Operon

Activator Operon

Repressor Operon

Topology

Landing Pad

Constitutive Promoter

• pArabLP was digested with EcoRI and NdeI
• BioBrick constitutive promoters were double digested EcoRI and NdeI.
• Digests were purified using QIAGEN Purification Kit
• Ligation was done overnight in 4⁰C.
• pArabLP-CP was then transformed into E.coli competent cells and grown overnight on LB+Amp+Cm plates in 37⁰C.
• Single colonies were isolated from the plates and grown in 5mL liquid culture containing LB+Amp+Cm overnight in 37⁰C.
• Plasmids from cells were isolated by using QIAGEN MiniPrep Kit
• Plasmids were then digested with EcoRI and SpeI overnight in 37⁰C (to ensure proper ligation).
• Analytical gel was then run to check fragment size of approximately 50bp.

NifA

• NifA was amplified from pRT22 using primers that had EcoRI, XbaI, NdeI and SpeI, PstI restriction sites flanking the 1560bp mutant sequence.
• The NifA sequence was then purified using the QIAGEN QIAquick PCR Purification Kit.
• NifA was double digested with NdeI and SpeI and left overnight in 37⁰C.
• pArabLP-CP was double digested with NdeI and SpeI also overnight at 37⁰C.
• Digests were purified using QIAGEN Purification Kit
• Ligation was done overnight in 4⁰C.
• pArabLP-NifA was then transformed into E.coli competent cells and grown overnight on LB+Amp+Cm plates in 37⁰C.
• Single colonies were isolated from the plates and grown in 5mL liquid culture containing LB+Amp+Cm overnight in 37⁰C.
• Plasmids from cells were isolated by using QIAGEN MiniPrep Kit
• Plasmids were then digested with EcoRI and SpeI overnight in 37⁰C (to ensure proper ligation).
• Analytical gel was then run to check fragment size of approximately 1650bp.

NifHp

• The NifH promoter (NifHp) was amplified from pRT22 using primers that had EcoRI, XbaI and NdeI, SpeI, PstI restriction sites flanking the 290bp sequence.
• The NifHp sequence was then purified using the QIAGEN QIAquick PCR Purification Kit.
• NifHp was double digested with EcoRI and SpeI and left overnight in 37⁰C.
• pLeuLP was double digested with EcoRI and SpeI also overnight at 37⁰C.
• Digests were purified using QIAGEN Purification Kit
• Ligation was done overnight in 4⁰C. Newly formed plasmid is renamed to pCLOCK1
• pCLOCK1 was then transformed into E.coli competent cells and grown overnight on LB+Amp+Cm plates in 37⁰C.
• Single colonies were isolated from the plates and grown in 5mL liquid culture containing LB+Amp+Cm overnight in 37⁰C.
• Plasmids from cells were isolated by using QIAGEN MiniPrep Kit
• Plasmids were then digested with EcoRI and SpeI overnight in 37⁰C (to ensure proper ligation).
• Analytical gel was then run to check fragment size of approximately 300bp.

NifL

• NifL was amplified from pIM18 using primers that had EcoRI, XbaI, NdeI and SpeI, PstI restriction sites flanking the 1590bp mutant sequence.
• The NifL sequence was then purified using the QIAGEN QIAquick PCR Purification Kit.
• NifL was double digested with NdeI and SpeI and left overnight in 37⁰C.
• pClock1 was double digested with NdeI and SpeI also overnight at 37⁰C.
• Digests were purified using QIAGEN Purification Kit
• Ligation was done overnight in 4⁰C. Newly formed plasmid is renamed to pCLOCK2
• pCLOCK2 was then transformed into E.coli competent cells and grown overnight on LB+Amp+Cm plates in 37⁰C.
• Single colonies were isolated from the plates and grown in 5mL liquid culture containing LB+Amp+Cm overnight in 37⁰C.
• Plasmids from cells were isolated by using QIAGEN MiniPrep Kit
• Plasmids were then digested with EcoRI and SpeI overnight in 37⁰C (to ensure proper ligation).
• Analytical gel was then run to check fragment size of approximately 2000bp.

GFP

• pClock2 was https://2008.igem.org/Team:Michigan/Notebook/DigestProtocol digested] with EcoRI and SpeI
• BioBrick BBa_E0040 that contained GFP was digested with EcoRI and XbaI in sequential digests.
• Digests were purified using QIAGEN Purification Kit
• Ligation was done overnight in 4⁰C.
• pCLOCK2 was then transformed into E.coli competent cells and grown overnight on LB+Amp plates in 37⁰C.
• Single colonies were isolated from the plates and grown in 5mL liquid culture containing LB+Amp overnight in 37⁰C.
• Plasmids from cells were isolated by using QIAGEN MiniPrep Kit
• Plasmids were then digested with EcoRI and SpeI overnight in 37⁰C (to ensure proper ligation).
• Analytical gel was then run to check fragment size of approximately 2700bp.