2 Where the FP came fromThere are 6 different FP used for this lab available through EBBEPOf the 6 proteins; 3 are from the Aequorea victoria jellyfish and 3 are from Discosoma striata anemoneAiquorea victoria jellyDiscosoma striata anemone
3 Structure of FP proteins All of the proteins used by EBBEP have a similar structure.They are all monomer barrel shaped molecules with a central chromatophoreSmall changes in theAmino acids in or directlyAround thechromatophore causechanges in color
4 How were the proteins made? All proteins were made by Mutagenesis of the natural occurring proteins found in types of Cnidarians.Proteins made fromanemone RFPTangerine (orange/pink)Cherry (Dark pink)Grape (purple)Proteins made fromJellyfish GFPEmerald (Green)Venus (yellow)Blue
5 Using evolutionSince the proteins come from two different “roots” of evolution you can see the inheritance in the sequences of the proteins
6 The FP plasmids Made from pRSET vector FP gene is less than 730bp Only difference in plasmids is the FP gene
7 Restriction enzymesType 2 restriction endonucleases are used in this lab. (most common)Cut DNA at a specific sequence.Enzymes usedHindIIIAhdI
8 Why are there restriction enzymes? Evolved by bacteria to protect against viral infectionOver 3000 known enzymes
9 Challenges in making the lab Needed an enzyme that cut green based genes different than red based genes.That’s easy! Several to choose fromFinding enzymes that cut the plasmid into a reasonable number of bands and have bands of an easily visible size on a gelNot so easy
10 Plasmid mapsProvided are 2 maps (red based and green based)
11 Reading the maps and predicting size HindIII only cuts the plasmid once making 1 linier piece the full size of the plasmid ~3623bp
12 Reading the maps and predicting size AhdI cuts 2 times producing 2 fragmentsSize of fragment 1= 2100bpSize of fragment 2 has 2 way to calculate= 1513bpOr= 1513
13 Teacher set-up tips Get labeling tubes early! DNA, Buffers, Water, loading dye, and marker/ ladder are directly aliquotted from stocks suppliedCan be completed a few days in advance lf you have a freezer. More if you have a frosted freezer
14 Teacher Set-Up tips Enzymes must be diluted. Receive a stock enzyme tube and a dilution tube for each enzyme.Add contents of one tube to the other (pre measured for you)Must be done no earlier than 24 hrs before or enzymes will not work as well
15 Teacher set up tipsSterile 1.5 snap cap tubes and filter pipette tips are ideal.Will work without the Sterile but you will possibly see fuzzier bands in the gel.Cap tubes before you give them out to help decrease the contamination
16 Teacher Set-Up tipsMake sure you plan for a long day. Staining gels with EtBr takes time.If you don’t have a digital imaging system then remember you digital camera.
17 Things to stress with students: Set up Digests on ICE!Concentrations are important (More is not better)Add reagents in Correct order.Water first ,Buffer, DNA, Enzymes are always last.Reason for Enzymes being last: Enzymes are sensitive to conditions outside of their normal range. Strong buffer solutions can effect the efficiency of the digest
18 Lab Procedure Each group of 2 students sets up 3 tubes. Negative control (no enzyme)An undigested plasmid shows as usually 2 or more bands (nicked/open circle, super super coiled,multimersTravelsFasterTravelsSlowerbs.kaist.ac.kr
19 Lab procedureHindIII digest-Should make 1 linier fragment. Shows true size of plasmidAhdI digest- determines if plasmids are from red (2 bands) or green (1band) line.2 Groups per gel with marker in middle.Best to give 1 green and 1 red to each gel.
20 Lab procedure Incubate at 37C. Freeze over night (fridge will work for 24hrs)Run gel. The bands are far enough apart you can run it pretty fast… if you can live with the smile bands on the gel
21 Keep it clean and don’t digest too long Contaminations of DNases can be devastating.Lane 8 has little to none DNase activityLane 1 has a large amount of DNase activitybiosyn.com
22 Things that can go wrong Impeded digestion due to incorrect set up. (too much or too little buffer etc.)Star digest activityUnder non-standard reaction conditions, some restriction enzymes are capable of cleaving sequences which are similar but not identical to their defined recognition sequence. This altered specificity has been termed star activity"
23 Star digestion example Examples:EcoRI is supposed to only cut GAATTC but, under extreme conditions, it might possibly cut CAATTC also.
24 Star digests Things that can cause Star digests Too much glycerol in reaction. (More is not better)Incorrect buffer of buffer concentrationExtended digest times. Don’t leave them over night
25 Expected resultsPlasmids made from the Green FP (jellyfish) digest into single bands for both HindIII and AhdIPlasmids made from the Red FP (anemone) digest into a single band for HindIII and 2 bands for AhdI
26 Sample gel from small FP plasmid digest lab MultimersLinierNicked circleSupercoiled2 distinct bands showing red protein originsRed HindIIIGreen AhdIRed AhdIGreen HindIIIUndigested green based plasmidUndigested red based plasmidMarker
27 Making a standard curve Measure to leading edge of each band in the marker/ ladder
28 Standard CurveGraph of lambda HindIII marker (base pair vs. Distance migrated)Using semi log graph paper