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From 2014.igem.org

NATIVE RECEPTOR LAB NOTEBOOK

Attributions: Alexa Garcia, Shinjini Saha

PLASMID ASSEMBLY PLAN

for Experiments 1 & 2

PLASMID ASSEMBLY PLAN

for Experiment 3

PLASMID ASSEMBLY PLAN

for Experiments 4 & 5

NOTEBOOK ENTRIES

June 15
AG, LA SS
Grew pENTR_LilrB2, pENTR_PirB

June 16
Liquid cultures didn't grow
SS
New liquid cultures made
AG, LA
NEGEM and High School presentations completed

June 17
Liquid culture still didn't grow BUT we figured out why
SS
Made new cultures and incubate them overnight
Made ggplates
LA
Prepare for a practice HEK293 transfection with Nelson
AG
Send ggDONR for sequencing
Split HEK293 cells

June 18
Liquid culture STILL didn't grow
New plates + old ggDONR grows and turn blue
We think something is wrong with the plates
Primers for fusion have arrived
LA
Prepared media for HEK293 in preparation for transfection transfection

June 19
Plates from our transformation grew blue and white colonies
PirB2 didn't grow any white colonies
SS
Liquid culture - to make stock - of ggDONR from troubleshooting experiment
golden gated again
AG
Made liquid culture of GGDonr and pENTR_L1_LilrB2_L2 for miniprep

June 20
SS
Took liquid cultures out of incubator and put in fridge.
AG, LA, SS
Attended NEGEM 3.1

June 22
LA
Transformed GoldenGated pENTR_L1_LilrB2_L2, pENTR_L1_PirB_L2
Plated
SS
Made cell stock of GGDonr and pENTR_L1_LilrB2_L2 (from previous GoldenGate)
Miniprepped GGDonr and pENTR_L1_LilrB2_L2 (from previous GoldenGate)
AG
Problem with HEK-293 cell culture - culture another plate

June 23
AG
Transformed Golden Gate product:
positive control didn't grow any colonies
blue and white colonies on LilrB2
only white colonies on PirB
picked colonies
SS
Digested previous pENTR_LilrB2 and GGDonr (BssSI, 3.1)
Could not figure out how to make the gel imaging station to work. But...
Gel did not fluoresce under UV, not even the hyperladder.

June 24
Liquid cultures from the second golden gate grew
SS
Liquid cultures miniprepped and DNA waiting to be verified

June 25
AG
Ran verification gels of the products of the minipreps - one very bright band for each lane
We suspect enzyme (BssSI) didn't work - DNA was super coiled. Old enzyme has been thrown out.
Split HEK293 cells. Prepare for practice transfection

June 26
SS
Digested LilrB2, PirB and GGDonr (again). EcoRI this time
AG
Practice transfection (fluorescent protein - 1 plasmid)

June 29
AG
Plated 3rd GG pENTR_LilrB2, pENTR_Pirb (Treatment group did control.)
Flow cytometry of practice transfection - high efficiency
Prepare for more complex transfection - 2 plasmids
June 30
LA
Picked colonies
cultures grew
found inactive cofilin plasmid
need to verify sequence and see if it has been used before
if suitable - will order today
started designing rest of fusions on geneious
AG
Transfection - 2 plasmids

July 1
SS
Miniprepped DNA
Restriction digested for verification
cofilin order on the plasmid page

July 2
SS
Ran gels of yesterday's digest
Did digests again on select pENTRs
40ul total volume instead of 20ul, same amount DNA (500ng)
Ran gel
AG
Flow cytometry - lower efficiencies (keep practicing).
Train team members in tissue culture

July 3-2
SS
Imaged gel that we ran yesterday

July 6
SS
Transformed LR Product (pEXPR hEF1a:PirB and hEF1a:LilrB2)
Picked colonies (3 each) and grew up overnight in liquid culture
Mini-prepped and nano-dropped
AG
Split HEK293 cells

July 7
LA
PCRs to make fusions started
50 ml liquid cultures for midi prep in incubator

July 8
AG
pEXPRs midiprepped
LR product digested and gel ran
LA
PCRs for fusions in progress
Digesting LR's
Hef1a:LirB2 BsrGI
Hef1a:PirB ClaI

July 9
Sequencing results received for pENTR
LilrB2 is correct
PirB: Qsite ligation with the C terminus gblock in pENTR
New PirB colonies picked
Restriction digest on pEXPR LilrB2 repeated because we suspected quality of digestion
LilrB2 = 1900 bps
PirB = 2500 bps (won't work because pENTR is not what we thought it was)
AG
Split HEK293 cells. Start 3 plate series so that splitting would occur every day.

July 10
SS
Digested pENTR_PirB with EcoRI.
Made primers for PirB, LilrB2, Cofilin fusion to YFP.
LA
Sent pENTR_PirB for sequencing for M13 F and R primers.
Sent pEXPR_hEF:LilrB2 from yesterday that seemed to work (L2, L3) for full sequencing.

July 11
LA
Send pEXPR hEF1a:LilrB2 and pENTR PirBfor sequencing (L2, L3)

July 14
AG
TEVp-cofilin fusion to be ordered as a gBlock
Split HEK293 cells.
LA
Sequencing results returned and analyzed
pEXPR hEF1a:LilrB2 didn't give us a good enough read - we're sending those out again
pENTR L1_PirB_L2 worked
SS
L1_PirB_L2 LRed

July 16
SS
Received primers - started PCRs
Done:
LilrB2 (eYFP)
PirB (eYFP)
LilrB2 (TCS-Gal4VP16)
PirB (TCS-Gal4VP16)
To be done:
Cofilin (eYFP)
Ran verification gel on PCR products

LA
streaked cofilin plasmid DNA
Sequencing results returned for LilrB2 pEXPR
LilrB2 insert is there: LilrB2 pEXPR verified
"No Priming" for second read in both samples?
AG
PirB pEXPR transformed
Split HEK293 cells.

July 17
SS
PirB LR colonies picked and liquid cultures prepared
LilrB2 PCRs repeated under 2 different (higher) annealing temperatures
Remaining PirB PCR product ran and band with the correct size DNA to be extracted
Gel with PCR products imaged
Expected sizes:
LilrB2: 2,000
PirB: 2,500

July 18
SS
PirB PCR bands extracted
Concentrations very low
around 5 ng/ul
PirB pEXPRs miniprepped
Restriction digest on PirB pEXPRs run
LA
PirB PCRs run again
LilrB2 PCRs done at higher annealing temperatures
Cofilin plasmid colonies picked
AG
Tissue culture

July 19
pEXRP_hEF1a:PirB 2 seems to give the correct band pattern,
but the bands are slightly lower than they should be.
Ran extraction gel for PCR products: HL1. LY65, LT67, LY68, LT70, PY, PT
Turns out the gel was upside down, so all the products diffused. Need to do all the PCR's again
SS
Cofilin plasmid miniprepped. Concentrations ~600ng/ul
Re-did PirB PCR's, Did Cofilin PCR. LilrB2 PCR's need to be redone
Current cell glycerol stocks: pENTR_PirB (verified), pENTR_LilrB2 (verified), pEXPR_hEF1a:LilrB2 (verified),
pEXPR_hEF1a: PirB (unverified), Cofilin gene in backbone.
AG
Tissue culture

July 20
SS
Did PCR's of LilrB2
Ran gel of all the PCR's
Ladder, LilrB2 YFP 65, LilrB2 YFP 68, LilrB2 TCSG4 67, LilrB2 TCSG4 70, PirB YFP, PirB TCSG4, Cofilin YFP

July 21
SS
Miniprepped new pEXPR_hEF1a: PirB (4-8) ~200ng/uL
Digested new pEXPR_hEF1a: PirB
Ran Gel
Looks like they all worked!
Sent pEXPR_hEF1a: PirB 2 (from saturday) for sequencing. Will not be good quality (did not have enough DNA)
Transform pEXPR_hEF1a: PirB (4-8)
Plate pEXPR_hEF1a:LilrB2 (cell stock)
AG
Tissue culture

July 22
SS
Liquid culture for hEF1a:(Receptor) midiprep grown up - to be midiprepped tomorrow
eYFP-cofilin golden gate transformed

July 23
SS
Miniprep pEXPR_hEF1a: LilrB2/PirB:
A couple came out to have around 700ng/ul. Others were around 400ng/ul
Midiprep Culture:
Picked 2 out of 5 pEXPR_hEF1a: PirB's to inoculate midiprep cultures, but kept the miniprep cultures of the other 3 just in case.
Also inoculated pEXPR_hEF1a: LilrB2.
Midiprep party tomorrow!
eYFP-Cofilin:
Plate had lots of white colonies and a few blue ones. Not expected. Expecting a lot of backbone re-ligation. Picked 10.
Will miniprep tomorrow.
AG
Tissue culture
July 24
SS
Miniprepped eYFP:Cofilin colonies
Ran digest gel (PstI, ApaI, Cutsmart)
2 might have worked
Sent for sequencing.
Turns out, didn't work. eYFP missing.

July 25
SS
Did midipreps. Very strange distribution of concentrations:
LilrB2 1: ~1200
LilrB2 2: ~700
PirB 7: ~4000
PirB8: ~300
Digesting midipreps just in case.
LilrB2: XbaI, ApaI
PirB: ApaI

LilrB2's are weird. Running undigested controls.
LirB2 DNA is contaminated!
Correct band pattern is most definitely there. Which means all parts: hEF1a promoter, LilrB2 gene and backbone are
there.
AG
Tissue culture
July 26
SS
Run DNA for gel extraction. Also look to see if other, verified DNA contaminated or not.
Pick colonies fusions
Transform
Transforming with L2 and L3 DNA (L24, L34).

July 27
SS
Minipreped a gazillion fusions.
4/5 cofilins seemed to work. Had 3 fragments. Means at least 2 of the goldengated parts are present. Will digest
with ApaI tomorrow to check if 3rd is present.
PirBs didn't seem to work, will pick more colonies.
LilrB2 didn't work
Will pick pEXPR_LilrB2 colonies.
AG
Tissue culture
July 28
SS
Cofilin 1, 3, 4, 5 worked, need to do another digest.
LilrB2:TCSG4 1, 4, 5 might have worked. Strange extra band on top. Will do different digest.
LilrB2: YFPL none worked. Will miniprep more.
PirB:TCSG4 none worked.
PirB:YFP none worked.
Cofilin worked! Sending all 4 for sequencing, 'cuz PCRs and mutations, ya know?
LilrB2:TCSG4 did not work. Will have to miniprep more.

Made glycerol cell stocks of all the cofilins, but due to some mistake, the labels got erased. So, unless they all
worked, I'll just re-transform one that worked.
Making glycerol cell stocks of pEXPR_hEF1a: LilrB2 (L2 and L3)

July 29
midipreped pEXPR_hEF1a:LilrB2.
All the eYFP:Cofilin worked! LRed today.
Did more golden gates.
Picked more colonies, too.
Inoculated midi culture for ggDonr

July 30
More fusions digested!
New goldengates of fusion proteins transformed
New LR of hEF1a:YFPCofilin transformed
LR of TREt:YFPCofilin done.

July 31
SS
LTs might have worked. Did digest again with different enzymes.
Did digest with some other interesting ones: LT12 and PT11 and PT12.
Transformed TREt:TEVpCofilin LRs
Picked hEF1a:TEVpCofilin LRs
Picked pENTR:YFPCofilin
Picked more LY and PY from new goldengates
Sent LT and PT for sequencing

LA
Transfected LilrB2 for membrane localization
Gel verification of LilrB2 and PirB PCR products ran

August 1
SS
Miniprep PY, LY, pENTR_TEVpCofilin
Verify Minipreps
Transform hEF1a:YFPCofilin
Pick TREt:YFPCofilin Colonies
All 4 pENTR_TEVpCofilin worked!
The others didn't work. Given the lack of blue colonies, I'm thinking backbone religation/only one part present
(probably YFP). Need to figure out something else.
Sequencing:
LilrB2TCSG4 seems to have worked! 11 and 13 might have point mutations (high G content, unlikely). Will proceed with
12.
PirBTCSG4 seems to have worked, too! 13 seems to have a point mutation (high G content, unlikely). Will proceed with
11.

August 2
SS
Transform all LRs (hEF1a:PT, hEF1a:LT, hEF1a:TC, TREt:TC)
Pick hEF1a:YC colonies
Miniprep TREt:YC if it worked. Otherwise, wait for Brian. Worked.
Midiprep inoculate TREt:YC
Digest LilrB2TCSG4 with NcoI and PstI seperately.
Digest TREt:YC with ApaI

TREt:YFP_Cofilin 1, 2, 4 verified. Will midiprep 2.
Ran PT (pENTR_PirBTCSG4) to make sure it isn't contaminated).
The gel for PT had an extra band, but the sequencing results show that the TCSG4 is present and goes into the PirB
region. That means everything is in order.

August 3
SS
Took pEXPR TREt YC midiprep out of incubator
Took pEXPR_hEF1a:YC miniprep out of incubator
Took plates out of the incubator
plates dried out. Picked colonies for TREt-TC

August 4
SS
Miniprep pEXPRs (TREt TC, hEF1a YC)
Check pEXPRs (TREt TC, hEF1a YC)
Inoculate Midipreps
Pick colonies for hEF1a LT, PT, TC
Digest TREtTC with ApaI Cutsmart
Digest hEF1aYC with BsrGI 2.1
Digest LT with BglI 3.1
Digest TC with PvuII with 3.1
Digest LT11 with BlpI with CS
Give up on hEF1aYC. Won't be using it anyway.

pEXPR_TREt:TEVpCofilin is wrong. Gives weird patterns. Need to pick again.
pENTR:LilrB2TCSG4 is wrong. Big chunk is actually missing. Need to pick again.

August 5
SS
Miniprep pEXPR (hEF1a: PirBTCS, hEF1a: TEVpCofilin)
Verify hEF1a: PirBTCS
Miniprep pENTR LilrB2TCS
Verify pENTR LilrB2TCS
Midiprep inoculate hEF1a: PirBTCS
Pick more TREt:TEVpCofilin
Plate more TREt:TEVpCofilin if possible.
Digest pEXPR_hEF1a: PirBTCSG4VP16 with BsrGI, buffer 2.1
Digest pENTR_LilrB2:TCSG4VP16 with XhoI +XbaI, buffer CS
Order: hEF1a PT 1, 2, 3, 4, LT16, 17, 18, 19
August 6
SS
Miniprepped more pENTR_LilrB2TCS
Verify hEF1a: PirBTCS. Else pick more colonies. Picked more colonies
Verify pENTR LilrB2TCS. Including LT16. Did not work
Verify pEXPR_TREt:TEVpCofilin. Worked!
Midiprep inoculate TREt:TEVpCofilin if it worked.
Midiprep inoculate hEF1a: PirBTCS if it worked. Didn't work
Sent pENTR_LilrB2 (04 2) for sequencing again...
Digest pEXPR_hEF1a: PirBTCS (1, 3, 4) with BsrGI, 2.1
Digest LT (21-25) with XbaI, XhoI, CS
Digest LT16 with NcoI, XhoI, CS
Digest pEXPR_TREt:TEVpCofilin with BsrGI, 2.1
hEF1a: PirBTCS did not work. Need to pick more

August 7
SS
Miniprepped more hEF1a: PirBTCSG4. Some worked!
Midiprep inoculating.
Also cell stock
PCR'd LilrB2 and YFP. LilrB2 band is too low. YFP didn't work.
Midiprepped TREt:YFPCofilin and TREt:TEVpCofilin.

August 8
SS
Midiprep pEXPR_PirBTCSG4VP16. The midiprep concentration was low, around 16. Probably because the culture didn't grow
very well.
Miniprep inoculating again.
Digest any YFP fusion minipreps we have to check for what segments are present and what went wrong.
Dilute midipreps.

August 12
AG
hEF1a:PirB-TCS-Gal4VP16 midiprepped
LilrB2 pENTR sent for sequencing

August 14
AG
pENTR LilrB2:
Blunt end cloning of gBlock

pEXPR hEF1a:PirB-YFP
Transform


August 22
AG
Miniprepped pENTR-LilrB2 (1-6)
Ran digests.
They look good.
Sent LilrB2 1 for full sequencing. Waiting till sequence verified for making cell stock.

August 23
SS
Transformed pEXPR_hEF1a:LilrB2 and pEXPR_TREt:LilrB2

August 24
SS
Got sequencing results back. The pENTR is sequence verified!
Picked colonies for pEXPRs.

August 25
Miniprep LRs
Digest LRs
Also digest pEXPR_hEF1a: PirBYFP
PCRing at 53 degress, 40 seconds.
Digesting with:
pEXPR_hEF1a: LilrB2 XhoI, CS
pEXPR_TREt: LilrB2 XbaI, CS
pEXPR_hEF1a: PirB_YFP ApaI, CS

Both LilrB2 pEXPRs worked!

August 26
SS
Midis didn't work. Had ~10ng/ul concentration.
So, taking the cell stocks and throwing them out.

August 27
SS
The pENTR_PirB_YFP did not actually have the YFP insert.

August 28
SS
Picked pENTR LilrB2_YFP/TCS colonies
Put pEXPR_LilrB2 midi culture in incubator (~8:45pm)

August 31
SS
Transformed pEXPR hEF1a:LilrB2 and TREt:LilrB2 (10pm)
Digesting pENTR LilrB2YFP and LilrB2TCS
LilrB2YFP: PstI 3.1
LilrB2TCS: XhoI + XbaI CS

Looks like LT4 worked. Will send for sequencing (M13F and R)
The LYs don't seem to have worked. May be a problem with the YFP PCRs.

September 1
SS
Inoculated midi for pEXPR(hEF1a/TREt):LilrB2

September 2
Midiprepped pEXPR(hEF1a/TREt):LilrB2
Concentrations around 1ng/ul.
pENTR_LilrB2TCS sequencing results came. Both components are there.

September 3
SS
LR'd LilrB2TCS (hef1a, tret)

September 4
SS
Transformed LRs
Tried midiprepping pEXPR_hef1a/tret:LilrB2. The vacuum manifold wasn't working.