Team:BostonU/TandemPromoters

From 2014.igem.org



Notebook: Tandem Promoters
This notebook will delineate the steps taken and the parts used to create a library of MoClo level 0 (AK, KB, AB fusion sites) tandem promoter parts as well as the level 1 and level 2 parts created to test them. It also includes the steps taken to add several new gene (CD fusion sites) and terminator (DF fusion sites) MoClo parts to our library. The following ladder was used in all of the gels to determine the size of each genetic part I was building:

June

Week of June 23
Plasmids from addgene:
   Genes: Bm3RI, BetI, Ph1F, SrpR, LrmA
   Terminators: L3S2P21, ECK120015170
   Destination Vectors: pICH89921, pICH82113    
  • Struck out genes and terminators onto Amp plates
  • Struck out Destination Vectors onto Kan plates
  • Picked two colonies from each plate- one for minipreps & one for frozen stocks
  • Ordered primers for genes and terminators
  • Miniprepped terminators and genes
  • Made frozen stocks for all plasmids
  • Diluted primers and did PCR reactions for genes and terminators

Poured LB, LB+Amp, and LB+Kan plates





Week of June 30
BM3RI, BetI, Ph1F, SrpR, LmrA, L3S2P21, ECK120015170    
  • Ran gel for previous PCR reactions
    1. Two terminators were too small to see well on gel
  • PCR in triplicate for all genes and terminators as well as negative control
  • PCR clean-up
    1. Didn't work for ECK120015170 because it was too small & fell through the filter
  • Level 0 MoClo reactions for BM3RI, BetI, SrpR, LmrA, L3S2P21
  • Redid PCR for ECK120015170
  • Level 0 MoClo reaction for ECK120015170
  • Transformations for all Level 0 MoClo reactions
    1. None of the transformations worked
    2. Accidentally used Pro cell strain with Cam resistance
  • Redid MoClo reactions and transformations for all parts
  • Ordered promoters for pBad, pTet, pA1LacO, and R0051
  • Used colony PCR to verify transformations worked

July

Week of July 7

This week I used the google glass for all protocols to give Wellesley College feedback on one of their software projects.
BM3RI, BetI, Ph1F, SrpR, LmrA, L3S2P21, ECK120015170    
  • Ran gel for colony PCR reactions
  • Picked colonies to grow overnight for the parts that turned out well on the gel
    1. BM3RI, BetI, SrpR, LmrA, ECK120015170
  • Redid transformations for Ph1F, L3S2P21, SrpR, and ECK120015170
    1. SrpR and ECK120015170 looked questionable on the gel so I redid them just in case
  • Miniprepped overnight cultures and sent for sequencing
  • Analyzed sequencing results and confirmed BM3R1, BetI, LmrA, and ECK120015170
    1. SrpR came back as LacZ, which means it wasn't properly transformed
  • Picked confirmed colonies from stab plate, grew overnight, and made frozen stocks
  • Miniprepped SrpR, Ph1F, and L3S2P21 and sent for sequencing
  • Analyzed sequencing results and confirmed SrpR and L3S2P21
  • Redid colony PCR for Ph1F because it had too low concentration for sequencing

pBad, pTet, pA1LacO, R0051
  • Diluted pBad, pTet, pA1LacO, and R0051 primers
  • PCR for pBad, pTet, and pA1LacO
    1. Held off temporarily on R0051 because we didn't have the R0051_Rev_B primer that we thought we had
    2. For each part we made AK and KB fusion site using the new fusion site, K, that we designed (ATGC)
  • Level 0 MoClo reactions in DVL0_AB
    1. pTet-pBad, pTet-pA1LacO, pBad-pTet, pBad-pA1LacO, pA1LacO-pBad, pA1LacO-pTet
  • Transformations for level 0 tandem promoter MoClo reactions in bioline cells

Week of July 14

In addition to my wetlab work this week I cleaned the lab, refilled stocks, and autoclaved backup supplies. I also talked to female high school students about iGEM and my experience with science and research to encourage them to pursue a science related field in university.
pBad, pTet, pA1LacO
  • Created stab plate and picked two colonies from each transformation plate
  • Miniprepped overnight cultures and sent level 0 tandem promoter MoClo parts in for sequencing
  • Analyzed sequences
    1. Only the pTet-pBad tandem promoter turned out correctly
    2. Noticed that the pA1LacO promoter has a large repeating sequence
      1. PCR temperature that I used was too high (too specific causing reverse primer to bind to the wrong part)
  • Redid PCR for pBad (AK) and pA1LacO (AK, KB)
  • Redid MoClo & Transformations for pBad-pTet, pBad-pA1LacO, pA1LacO-pTet, pA1LacO-pBad, pTet-pA1LacO

R0051
  • Received and diluted R0051_Rev_B primer

L3S2P21, SrpR
  • Made frozen stocks from the confirmed colonies



Week of July 21

pBad, pTet, pA1LacO
  • Colony PCR for pBad-pTet, pBad-pA1LacO, pA1LacO-pTet, pA1LacO-pBad, pTet-pA1LacO
  • Miniprepped and sent for sequencing
  • pBad-pTet confirmed
  • pA1LacO still not amplifying correctly
    1. Redid PCR
    2. 3% gel
    3. Redid PCR again
  • Made frozen stocks for pBad-pTet and pTet-pBad

PhlF
  • Redid PCR & ran gel
  • Redid MoClo and transformations
  • Miniprepped and sent for sequencing
    1. PhlF confirmed!

Week of July 28

Level 1s
  • Struck out J23104-BCD2-C0080-B0015 (PJ04B2C80_EF)
  • MoClo and Transformations for TetR Homologs (J23104-BCD2-Srpr,LmrA,PhlF,BetI,BM3R1-B0015) in DVL1_FG and bioline comp cells
    1. Nothing grew for transformations
    2. Redid MoClo and transformations
    3. Sequenced, but failed
  • MoClo level 1s (AE) and transformations for pBad-pTet,pTet-pBad-BCD2-RFP-B0015
    1. Miniprepped and sequenced
    2. pBad-pTet and pTet-pBad level 1s confirmed!

PhlF
  • Made frozen stock

pA1LacO
  • Redid PCR and ran 2.5% gel
    1. KB band looked correct
    2. Gel extraction



August

Week of August 4

NEGEM!

TetR homolog level 1s
  • Redid MoClo and transformations
  • Minipreps and sent for sequencing
    1. Sequencing failed again
    2. Only DVL1 backbone detected for BetI and PhlF
    3. Nothing detected for BM3R1
    4. SrpR gene and BCD2 were detected

pA1LacO
  • Struck out and miniprepped DVL0_AB
  • MoClo and transformations with pTet_AK & pBad_AK
  • Also tried redoing PCR with 20ul instead of 50ul and used gradient with 57C and 61C
    1. Ran gel and both AK and KB looked good
  • Did MoClo, transformations, and minipreps

Other accomplishments
  • Streaked out and grew J23104_FB and B0015_DG because we will need them later for priority encoder

Week of August 11

This week I cleaned the lab. We also temporarily lost our ability to send samples for sequencing due to a problem with our PO box.

Tandem Promoters
  • Built Level 1 J23104-BCD2-TetR-B0015 to test pBad-pTet and pTet-pBad level 1s
    1. I'll test using pro cells and also using a level two testing device
  • I designed and ordered new primers for pA1LacO_AK and pA1LacO_KB
  • Since the PCR reactions for pA1LacO appear to be working, I tried changing the transformation conditions
    1. Tried changing the recovery and overnight temperature to 30 degrees C
    2. Transformations didn't grow in 30 so I switched them over to the 37
    3. Ran colony PCR (1.5% gel) and used incorrect sequenced tandem promoters as controls to compare them to
  • The gel looked potentially correct, so I grew overnight
  • Ran digest
    1. pA1LacO-pBad looked correct, but it's hard to tell since there's only a 34 bp difference between correct and the incorrect ones I've been getting



Week of August 18

Tandem Promoters
  • Redid MoClo reactions and transformations
  • After 24 hours, the transformations didn't grow in 30
  • Redid transformations with one 30 plate and one 37 plate for each tandem promoter
  • Colony PCR for TetR level 1 and tandem promoters
  • Redid 30 transformations
    1. Transformations looked good so I miniprepped adn sent for sequencing
    2. Transformed pBad-pTet and pTet-pBad level 1s into pro cells


New Backbones
  • I will be creating AK, KB backbones as well as DVL2_AE backbone with Amp resistance
  • PCR reaction to get LacZ_AK, LacZ_KB, LacZ_AE
  • Ran gel for LacZ fragments
  • Gel looked good, so I did PCR cleanup for them
  • Restriction digest for CAM and AMP backbones and LacZ fragments
  • Ran 1.5% gel for digests
  • Gel extraction and ligations
  • Transformed backbones


Other Accomplishments this Week
  • Designed primers for promoters and terminators we will need in the priority encoder:
    1. PhlF_GB, PhlF_EB
    2. BM3R1_FB, BM3R1_EB
    3. BetI_EB, BetI_KB
    4. LmrA_FK, LmrA_EB
    5. SrpR_KB, SrpR_EB
    6. ECK120015170_DG, ECK120015170_DH
    7. L3S2P21_DG, L3S2P21_DH
  • Wrote abstract for UROP (the program that funded me over the summer)
  • Worked on project description for our wiki
  • Made DH5alpha strain competent cells

Week of August 25

New Backbones
  • DVL2_AE didn't grow, but picked colonies for DVL0_AK, DVL0_KB
  • DVL0_KB didn't grow overnight
  • Sent DVL0_AK for sequencing
  • Retransformed DVL2_AE
  • DVL0_AK came back with AE fusion sites, so I might've mixed up labeling
  • Redid ligations for all backbones
  • Redid transformations in bioline cells
  • Miniprepped and sent for sequencing


Tandem Promoters
  • Incorrect sequencing again
  • Possibly a toxicity problem
    1. plan on transforming into DH5alpha pro cells (represses pBad/pTet/pA1LacO promoters)
  • Redid transformations in pro cells
  • Also transformed known plasmid to calculate efficiency of pro cells
    1. After recovery, there was barely any pellet for the promoters
    2. Transformations didn't grow
  • Redid transformations again using two different sets of pro cells
    1. Didn't grow again

September

Week of September 2 and Week of September 9
New Backbones
  • DVL0_AK and DVL0_KB were correctly sequenced
    1. LacZ was upside down
    2. Checked sequence for reverse complement


Tandem Promoters
  • Set up Flow Experiment:
    1. Controls: DH5alpha, J04B2RM, J04B2GM, COXGR, COXRG
    2. Construsts for Testing: pBad-pTet - BCD2 - E1010m - B0015, pTet-pBad - BCD2 - E1010m - B0015
    3. Followed the Flow Cytometry protocol
    4. The results for both turned out well
    5. For detailed results, look at the data collected page
    6. Decided a higher concentration for arabinose and lower concentration for atc is needed for next experiment
    7. 5000 and 10,000 ng/ul atc killed the cells

Week of September 16
Tandem Promoters
  • Struck out the following from -80 and -20 frozen stock
    1. J04B2RM
    2. J04B2GM
    3. COXGR
    4. COXRG
    5. DH5alpha
    6. pBad-pTet level 1
    7. pTet-pBad level 1
  • PCR reactions for new promoter and terminator primers:
    1. pA1LacO_AK(NEW)
    2. pA1LacO_KB(NEW)
    3. pBetI_KB
    4. pBetI_EB
    5. pLmrA_FK
    6. pLmrA_EB
    7. pSrpR_KB
    8. pSrpR_EB
    9. pBM3R1_FB
    10. pBM3R1_EB
    11. pPhlF_GB
    12. pPhlF_EB
    13. ECK1200131570_DH
    14. ECK120015170_DG
    15. L3S2P21_DH
    16. L3S2P21_DG

Week of September 23
Tandem Promoters
  • Flow Cytometry set up for pTet-pBad with new small molecule concentrations
  • Somehow plates got mixed up, so flow results were not correct
  • Flow graphs showed GFP expression instead of RFP expression
  • Struck out -20 and -80 frozen stocks for pBad-pTet level 1 and pTet-pBad level 1 to give to WPI
  • Also made small molecule media to give to them
  • They will do confocal microscopy with tandem promoters


NEGEM!

Week of October 7
  • Struck out the following parts:
    1. DVL0_EB
    2. DVL0_DG
    3. DVL0_AB
  • Miniprepped those parts
  • MoClo for new promoters, tandem promoters, and terminators (mentioned in Week of September 16)
  • New tandem promoters:
    1. pBad-pBetI
    2. pLmrA-pSrpR







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