iGEM Michigan

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# Results

Experimental Outcomes

---

##### Contents

# Inverse Fusion PCR

We designed primers to insert a linker and alpha factor on the carboxyl termini of our encapsulin protein, which is known to be on the protein's exterior surface. This was done to facilitate endocytosis in yeast. To ensure unique binding of our cloning primers, we added alpha factor at the end of a 6 residue glycine-alanine linker. Retrospectively, we realized that this sequence seems a bit hydrophobic to serve as a good linker, however, it is surrounded by hydrophilic alpha factor and spytag which may mitigate its hydrophobic tendencies.

Primer design was based on a published protocol for inverse fusion PCR (citation). Three primers are required for this procedure: a forward primer complementary to vector nucleotides immediately following the desired insertion site, a reverse primer complementary to vector nucleotides before the insertion point and containing the insertion sequence, and a second reverse primer complementary to the end of the insert.

This was designed to allow two reactions in the same pcr experiment. First, the primers with forward and reverse complements to the vector insert add the insertion sequence to the vector and linearize it. Second, the forward primer and the second reverse primer amplify the vector-insert fusion. Finally, the phosphorylated forward primer allows ligation of the fused sequence.

All primers were designed with a melting temperature of ~58 degrees, as recommended in the literature and were checked with IDT oligo analyzer for hairpins, homodimers, and heterodimers. Unique binding was confirmed with Snapgene.

Repeated efforts to implement inverse fusion pcr were unsuccessful, likely due to incorrect primer concentrations in PCR.

# Encapsulin Protein Purification

Using encapsulins as a cell type specific drug delivery platform requires their endocytosis to be regulated by an engineered ligand. To show that the encapsulin is not universally endocytosed in the absence of an added ligand, we purified bare encapsulins, lacking the added alpha factor ligand.

The protein was successfully expressed in BL21 E. Coli, and purified with a nickel column via a his-tag on the exterior of the encapsulin. The successful purification demonstrates the self assembly of the encapsulin loaded with protein cargo, showing the ease of encapsulin creation for protein-based therapeutics.

SDS Page Gel confirmed successful purification. This was evaluated based on the expected size of the encapsulin, which was approximately 37 kDa.

![SDS Page Results](https://static.igem.org/mediawiki/2021/6/6b/T--Michigan--img--n13.png)

Further fluorescent microscopy showed possible encapsulin clusters.

![Fluorescence Microscopy (possible encapsulin clusters circled in white)](https://static.igem.org/mediawiki/2021/1/12/T--Michigan--img--n14_2.png)

Figure 2: Fluorescence Microscopy (possible encapsulin clusters circled in white)

Protein concentration was measured with a NanoDrop to be on the order of 3 ng/ul.

# Encapsulin Endocytosis

An endocytosis protocol was run on the encapsulin (without alpha factor) into yeast cells. The purpose of this experiment was to serve as a negative control in which endocytosis fails to occur without the alpha factor present to mediate endocytosis. The following images show pure yeast and yeast with the encapsulin added. While it’s possible that endocytosis failed, the images themselves make a definitive conclusion difficult to draw.

![Pure Yeast](https://static.igem.org/mediawiki/2021/a/ad/T--Michigan--img--n15.png)

Figure 3: Pure Yeast

![Yeast + Encapsulin](https://static.igem.org/mediawiki/2021/e/ef/T--Michigan--img--n16.png)

Figure 4: Yeast + Encapsulin

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