SBB10Ntbk-JingLuo

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 Construction of sbb20: FokI Cleavage Domain - <FokI-!
 PCR gh1000F/gh1001R on BBa_K243001   (261 bp, gp = A)
 PCR gh1001F/gh1003R on BBa_K243001   (264 bp, gp = B)
 PCR gh1003F/gh1000R on BBa_K243001   (144 bp, gp = C)
 ---------------------------------------------------
 PCR gh1000F/gh1000R on A+B+C           (622 bp, EcoRI/BamHI)
 Digest pBjk2741                     (EcoRI/BamHI, 2170+910 L)
 Product is pBjk2741-sbb20
 ---------------------------------------------------
 gh1000F  Forward for part <FokI-!    ccagtGAATTCatgAGATCTCAGCTGGTTaaatctgaactggaggag
 gh1001F  Making internal mutation 1+2 cgttggttccccgatcgattatggcgttatcgtggAcacaaaagc
 gh1001R  Making internal mutation 1+2 cgccataatcgatcggggaaccaacggtataaatggcaccGTctggtttac
 gh1003F  Making internal mutation 3   ccatatcaccaatTgcaatggggcagtgctgag
 gh1003R  Making internal mutation 3   ccattgcAattggtgatatgg
 gh1000R  Reverse for part <FokI-!       gcaaaGGATCCTTAaaaattgatctcgccattgttg

Part:  GATCTCAGCTGGTTaaatctgaactggaggagaaaaaatccgagctgcgccacaaactgaaatatgtgcctcacgagtatatcgaactgatcgagatcgcccgtaatagtacccaagaccgtatcctggaaatgaaagtgatggagttcttcatgaaagtctatggctatcgtggcaaacatctgggtggtagccGTAAACCAGACGGTGCCATTTATACcgttggttccccgatcgattatggcgttatcgtggAcacaaaagcgtattctggcggttataatctgccgattggtcaggctgatgagatggaacgttatgtggaagagaatcagacccgtaacaaacatctgaacccgaacgaatggtggaaagtgtatccgtcaagtgtcaccgagttcaaatttctgttcgtgagcggccactttaaaggcaactataaagcccagctgactcgtctgaaccatatcaccaatTgcaatggggcagtgctgagtgttgaggaactgctgatcggtggagaaatgatcaaagcaggcaccctgactctggaagaagttcgccgtaaattCAACAATGGCGAGATCAATTTTTAAG
Construction of sbb35: P9 rep Protein - {rbs.ColE2 RepA}
Part already exists, only requires a EcoR1/BamH1 transfer into pBca9145-Bca1144.

Gel ladder that was used for all the experiments
Image:Generuler1kbplus.jpg
General Experiment Outline:

  • Generate the insert
  • Restriction enzyme digest the insert and vector
  • Purify necessary fragments using preparative gel and zymo gel clean
  • Ligate insert and vector together
  • Transform with correct cells
  • Plate onto appropriate selective antibiotic media
  • Pick colonies and culture
  • Miniprep colonies
  • Analytical digest mapping
  • Sequencing of colony that looked correct via digest mapping

Inventory (Inside Box C)

  • y A(star) - part A for SOEing
  • y B(star) - part B for SOEing
  • y C(star) - part C for SOEing
  • JHL Gel frag - preparative gel fragments of PCR A, B, C dissolved in ADB buffer
  • PCR Zc ABC - Zymo gel clean preparative gel fragments of PCR A, B, C
  • sbb20 Sew - SOEing with external primers, first attempt
  • sbb20 SOE H2O - 2nd round of SOEing of PCR products A, B, C
  • sb20 digest zc - Digested w/ EcoR1/BamH1 and zymo cleaned of the Round 2 SOEing reaction
  • 20 digest prep gel - sbb20 from preparative gel after digestion
  • Xfer digest zc - Eco/Bam transfer digest and zymo cleaned
  • MP20 #1 - Miniprepped sbb20 colony from transformed cells w/ part
  • MP20 #2 - Miniprepped sbb20 colony from transformed cells w/ part
  • MP20 #3 - Miniprepped sbb20 colony from transformed cells w/ part
  • MP20 #4 - Miniprepped sbb20 colony from transformed cells w/ part
  • sb35 1 - Miniprepped sbb35 colony from transformed cells w/ part
  • sb35 2 - Miniprepped sbb35 colony from transformed cells w/ part
  • sb35 3 - Miniprepped sbb35 colony from transformed cells w/ part
  • sb35 4 - Miniprepped sbb35 colony from transformed cells w/ part

Contents

2/17

Part sbb20:
Purpose: Create sbb20 insert -- Using the designed oligos to clone the fragments of sbb20, which will later be SOE'd together.
Protocol:

  • Using the 2K55 temperature program, cloned the necessary parts for SOEing using the protocol: Cloning by PCR

Results

  • The next step is to run a preparative gel to determine the results of the PCR.

Products labeled:
- y A(star) (sbb20A), y B(star) (sbb20B), y C(star) (sbb20C)

2/22

Part sbb20:
Purpose: Create sbb20 insert -- Identify the presence or not of the appropriate sequence, extracting it and purifying it from the gel.
Protocol:

  • Ran a preparative gel on the PCR products from 2/17 (sbb20A,sbb20B,sbb20C)

Results

  • Results of the preparative gel looked good. Lanes 9, 10, 11 (Part A, B, C respectively): Gel pic
  • part a = 261 bp
  • part b = 264 bp
  • part c = 144 bp

Image:XYL022210gel1.jpg
Lanes

  1. ladder
  2. sbb04A
  3. sbb04B
  4. sbb04C
  5. RH 33A
  6. RH33B
  7. blank
  8. blank
  9. CD1 <-- Part A
  10. CD2 <-- Part B
  11. CD3 <-- Part C
  12. sbb19A
  13. sbb19B
  14. ladder

Products labeled:
- Preparative gel: JHL Gel frag
- Post zymo clean: PCR Zc ABC

2/24

Part sbb20:
Purpose: Create sbb20 insert -- Using the external primers to combine the three parts together into sbb20.
Protocol:

Results

  • Next step is to run an analytical gel to determine the results of the SOEing reaction.

Products labeled:
- sbb20 sew

3/1

Part sbb20:
Purpose: Create sbb20 insert -- Identify whether or not the SOEing reaction produced the full length sequence.
Protocol:

  • Ran an analytical gel

Results

  • Results of analytical gel showed SOEing PCR failed to produce any product: Gel pic
  • Full SOEing sequence = 622 bp

Image:MN030110.jpg

  1. Ladder
  2. RH33-2K45
  3. RH33-2K45 w/ DMSO
  4. sbb02
  5. sbb01
  6. sbb03
  7. JL20 <-- SOEing reaction
  8. PiggyBacA
  9. Ladder
  • Reattempting the SOEing, by repeating with a modified SOEing PCR to use DMSO or H2O and temperature program 45: SOEing by PCR

Products labeled:
- sbb20 SOE H2O
- sbb20 SOE DMSO

3/3

Part sbb20:
Purpose: Create sbb20 insert -- Identify the presence or not of the appropriate sequence from the SOEing reactions and extract it.
Protocol:

  • Ran on preparative gel.

Results

  • The full length sequence was present: Gel pic
  • Full SOEing sequence = 622 bp

Image:Ak3-3-10.png

  1. Ladder
  2. sbb20 Digest <-- sbb20 SOE H2O
  3. sbb20 Digest <-- sbb20 SOE DMSO
  4. KRM27 1
  5. KRM27 2
  6. KRM27 3
  7. KRM27 4
  8. KRM16 1
  9. KRM16 2
  10. KRM16 3
  11. KRM16 4
  12. FK114
  13. Ladder

Products labeled:
- 20 digest prep gel

3/8

Part sbb20:
Purpose: Create the full correct plasmid (sbb20 part + vector) -- Combine the digested insert (sbb20 part) with the vector, transform into cells, and plate.
Protocol:

Results

  • The next step is to see if any colonies grow up on the selective resistance plates, if so pick colonies and culture them.

Products labeled:
- JHL sbb20 plate

Part sbb35:
Purpose: Create the full correct plasmid (sbb35 part + vector) -- Transfer the part from one vector to another, starting with digestion of the sbb35 part.
Protocol:

Results

  • The next step is to digest the vector and ligate the two together, whether or not this works will be determined once the cells are miniprepped and a analytical digest is run.

Products labeled:
- Xfer digest zc

3/9

Part sbb20:
Results:

  • Colonies did grow and the GSI's picked out colonies and cultured them using protocol: Picking of Colonies

3/10

Part sbb20:
Purpose: Create the full correct plasmid (sbb20 part + vector) -- Extract the DNA from the colonies so that it can be analyzed.
Protocol:

Results

  • Need to determine whether or not the cells successfully took the full plasmid, this will be done next time by analytical gel.

Products labeled:
- MP20 #1, MP20 #2, MP20 #3, and MP20 #4

Part sbb35:
Purpose: Create the full correct plasmid (sbb35 part + vector) -- Combine the insert with the vector, transform into cells, and plate.
Protocol:

Results

  • The next step is to see if any colonies grow up on the selective resistance plates, if so pick colonies and culture them.

Products labeled:
- JHL sbb35

3/11-15

Part sbb35:

3/15

Part sbb20:
Purpose: Create the full correct plasmid (sbb20 part + vector) -- Determine if the transformed cells DNA contain the full plasmid.
Protocol:

Results

  • Results of the analytical gel showed that it succeeded. The cells have an approximately full length plasmid: Gel pic
  • Insert and vector = 622 bp + 2170 bp

Image:Jing gel2.jpg

  1. Ladder
  2. sbb36 Clone 1
  3. sbb36 Clone 3
  4. sbb20 Clone 1 <--
  5. sbb20 Clone 2 <--
  6. sbb20 Clone 3 <--
  7. sbb20 Clone 4 <--
  8. Ladder
  • The next step is to sequence the plasmid to make sure that it is 100% what we wanted and no mutations.

Part sbb35:
Purpose: Create the full correct plasmid (sbb35 part + vector) -- Extract and purify the DNA from the cells
Protocol:

Results

  • The next step is to run the DNA on an analytical gel, this will allow us to see roughly whether or not the full sequence is present.

Products labeled:
- sb35 1, sb35 2, sb35 3, sb35 4

3/17

Part sbb20:
Purpose: Create the full correct plasmid (sbb20 part + vector) -- Check sequence of plasmid to target plasmid.
Protocol:

  • Sequencing

Results

  • Will receive the results later.
  • Edit later time: Sequence for [Plate A:B6] has numerous mutations and it not usable at all. See Sequence log

Products labeled:
- Took the B6 well for sequencing of the sbb20

Part sbb35:
Purpose: Create the full correct plasmid (sbb35 part + vector) -- Determine if the full sequence is present.
Protocol:

  • Ran an analytical gel
  • The result of the analytical gel showed that it failed. The plasmids turned out to be super huge: Gel pic 1+2 Gel pic 3+4

Image:Imageprepgeljhl.jpg

  1. Ladder
  2. sbb07 digest
  3. JL clone 1 <--
  4. JL clone 2 <--

Image:Mb 03 17 10.jpg

  1. ladder
  2. JL3 <--
  3. JL4 <--
  4. empty
  5. sbb23-1
  6. sbb23-3
  7. sbb23-4
  8. ladder
  • Ran out of time to finish this part and is left as is.

Team 4: Toxicity and Expression

[Link to Team Notebook]

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