SBB10Ntbk-JingLuo

 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

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

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

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

• Ran a gel purification of the preparative gel fragments using protocol: Zymo gel purification

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:

• Ran a SOEing reaction using protocol: SOEing by PCR
  

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

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

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

• It was extracted and run through gel purification using protocol: Zymo gel purification.
• Digested the product with restriction enzymes using the protocol: EcoR1/BamH1 Digest of PCR products

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:

• Ligated the part with the pBjk2741 vector using protocol: Ligation of EcoR1/BamH1 Digests
• Transformed into JTK086 spec cells using protocol: Transformation by Heat Shock
• GSI's plated out onto spec antibiotic plate.

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:

• Began a Eco/Bam transfer using protocol: EcoR1/BamH1 Part Transfer
• stopped after the restriction enzyme digestion that was done using protocol: EcoR1/BamH1 Digest of PCR products
• performed a regular zymo clean up on the part using protocol: Regular zymo cleanup

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:

• Minipepped cells using protocol: Macherey-Nagel Minipepped

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:

• The pBca1256 vector was pre-digested by the GSI's using protocol: EcoR1/BamH1 Digest of PCR products
• Ligated the part with pBca1256 vector instead of pBca9523 using protocol: Ligation of EcoR1/BamH1 Digests
• Transformed into DH108 spec cells using protocol: Transformation by Heat Shock
• Plated out onto spec antibiotic plate

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:

• GSI's picked a colony and cultured it according to protocol: Picking of Colonies

3/15

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

• Ran an analytical gel with a portion of the miniprepped DNA using protocol: Analytical digest mapping

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

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:

• The cells were miniprepped using protocol: Macherey-Nagel Minipepped

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

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

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]