Team:BostonU/NotebookML

From 2013.igem.org



Characterization Notebook

Goal: Develop a well-characterized library of parts for the MoClo Assembly Method

Subgoals Summary:
  • Assemble level 0 parts needed
    • Constitutive Promoter Characterization Project:
      • J23100_AB, J23113_AB, J23115_AB, J23115_EB, J23116_AB, J23116_EB, J23117_AB, J23118_AB, J23119_AB
      • BCD1_BC, BCD2_BC, BCD8_BC, BCD12_BC, BCD13_BC, BCD16_BC completed
    • Repressible Promoter Characterization Project:
      • C0012 (LacI), C0071_CD (rhlR_CD), pLacI (R0010_FB) completed
  • Assemble level 1 circuits for characterization of Constitutive Promoter Library
    • 160 circuits that have different combinations of J231XX promoters and RBS’s: red fill indicates that the construct has been made
    • 1 set with all promoters + BCD2 completed
    • 1 set with all promoters + B0034m1 completed
    • 1 set with all RBS’s+J23100 completed
    • 1 set with all RBS’s+J23104 completed
  • Assemble level 1 circuits for characterization of Repressible Promoter in progress, need to verify and run flow cytometry
  • Run flow cytometry on the promoter characterization circuits completed
  • Assemble level 2 inverter as an example of the library’s use completed
  • Switch the backbone in some existing level 1 parts to study how different antibiotics affect the flow cytometry data completed, need to analyze data in TASBE suite

May

Week of May 12, 2013
  • Learned about synthetic biology, MoClo, and developed plans for our summer projects, specifically quorum sensing.
  • Learned about Clotho software suite and learned how to use Eugene, RavenCAD, and Pigeon from other members of the CIDAR lab.
  • Familiarized ourselves with protocols and lab etiquette
  • Stocked up on lab supplies (plates, broth, plasmids)

Week of May 19, 2013
  • Began to create new bicistronic design RBS’s (BCDs): used PCR on BCD2 template to make BCD1, followed with LV0 reaction
  • Began to restock low concentration and low volume plasmid for level 0 parts

Week of May 26, 2013
  • Sent BCD1 made last week for sequence verification
  • Assembled Lv1 circuits to test out new fluorescent proteins (CyPet, DsRed, mCitrine, mOrange) and for flow cytometry training
  • Ran flow cytometry with Traci for our training
  • Continued to restock low concentration and low volume parts

June

Week of June 2, 2013
  • Continued creating new BCD set from BCD2 template, ran PCR to make BCD8, BCD12, BCD13, BCD16, followed with LV0 Rxn, and sequence verification. All reactions yielded original BCD2, likely due to preparing buffer the night before. Retried the same LV0 experiment later in the week.
  • Met with Dr. Jake Beal from BBN Technologies. He showed us how to use BBN’s Synthetic Biology Tools to analyze our future flow cytometry data.
  • Performed ligation PCR in order to generate the following constitutive promoters: J23110_AB, J23113_AB, J23115_AB, J23115_EB, J23116_AB, J23116_EB, J23117_AB, J23118_AB, J23119_AB
  • Met with Wellesley HCI team for a synthetic biology training session and to learn about each other’s projects. We also held Eugene tutorials and practiced making Eugene scripts for some famous experiments. We also had our first social event, bowling at Jillian’s!

Week of June 9, 2013
  • Tried to PCR apFAB46_AB, apFAB57_AB, apFAB61_AB, apFAB70_AB, apFAB71_AB promoters but unfortunately it failed
  • Tried PCR for R0079_EB, R0079_FB, C0012_CD, and C0062_CD but the transformations proved unsuccessful
  • Continued the second try on BCD Lv0 Parts, also failed: only produced BCD2 template. Probably too much DNA template in the PCR reaction. Retried entire protocol starting from PCR. BCD16_BC was successful
  • Archived frozen stocks and plasmids of BCD1_BC and BCD1lox_BC
  • Travelled to Wellesley to brainstorm some ideas for improving the visualization of the Eugene language
  • Attempted Level 1 reactions using J23100_AB, BCD1_BC, BCD2_BC, B0034_BC. B0034m1_BC, B0034m2_BC, E1010m_CD, B0015_DE, DVL1_AE; 80% of reactions failed. Discovered that BCD1_BC, BCD2_BC, B0034_BC, B0034m1_BC, and B0034m2_BC might be contaminated. Did Zymo transformation with RBS and BCDs that failed due to following an older protocol. Did Bioline transformation of RBS and BCDs

Week of June 16,2013
  • Continued third try on unsuccessful BCD Lv0 parts. This time we gel extracted the PCR products and used a higher quality ligase in the reaction mixture
  • Performed backbone switch for RFP transcriptional units from KAN to CAM and AMP
    • pJ004R_AE, pJ024R_AE, pJ034R_AE, pJ044R_AE, pJ054R_AE, pJ064R_AE, pJ074R_AE, pJ084R_AE, pJ094R_AE in both DVL0_GB and DVL2_AF
  • Attempted 6 Lvl 1 reactions: pJ004C40_AE, pJ004C79_AE, pJ00B1C40_AE, pJ00B1C79_AE, pJ00B2C40_AE, and pJ00B2C79_AE. Unfortunately the transformations did not result in colonies. It was later found that the terminator used B0015_DF was contaminated
  • Began PCR for B0015_DH, an essential terminator for our inverter constructs that has proven difficult to generate in the past
  • Did level 1 reactions to try and construct transcriptional units to impart resistance to spectinomycin, ampicillin, and kanamycin. These are referred to as pJ002maadA_EF, pJ002mbla_EF, and pJ002mkanR_EF, respectively
    • All level 1 reactions produced no insert when run on the gel. This was determined to be because the B0015_DF was actually B0033m_BC. A new PCR and streak out of B0015_DF from the -80 were started
  • Sequence confirmed J23110_AB, J23116_EB, J23117_AB, and J23118_AB and archived them. J23113_AB and J23115_AB were not sequence confirmed and will be restarted in the future
  • Transformed the BioBrick plasmids pSB1AT3 and pSB3T5 and miniprepped them. Both were then used as template in a PCR reaction to produce the tetA_CD level 0 part. The first level 0 reaction failed, but the second produced the appropriate band on a gel and was eventually sequence confirmed
  • Bioline transformation of RBS and BCDs from previous week was a success. Picked new colonies for overnight cultures and sent in samples for sequencing

Week of June 23, 2013
  • Sent BCD8, 12, and 13 for sequence verification. All were successful
  • Started workflow for Lv0s needed for pRepressible Library: C0012_CD, C0062_CD, C0071_CD, R0010_FB, R0079_FB by streaking out BioBrick cultures for the respective parts
  • Tested all Lvl 0, 1, and 2 destination vectors for possible biobrick backbone contamination. DVL0_DH was missing a SpeI site and DVL0_FB was missing 4 base pairs from its Lac Z insert
  • Did sequence analysis of BCD1_BC, BCD2_BC, B0034_BC, B0034m1_BC, B0034m2_BC. Sequences were correct; archived and stored new frozen stocks and plasmids

Week of June 30,2013
  • Added fusion sites to C0071, C0062, C0012, R0010, R0079 using PCR, followed with clean up, Lv0 Reaction, and sequence verification. R0010_FB, R0079_FB, and C0071_CD were successful. C0012_CD and C0062_CD were not. We think the DVL0_CD backbone is contaminated with empty biobrick backbone, causing an inefficient reaction and transformation. Tried to pick more colonies from plate to see if we could find some that contained the desired plasmid.
  • Struck out B0015_DF from the -20C and -80C frozen stocks to sequence since we think these samples are incorrect
  • B0015_DH was sequence confirmed but with only one SpeI site. It will function as a MoClo part but should not be used as a template in the future

July

Week of July 7, 2013
  • From the additional colonies, we picked for C0012_CD, C0062_CD; C0012_CD was successful. Redid the C0062_CD Lv0 Rxn
  • Started Lv1 reactions for our arabinose-tetR inverter that will serve as an example of how the library can be used
  • The PCR reactions and level 0 reactions for J23113_AB, J23115_AB, J23115_EB, J23116_AB, J23119_AB, and DVL0_DH were done and all were sent for sequencing.
  • Level 1 MoClo reactions were done for the pConstitutive library with different promoter and RBS combinations but all involving E1010m_CD, B0015_DE, and DVL1_AE. All were successfully archived.
    • PromoterRBS
    J23100_ABBCD1_BC
    J23100_ABBCD8_BC
    J23100_ABBCD12_BC
    J23100_ABBCD13_BC
    J23100_ABBCD16_BC
    J23101_ABBCD2_BC
    J23102_ABBCD2_BC
    J23103_ABBCD2_BC
    J23104_ABBCD2_BC
    J23105_ABBCD2_BC
    J23106_ABBCD2_BC
    J23107_ABBCD2_BC
    J23108_ABBCD2_BC
    J23109_ABBCD2_BC
    J23110_ABBCD2_BC
    J23111_ABBCD2_BC
    J23112_ABBCD2_BC
    J23117_ABBCD2_BC
    J23118_ABBCD2_BC
    J23119_ABBCD2_BC
  • Here is a sample of what one of the transformations looked like. A white colony was picked, cultured, plasmid purified, and confirmed.

Week of July 14,2013
  • Level 1 MoClo reactions were done for the pConstitutive library with different promoter and RBS combinations but all involving E1010m_CD, B0015_DE, and DVL1_AE. Because X-GAL ran out, we could not transform the following:
    • PromoterRBS
    J23101_ABB0034m1_BC
    J23102_ABB0034m1_BC
    J23103_ABB0034m1_BC
    J23104_ABB0034m1_BC
    J23105_ABB0034m1_BC
    J23106_ABB0034m1_BC
    J23107_ABB0034m1_BC
    J23108_ABB0034m1_BC
    J23109_ABB0034m1_BC
    J23110_ABB0034m1_BC
    J23111_ABB0034m1_BC
    J23112_ABB0034m1_BC
    J23113_ABB0034m1_BC
    J23114_ABB0034m1_BC
    J23115_ABB0034m1_BC
    J23116_ABB0034m1_BC
    J23117_ABB0034m1_BC
    J23118_ABB0034m1_BC
    J23119_ABB0034m1_BC
  • C0062_CD reaction contaminated with biobrick backbone, but the DVL0_CD sequence looked clean. Will streak out a new plate of DVL0_CD when we have more x-gal.
  • Made glycerols and archived successful Level 1 parts for inverters

Week of July 21, 2013

  • For pConstitutive library, confirmed following reactions:

    Promoter

    • Started a set of level 1 transcriptional units that will be used to characterize the repressible promoters pTetR and pLacI. LV1s pLacI-1 and pTetR-1 already exist. An example of the LV2 circuit that the pTetR-1,-2,and -3 transciptional units is shown below:

    • We had issues with pConstitutive constructs containing the promoters J23114 and J23115 with BCD2_BC. The promoters were sent for sequencing but were confirmed so they are not the issue.

    Level 1 Devices

    Shorthand

    Fusion Sites

    pLacI

    pLacI-1

    J23104+BCD2+E1010m+B0015

    pJ04B2Rm_AE

    AE

    pLacI-2

    J23104+BCD2+C0012+B0015

    pJ04B2C12_EF

    EF

    pLacI-3

    R0010+BCD2+E0040+B0015

    pR10B2G_FG

    FG





    pTetR

    pTetR-1

    J23104+BCD2+E1010m+B0015

    pJ04B2Rm_AE

    AE

    pTetR-2

    J23104+BCD2+C0040+B0015

    pJ04B2C40_EF

    EF

    pTetR-3

    R0040+BCD2+E0040+B0015

    pR40B2G_FG


    Week of July 28, 2013

    • Added Betaine, BSA, or DMSO to reactions that have not worked after 3 tries. These reagents have been know to be helpful in PCR reactions that are not working. We will see if they have any effect on the MoClo reaction yield.

    • Optimized reactions by reducing the total volume of the reaction and the relative volumes of the enzymes. ANY CONCLUSIONS?


    August

    Week of August 4, 2013

    • Obtained gel results and sequence results for pLacI-2,3 and pTetR-2,3. The -3 reactions did now show correct bands on the gel. The -2 reactions were gel confirmed, but the sequence showed there was an error in the J23104 promoter. There is an error in the part’s sequence. We will have to re-do this reaction.

    • Re-tried the reaction for pLacI-3 and pTetR-3 using a new strategy: only two parts are combined at once in the reaction. The promoter and RBS level 0 plasmids were added to a master mix of the T4 ligase, ligase buffer, BsaI enzyme, and water. Then they were put in the thermocycler to run 10 cycles of MoClo (37 C for 3 minutes, 16 C for 1.5 minutes). Then the next part was added, and the reaction mixture was run for another 10 cycles. This process was repeated for the terminator and the destination vector.

    • Nicked new prep of DVL0_CD with PstI to check for contamination of biobrick empty vector since there have been previous problems. Will use for LV0 reactions for C0062_CD, C0061_CD, C0080_CD, and new GFPm_CD


    Week of August 11, 2013

    • C0080_CD was sequence verified as level 0 part


    Week of August 18, 2013

    • Did reaction for last transcriptional unit of example inverter: pJ00B2C80_EF

    • J23104_EB confirmed and used in the coexpression, pLacI-2, pTetR-2, pBAD-2

    • GFPm_CD sequences were not correct


    Week of August 25, 2013

    • Built Level 2 Inverter consisting of:

      • I13453_AB+BCD2_BC+C0040_CD+B0015_DE+DVL1_AE
      • J23100_EB+BCD2_BC+C0080_CD+B0015_DF+DVL1_EF
      • I13453_FB+BCD2_BC+E0030_CD+B0015_DG+DVL1_FG
      • I13453_GB+BCD2_BC+E1010m_CD+B0015_GH+DVL1_GH

    • Completed pJ00B12Rm_AE and pJ11B16Rm_AE, missing devices from the pConstitutive library

      Here is the image of the transfomation

    • Began redoing I13453_GB, J23111_AB, and J23119_AB because their sequence files revealed either a gap or a handful of incorrect base pairs


    Week of September 1, 2013

    • Confirmed pJ04B2C62_FG

    • E0040m_CD has continued to present issues. Running PCR product on a gel produces a 550 bp band and a 1000 bp band when it should be 550 bp and 150 bp

    • R0062_AB, R0062_EB, and R0062_GB sequence confirmed

    • Confirmed the following for intended level 2 repressible constructs:

      • pR10B2Y_FG = R0010_FB+BCD2_BC+E0030_CD+B0015_DF+DVL1_FG
      • pR40B2Y_FG = R0040_FB+BCD2_BC+E0030_CD+B0015_DF+DVL1_FG
      • pI134B2Y_FG = I13453_FB+BCD2_BC+E0030_CD+B0015_DF+DVL1_FG



      • Week of September 8th, 2013

      • confirmed a second inverter, referred to as 13iGEM2_AH. It is an arabinose-tetR inverter that changes from yellow to red with the addition of arabinose

      • Converted the following MoClo level 1 reporter devices using PCR and XbaI/PstI:

      • pJ04B2mCit_EF
      • pJ04B8Rm_AE
      • pJ04B1Rm_AE
      • pJ04B12Rm_AE
      • pJ04B13Rm_AE
      • pJ04B16Rm_AE
      Week of September 15th, 2013 Week of September 22nd, 2013

      • Realized that the frozen stocks for pJ01B2Rm_AE-->pJ10B2Rm_AE do not correspond to the locations in the registry. After sequencing mini preps from the glycerols locations were fixed, but realized that the level 1’s with J23101, J23103, J23106, J23107, and J23110 need to be redone.

        confirmed redone pJ06B2Rm_AE and pJ07B2Rm_AE

      • Tried to redo the CoxRY_AF level 2 control device. The sequence verification reactions failed, and the minipreps had concentrations of only 15ng/uL

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