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: Results Products labeled: - y A(star) (sbb20A), y B(star) (sbb20B), y C(star) (sbb20C)
 * Using the 2K55 temperature program, cloned the necessary parts for SOEing using the protocol: Cloning by PCR
 * The next step is to run a preparative gel to determine the results of the PCR.

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: Results Lanes Products labeled: - Preparative gel: JHL Gel frag - Post zymo clean: PCR Zc ABC
 * Ran a preparative gel on the PCR products from 2/17 (sbb20A,sbb20B,sbb20C)
 * 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
 * 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

2/24
Part sbb20: Purpose: Create sbb20 insert -- Using the external primers to combine the three parts together into sbb20. Protocol: Results Products labeled: - sbb20 sew
 * Ran a SOEing reaction using protocol: SOEing by PCR
 * Next step is to run an analytical gel to determine the results of the SOEing reaction.

3/1
Part sbb20: Purpose: Create sbb20 insert -- Identify whether or not the SOEing reaction produced the full length sequence. Protocol: Results
 * Ran an analytical gel
 * 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

Products labeled: - sbb20 SOE H2O - sbb20 SOE DMSO
 * Reattempting the SOEing, by repeating with a modified SOEing PCR to use DMSO or H2O and temperature program 45: SOEing by PCR

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: Results Products labeled: - 20 digest prep gel
 * Ran on preparative gel.
 * 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

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 Products labeled: - JHL sbb20 plate
 * 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.
 * The next step is to see if any colonies grow up on the selective resistance plates, if so pick colonies and culture them.

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 Products labeled: - Xfer digest zc
 * 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
 * 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.

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 Products labeled: - MP20 #1, MP20 #2, MP20 #3, and MP20 #4
 * Minipepped cells using protocol: Macherey-Nagel Minipepped
 * Need to determine whether or not the cells successfully took the full plasmid, this will be done next time by analytical gel.

Part sbb35: Purpose: Create the full correct plasmid (sbb35 part + vector) -- Combine the insert with the vector, transform into cells, and plate. Protocol: Results Products labeled: - JHL sbb35
 * 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
 * The next step is to see if any colonies grow up on the selective resistance plates, if so pick colonies and culture them.

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: Results
 * Ran an analytical gel with a portion of the miniprepped DNA using protocol: Analytical digest mapping
 * 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: Results Products labeled: - sb35 1, sb35 2, sb35 3, sb35 4
 * The cells were miniprepped using protocol: Macherey-Nagel Minipepped
 * 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.

3/17
Part sbb20: Purpose: Create the full correct plasmid (sbb20 part + vector) -- Check sequence of plasmid to target plasmid. Protocol: Results Products labeled: - Took the B6 well for sequencing of the sbb20
 * Sequencing
 * 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

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]