pARA-R Sequence LABS 2a and 4a RFP Expression Sequence biotechnology lab program LABS 2a and 4a pARA-R Sequence RFP Expression Sequence Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Kristi Schramm Pierce College, Woodland Hills, CA V.1.2.1

Engineering the Plasmid: ligation of rfp gene into p-ARA sticky end BamH I sticky end Hind III sticky end Hind III sticky end BamH I

Recombinant plasmid of interest Restriction digest of pARA-R Sites labeled BamH I and Hind III represent restriction sites for these restriction enzymes Recombinant plasmid of interest pARA - R contains a gene for ampicillin resistance, ampr pARA-R 4720 bp rfp 702bp BamH I Hind III

Red sea anemone aids biomedical research Red sea anemone aids biomedical research. A new red fluorescent protein derived from a bright red sea anemone discovered in the tropical ocean.

The fluorescent protein gene was originally isolated from Discosoma , a sea anemone, which is a Eukaryotic organism. Therefore, the recombinant DNA plasmid, pARA-R has elements of both Prokaryotic and Eukaryotic Organisms. The pARA-R has been engineered into the bacterial plasmid to express the rfp gene to produce a mutant RED FLUORESCENT PROTEIN (mFP); known as GENE EXPRESSION

Restriction fragments after digest with Hind III and BamH I restriction analysis of pARA-R Restriction fragments after digest with Hind III and BamH I BamH I Hind III 4018 bp Hind III BamH I 702bp

Prediction for restriction gel restriction analysis of pARA-R Prediction for restriction gel M A+ A- M A+ A- 500 1000 1500 2000 3000 4000 5000 8000 10000 4.018kb 0.702kb

Lab 5a - Recombinant plasmid of interest pARA-R construct Lab 5a - Recombinant plasmid of interest pARA-R 4720 bp BamH I rfp 702bp Hind III

LAB 5A – Transformation of Escherichia coli (E. coli) A REVIEW  Recombinant DNA Plasmid Bacterial transformation involves transfer of genetic information into a cell by direct uptake of the DNA. Transformation can occur naturally but the incidence is very low. These bacterium can take up DNA only during the period of logarithmic growth. At this time the cells are said to be competent. Once competent, the cells are ready to accept DNA. (see clip) Restriction enzymes (Endonucleases) can be used to cut and insert pieces of foreign DNA into the plasmid vector - a self replicating DNA molecule. ** Gene Cloning Using Plasmids - reading assignment

preparing competent cells for transformation Lipid bilayer (inner) Adhesion zone Peptidoglycan layer Lipid bilayer (outer) Calcium ions

transforming Escherichia coli with pARA-R Competent Cells pARA-R Recombinant Plasmids

transforming Escherichia coli with pARA-R Lipid bilayer (inner) Adhesion zone Peptidoglycan layer Lipid bilayer (outer) Calcium ions pARA-R

Factors that determine transformation The larger the plasmid, the less likely it will be taken up by the bacterium. supercoiled form is the easiest. Shape of the plasmid - supercoiled, nicked circle, multimer. Supercoiled form is the easiest to pass through the membrane. Making Competent Cells Suspend (soak) in calcium chloride to neutralize the negative electrical charges of the plasma membrane. Create pressure difference between the inside and outside of the bacterial cell - “heat shock”, really hot and then really cold.

The bacterial plasmid ampr - ampicillin resistant gene - prevents bacteria from fully forming their cell walls. Arabinose - a simple sugar, is needed by the bacterium to express the rfp gene. PBAD - where the RNA polymerase needs to bind to initiate transcription

Answer the following questions before beginning the lab. What is transformation? What is a competent cell and how are competent cells produced? What is heat shock? What is a recombinant plasmid? What is the function of Ampicillin? What is the function of arabinose? Making Competent Cells 1.

Preparing Competent Cells for Transformation (re-suspended cells) pARA-R (plasmid) *AFTER 15 minutes in ice LB broth “LB” P+ 50 Microliters 0-5-0 (P-200) 10 Microliters 1-0-0 (P-20) 150 1-5-0 P- Negative control 50 Microliters 150 Microliters

PLATE PREPARATION P- P+ P- P+ P+ LB LB/amp LB/amp/ara Draw a line on the bottom of the LB and LB/amp plates - PREPARE PLATES DURING ZERO PERIOD

Results P+ P- P+ P- P+ _ + + + + LB LB/amp LB/amp/ara

growth of transformed bacteria on various plates P+ plates LB LB/amp LB/amp/ara P- plates No growth LB LB/amp

LAB 6 - Colony isolation and culture preparing an overnight culture of E. Coli for RFP expression LAB 6 - Colony isolation and culture LB/amp/ara broth

RFP expression araC protein prevents RFP transcription by causing a loop to form in the region of the fp gene r araC gene araC protein PBAD rfp gene

RFP expression araC gene PBAD rfp gene Transcription mRNA Translation araC protein

arabinose – araC protein RFP expression RFP (red fluorescent protein) Arabinose – araC protein complex prevents DNA looping and helps to align RNA polymerase on the promoter site (PBAD). arabinose Translation RNA polymerase arabinose – araC protein complex araC protein mRNA Transcription araC gene rfp gene PBAD

RFP

RFP

purification of RFP from an overnight culture Cell pellet with RFP Lysed cells Pellet cell debris RFP with binding buffer