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Team:UC Santa Barbara/Notebook
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7/14/14 - Project began. Started planning project timeline and methods/materials needed to complete project. 7/16/14 - Ordered primers for CysK-T25 OE PCR project, 4 primers total:
Primer #1: CysK-EcoRI Forward-tttgaattcATGAGTAAGATTTTTGAAGATAACTCG
Primer #2: CysK-OE reverse - cccgcttgctccagtcccCTGTTGCAATTCTTTCTCAGTG
Primer #3: T25-PstI reverse - tttctgcagaTTATATCGATGGTGCAGCC
Primer #4: T25-OE Forward - gggactggagcaagcgggCAGCAATCGCATCAGGC
7/22/14 Hiro + Zack - Began PCR of OE fragments for CysK-T25
Enzyme: PFU Polymerase
PCR Temps: 95 degrees C for 1 minute
50 degrees C for 1 minute 30 seconds
72 degrees C 3 minutes 30 seconds
7/23/14 -John + Zack - Digest of CysK-T25 with EcoRI and PstI
Protocol: -5uL PCR product, 2uL Buffer 2.1, 12uL H2O, 0.5uL of EcoRI and PstI in an eppendorf tube
-5uL Vector, 2uL buffer 2.1, 12uL H2O, 0.5uL of EcoRI and PstI in an eppendorf tube
-Set both in warm room for 90-120 minutes
After Digest: Run the digest products out on a gel, cut relevant bands out, and purify DNA from gel
Gel Purification Protocol:
*Add 600uL Gel Dissolve to eppendorf tubes containing gel
*Heat tubes in 60 degree waterbath until gel dissolves
*Add 200uL isopropanol once gel dissolves
*Transfer contents of eppendorf tube to gel purification column, let sit for 30 seconds
*Turn on vacuum, then in order add:
500uL PB
750uL PE
250uL PE
*Take gel purification column off vacuum, and spin column in centrifuge at 15K rpm for 2 minutes to collect waste product
*Transfer gel purification column to eppendorf tube, add 50uL elution buffer, spin at 8K rpm for 30 minutes to collect DNA
After gel purification, we ran a ligation to ligate the vector and insert together
Ligation Protocol:
*In a microfuge tube, add 16uL vector DNA, 2uL Insert DNA, 2uL 10x ligase buffer, 0.5uL T4 Ligase
*Control Tube: 16uL H2O, 2uL insert DNA, 2uL 10x ligase buffer, 0.5uL T4 Ligase
*Let sit at room temperature for 1 hour or more
After Ligation: Transform into X90 Cells
Transformation Procedure:
*Take cells out of -80 freezer, let thaw on ice
*Aliquot 2 eppendorf tubes with 50uL of cells each
*Add 10uL of vector + insert ligation to one and 10uL of H2O + insert ligation to the other
*Let the eppendorf tubes sit for 15 minutes on ice
*Put eppendorf tubes in 42 degrees celsius water bath for 45 seconds exactly (heat shock)
*After heat shock, add 1mL LB Broth growth medium to tubes using sterile technique and set mixtures in warm room for 1 hour to let cells recover
*Warm up antibiotic-laced agar plate at the same time
*Take cells out, spin at 15K RPM for 2 minutes
*Remove 950uL of LB media leaving 50uL behind, and then resuspend cells in the leftover 50uL
*Transfer 50uL to agar plate, spread cells, put in warm room overnight.
7/24/14 John + Hiro - Colonies formed! - We must check to make sure correct plasmid was transfected
Take samples of colonies and grow them overnight in liquid LB medium
*Add 2mL LB broth to test tube w/ 4uL of ampicillin (pTrc99A is Amp resistant)
*Set in warm room to grow overnight
7/25/14 John + Hiro - Miniprep plasmid from cultures, digest and check for correct sized drop out band
Miniprep protocol:
*Take 1mL of overnight culture from heat shock, spin down at 15K for 2 minutes and remove all LB
*Add 250uL P1 buffer, 250uL P2 buffer (to lyse cells), 350uL N3 buffer to precipitate proteins
NOTE: Be careful not to mix too hard or chromosome will shear and contaminate plasmids
*Spin down at 15K RPM for 10-12 minutes
*Transfer supernatant to miniprep column, put miniprep column on vacuum collection apparatus
*Vacuum through, add 500uL PB, 750uL PE, 250uL PE again.
*Take miniprep column off, spin at 15K RPM in waste collection tubes
*Take out, transfer to eppendorf tubes, add 50uL elution buffer
*Spin down at 15K for 30 seconds, collect DNA in eppendorf tubes.
Once DNA is collected, run digest with EcoRI and PstI and run on a gel
-CysK-T25 insert is ~1.8 kilobase pairs in size
Results:
8/4/14 John - Restart OE-PCR of CysK-T25 and re-transform into cells
Today: Set up overnight Overlapping-extension (OE) PCR - Use TAQ, PFU, and VENT polymerase this time, change to 5 minute extension period (72 degrees)
8/11/14 John + Travis - Digest PCR product from TAQ, check for PCR quality with gel, digest pTrc99A, gel purify DNA - Left overnight in 4 degree refrigerator.
8/12/14 John - Transform ligated PCR product into X90 cells, plate them out.
Set in 37 degree warm room overnight
8/13/14 John - Miniprep overnight cultures, digest, and check for proper plasmid transformation
Results:
8/14/14 John - transform cells into X90 Cells, culture overnight in warm room 8/15/14 John - Miniprep cells, digest resulting plasmid and check for correct plasmid transfection. Results: 8/16/14 John (Happy Saturday!) - Restart PCR, this time using VENT polyermase 8/18/14 John - Digest PCR product, Purify from gel, ligate 8/19/14 John - Transform PCR Product into X90 cells, Grow overnight 8/20/14 John - Colonies! Grow them up overnight in Liquid LB medium 8/25/14 John - Miniprep clones and digest with EcoRI and PstI, run out on gel. Results:
8/26/14 Travis - Send plasmids to UC Berkeley for sequencing to verify correct insert
Result: Indeed correct plasmid was inserted (yay!)
9/3/14 John - Subclone CysK-T25 from pTrc99A to pCH450 (pTrc99A is Amp resistant, pCH450 is Tetracycline resistant)
Purpose is to make sure that plasmid backbone for our multiple plasmids don't have interfering antibiotic resistances.
Result:
9/3/14 - 10/5/14 - Begin planning process for making cognate I-T18 plasmid
I-T18 only existed as part of a larger cassette, so we must PCR off the section we want
Also finals were this time and a 2 week vacation was taken.
10/6/14 John - Begin PCR to extract I-T18 sequence from plasmid. Also, subclone CysK-T25 into pSB1C3 to send to iGEM HQ for judging.
Subclone Results:
10/8/14 Travis + John - Digest I-T18 PCR product and ligate into pTrc99A (Amp Resistant)
10/9/14 John - Begin making CyA- knockout bacteria that will house all of our plasmids. To generate a reporter for cAMP production, we need to introduce a GFP downstream of a pPap promoter (respondent to cAMP)
We must use a phage to transduce GFP to the chromosome of our background cell line.
-Start by culturing 3 GFP mutant strains overnight
Also today - Transform I-T18 ligation product into X90 cells, and miniprep CysK-T25 SB1C3 cell lines again to check for correct ligation.
CysK-T25 second subclone results:
10/9/14
This is where our journal ends. We will continue the phage transduction and I-T18 cloning, and plan on presenting our full system to the iGEM 2014 International Jamboree. See you there!
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Insert in every lane was >1.8KBP, so incorrect plasmid was transformed. Possibly contamination?
So it worked, although the blurriness indicates low quality PCR with many incomplete extension segments. Used lanes 1, 2 (TAQ), 4, 5 (VENT) for ligation step.
Insert is >1.8KBP, must re-do PCR again.
Success! Lanes 1 and 6 display the correct ~1.8kbp dropout! It Worked!
Success!
No success.
