SBB10Ntbk-Mesa

Info

Profile
Project
Protocols

Parts Project

Construction Files

sbb27 (KRM27)

sbb27: CA42 rep Protein
PCR KRM001 and KRM002 on pEC22-CA42  (987 bp, EcoRI/BamHI)
Sub into pBca9523-Bca1144            (EcoRI/BamHI, 2472+910, L)
Product is pBca9523-sbb27         {rbs.repCA42!}
----------------------------
KRM001   Forward oligo for cloning of rbs.repCA42!   
ccataGAATTCatgAGATCTcccggagagaatcgtaacac
KRM002   Reverse oligo for cloning of rbs.repCA42!    
ctgatGGATCCctattttccgcttttccag

sbb16 (KRM16)

sbb16: phiC31 attP
Wobble KRM003/KRM004            (115bp, EcoRI/BamHI)
Sub into pBjk2741-Bca1144         (EcoRI/BamHI, 2170+910, L)
Product is pBjk2741-sbb16     {phiC31 attP}
----
KRM003   Forward construction of phiC31 attP basic part
ccataGAATTCatgAGATCTagaagcggttttcgggagtagtgccccaactggggtaacctttgag
KRM004   Reverse construction of phiC31 attP basic part
ctgatGGATCCgtgtcatgtcggcgaccctacgcccccaactgagagaactcaaaggttaccccagttg

2/17/2010

Received Oligos: KRM001, KRM002, KRM003, KRM004

Diluted all oligos to a 100μM concentration

Made 10μM dilutions of KRM001 and KRM 002

9μl H2O
1μl 100μM oligo

PCR and Wobble Reaction

Prepared one wobble and one PCR reaction, KRM16 and KRM27 respectively.

Wobble Reaction (KRM16)
29μl ddH2O
5μl 2
5μl dNTPs
5μl KRM003 (100μM)
5μl KRM004 (100μM)
0.75μl 1

PCR Reaction (KRM27)
24μl ddH2O
3.3μl 2
3.3μl dNTPs
1μl KRM003 (10μM)
1μl KRM004 (10μM)
0.5μl 1
0.5μl Template DNA (pEC22-CA42)

2/22/2010

Zymo Clean Up on PCR (KRM27)

Elute in 33μl

Small Fragment Zymo on Wobble (KRM16)

Elute in 50μl

PCR and Wobble Analytical Gels

Ran an Analytical Gel for Purified PCR (KRM27) and Wobble (KRM16) samples

3μl Purified PCR (KRM27)
7μl Loading Buffer
3μl Purified Wobble (KRM16)
7μl Loading Buffer

Purified Samples were stored in140L Box A

Gel Results

KRM27 expected size is 971bp (Second to far right lane)
KRM16 expected size is 99bp (Far right lane)

2/24/2010

EcoRI/BamHI Digests

PCR EcoRI/BamHI Digest on Purified PCR (KRM27)

Remaining Purified PCR product in tube labeled->KRM27, Purified PCR product, 2/22/10

Wobble EcoRI/BamHI Digest on Purified Wobble (KRM16)

All of Purified Wobble product was used in digest

Small Fragment Zymo on Wobble Digest (KRM16 Digest)

Elute in 50μl
Wobble digest product in tube labeled->KRM16 Digest, Purified Digest, 2/24/10

Preparative and Analytical Gels for Digests

Ran a Preparative and an Analytical Gel for PCR Digest (KRM27 Digest) and Wobble Digest (KRM16 Digest) samples, respectively

9μl PCR Digest (KRM27 Digest)
1μl 6X Loading Buffer
3μl Wobble Digest (KRM16 Digest)
7μl Loading Buffer

Cut out PCR Digest from preparative gel, place in 1.5ml tube Add 600μl ADB buffer @ 55°C

Gel Results

KRM16 Digest expected size is 99bp (Far right lane)

Samples were stored in 140L Box A

3/1/2010

Zymo Gel Purification on KRM27 Digest

Elute in 33μl

Ligation Reactions

KRM27 Digest into pBca9523-Bca1144
KRM16 Digest into pBjk2741-Bca1144

Negative Control Ligations

pBca9523-Bca1144 (no insert)
pBjk2741-Bca1144 (no insert)

Transformation

KRM27/pBca9523 => KRM27 Digest into pBca9523-Bca1144
KRM16/pBjk2741 => KRM16 Digest into pBjk2741-Bca1144

3/3/2010

Minipreps

Miniprep samples:

sb0027: KRM27-1,KRM27-2,KRM27-3,KRM27-4
sb0016: KRM16-1,KRM16-2,KRM16-3,KRM16-4

Mapping

Miniprep Digestion with EcoRI/BamHI on all miniprep samples

Gel Results

KRM27-1,KRM27-2,KRM27-3,KRM27-4 expected insert size is 971bp
KRM16-1,KRM16-2,KRM16-3,KRM16-4 expected insert size is 99bp

1. Ladder
2. sbb22 Digest
3. sbb20 Digest
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

3/8/2010

Remapping of sb0016 Samples

Digested KRM16-1,KRM16-2,KRM16-3,KRM16-4 with BamHI/XhoI

Gel Results

KRM16-1,KRM16-2,KRM16-3,KRM16-4 expected insert size is 783bp

All samples had an insert at the proper band size. Gel Pic

3/10/2010

Sequencing

KRM16-1,KRM16-2 and KRM27-1,KRM-2 were chosen to be sequenced.
Samples were given to Chris and were loaded as [Plate A:C1],[Plate B:C1] and [Plate A:D1],[Plate B:D1], respectively.

Sequence of sbb16

Sequence for [Plate A:C1] is a perfect read. Construct is pBjk2471-sbb16.
See: Sequencing Log.

3/16/2010

Sequence of sbb27

Sequence for [Plate A:D1] has a silent mutation (349bp A->G) Ala->Ala, but part is still good. Construct is pBca9523-sbb27.
See: Sequencing Log.

Team Assay Project

3/31/2010

Assay Team 3

Origin of Stability and copy number

Our group will first be characterizing the orthogonality of 3 colE2 replicons; CA42, 099, P9.
And secondly we will be measuring the regulatory effects of using the natural 5' regulatory elements of these replicons systems in E. coli.