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From 2010.igem.org

Contents 1 Hassan 2 Andreas 2.1 Assembly of CPP⋅protein⋅His constructs 2.1.1 Assembly of His⋅SOD/yCCS and SOD/yCCS⋅His into pSB1K3 2.1.1.1 Transformation results 2.1.1.2 Colony PCR 2.1.1.3 Gel verification 2.1.2 Transfer of RFP to pEX 2.1.2.1 Colony PCR 2.1.2.2 Gel verification 2.1.2.3 Colony re-streak

Hassan

found some extra articles for gene-regulatory networks: [http://www.ncbi.nlm.nih.gov/pubmed/17504165] and [http://www.biomedcentral.com/1471-2105/8/S6/S9]

Andreas

Assembly of CPP⋅protein⋅His constructs

Assembly of His⋅SOD/yCCS and SOD/yCCS⋅His into pSB1K3

Step I in cloning strategy (19/8)

Transformation results

• pSB1K3.SOD⋅His
• pSB1K3.yCCS⋅His
• pSB1K3.His⋅SOD
• pSB1K3.His⋅yCCS

Good yield on all plates.

Colony PCR

Three white colonies picked from each plate for verification.

• pSB1K3.SOD⋅His: 1=A1, 2=A2, 3=A3
• pSB1K3.yCCS⋅His: 1=A4, 2=A5, 3=A6
• pSB1K3.His⋅SOD: 1=A7, 2=A8, 3=A9
• pSB1K3.His⋅yCCS: 1=A10, 2=A11, 3=A12
• Blank: A13

Procedures as described in colony PCR protocol.

• Forward primer: VF2
• Reverse primer: VR
• Elongation time: 2:00

Gel verification

Transfer of RFP to pEX

Continued from 19/8

Colony PCR

Since the gel results from 20/8 only verified pMA.His, a new colony PCR was run to verify pEX and pEX.RFP. Two new clones (5 & 6) of pEX.RFP were picked and run with clones 2 and 3 from 20/8. To test whether the red colonies could possibly be from pSB1K3 (i.e. if the selection didn't work properly), pEX.RFP 2 was also amplified with pSB primer VF2 and VR.
Also, pEX plasmid was amplified in parallel with pEX plasmid to compare the insert sizes.

• J1: pEX.RFP 2
• J8: pEX.RFP 3
• J2: pEX.RFP 5
• J3: pEX.RFP 6
• J4: pEX clone
• J5: pEX plasmid
• J6: pEX.RFP 2 (w/ pSB primers)
• J7: Blank

Procedures as described in colony PCR protocol:

• Forward primer: pEXf or pSB VF2
• Reverse primer: pEXr or pSB VR
• Elongation: 2:00

Gel verification

To be added

Colony re-streak

In order to verify that our red colonies are indeed carrying pEX and not pSB1K3 (i.e. the Amp selection has failed), resulting in false-positives for transfer of RFP from pSB1K3 to pEX, clones 2, 3, 5 and 6 were streaked on quarters of Amp 100 and Km 50 plates. Plates incubated 48 h in 37 °C.
If RFP has been successfully transfered to pEX, the clones should only grow on Amp 100 plates.