Viability of Flp Site-Specific Recombinase in DNA Recombination Jonathan M. Howard The University of Texas at Austin 2002 Abstract The purpose of this experiment was to determine if the Flp protein and various mutant Flp proteins are viable by performing both in vivo and in vitro recombination assays. Each experiment used control plasmid pIG243 and coded plasmids pBU1 (kanamycin resistant) and pBU11 (ampicillin resistant). Both experiments also used a wild-type Flp and a mutant Flp (K82M). The in vivo assay was used to determine if the Flp protein could cleave the coding for a color inducing protein (lacZα) in the plasmids and recombine the DNA so that the plasmids would be resistant to both antibiotics. Results, determined by growth and color of colonies expressed on X-gal plates, indicated that in vivo recombination was successful. The in vitro assay used the same plasmids, but recombination was determined by DNA electrophoresis. The in vitro assay was performed three times, the first assay showed up positive, the second assay was negative, and the third assay returned positive results. Final conclusions are that the Flp used, both wild-type and mutant, are viable.

Viability of Flp Site-Specific Recombinase in DNA Recombination

Viability of Flp Site-Specific Recombinase in DNA Recombination Introduction Flp is a protein originating from the strain of yeast known as Saccharomyces cerevisiae and is a member of the integrase/tyrosine family. To increase production, Escherichia coli (strain DH10B) has been genetically engineered to create Flp. In order to test Flp and various mutant forms of Flp, the Flp recombination target (FRT) has been mutated (mFRTs). In the dual reporter screening method (in vivo), either lacZα or the red fluorescence protein (RFP) is flanked by mFRTs. The plasmids produced for both in vivo and in vitro assays are pBU1, pBU11, p33Rdw2 and p33Rdm11, which are coded FRT-lacZα-FRT, mFRT11-lacZα-mFRT11, FRTw2-RFP-FRTw2, and mFRT11-RFPmFRT11, respectively (for base pair sequencing of mFRTs, see Appendix A). Production of the reporters for blue/white colony color began with pUC18 being digested by SacI and HindIII, treated with Klenow polymerase and circularized by selfligation. This eliminates all restriction sites in the multiple cloning sites except for EcoR1. The lacZα gene from the resulting plasmid is the PCR-amplified using oligonucleotides. It was then cloned in pBAD24 (ampicillin resistant) and digested with SphI and HindIII to obtain pBU1 and pBU11, respectively. The reporters for red/white screening was constructed by PCR-amplifying the coding region of the RFP gene from plasmid pDsRed1-N1 (Stratagene, La Jolla, CA) and cloned into pUC18 digested with EcoRI and NdeI. Then the RFP region was PCR-amplified using primes and cloned into pBAD33 (chloramphenicol resistant) digested with SphI and HindIII to obtain p33Rdw2 or p33Rdm11, respectively (1). In each in vitro assay a different form of Flp was used, including wild-type, His tagged, and K82M∗ (for amino acid list and abbreviations see Appendix B and see Appendix C for Flp protein sequence). The FLP gene in the Flp expression plasmid p4BFlp was placed under control of the arabinose-inducible PBAD promoter (1). The DNA fragment spanning the araC-rrnB region from pBAD33 was inserted into pBBR1MCS-2 (kanamycin resistant) digested with NsiI and Bsu36I. The mob gene present in on the resulting plasmid was inactivated by digesting it with SfiI and BspHI, treating it with Klenow polymerase and re-circularizing it by self-ligation to get expression vector p4B. Wild-type or variant FLP genes were placed into p4B between the SacI and HindIII sites to obtain p4B-Flp or p4B-mFlp, in which the Flp expression can be rapidly induced by Larabinose and rapidly repressed when L-arabinose is removed from the medium. The wild-type Flp used in these experiments is Flpe, which contains P2S, L33S, Y108N and S294P substitutions (1).

Preparing Cell Cultures 1. A cell sample, produced by Dr. Yuri Voziyanov, was isolated and transferred to a sterile Falcon tube containing 10ml of LB (10g/l NaCl, 10g/l tryptone peptone and 5g/l yeast extract). Then, depending on which the cell sample was resistant to, 10µl of ∗

The number indicates the position in of the amino acid in the protein sequence. The first letter indicates the original amino acid and the end letter indicates the new amino acid.

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Viability of Flp Site-Specific Recombinase in DNA Recombination

either ampicillin (Amp), kanamycin (Km) or chloramphenicol (Cm) was added. Let culture incubate at 37°C overnight. (Note: Only a 1-5ml overnight culture should be made for plasmid purification) 2. When preparing to inoculate the large cell culture for protein purification, dilute the overnight culture 100 fold in LB. To inoculate a 200ml culture, use 198ml LB and 2ml of the overnight culture. Return to 37°C incubation until optical density (OD) is ~1.2 (~108cells/ml).

Plasmid Purification 1. We took a 1-5ml overnight culture and spun down the cells in the centrifuge. The supernatant was removed and the pelleted bacterial cells resuspended in 250µl Buffer P1 and transferred to a microcentrifuge tube. 250µl Buffer P2 was added and the tube gently inverted 4-6 times until solution was viscous and clear. The lysis reaction should not be allowed to last more than five minutes. 2. The supernatant was added to a QIAprep column by pipetting and centrifuged for 30-60 seconds and then the flowthrough was discarded. We washed QIAprep column by adding .5ml Buffer PB and centrifuging for another 30-60 seconds. The flowthrough was discarded. The QIAprep column was washed by adding .75ml Buffer PE and centrifuging for another 30-60 seconds. The flowthrough was discarded and the sample was centrifuged and additional 1 min. to remove residual buffer. 3. We then placed QIAprep column in 1.5ml microcentrifuge tube and eluted the DNA by adding 50µl Buffer EB (10mM Tris-Cl, pH 8.5) to the center of each QIAprep column, letting it stand for 1 min. and then centrifuging for 1 min. Finally, we stored the plasmid.

Protein Purification: No His Tag 1. We prepared a 500ml culture of E. coli (p4B-1034 producers, K82M). Arabinose was added to a final concentration of .2% in order to induce Flp production and shaken at the bench for approx. 4-5 hrs. The cells were spun down at 3000 rpm for 30 min at 4°C. The cells were then washed with cold dH2O 3 times. The cells were then frozen and stored at -70°C. 2. Cells were later resuspended in Buffer A0.25 (~10ml/g of cells). 1 mini-tablet of Protease Inhibitor Cocktail Tablets was added per 10ml of solution and was incubated on ice for 30 min. The solution was then frozen in liquid N2 and thawed in water three times or until the cells were broken and the solution had gained a viscous consistency. This means that the chromosomal DNA and protein are floating freely in the solution. The solution was then sonicated on ice for 15 second intervals at level 5 until the viscous consistency was lost. This is done to break apart the chromosomal DNA so that it can be separated from the protein. 3. The solution was then added to centrifuge tubes and spun down at 35,000 rpm for 60 min at 4°C. This removes the large chromosomal DNA. The supernatant was then saved and added to a beaker. 4. We then slowly added a 20% Streptomycin sulfate solution (in A0.2) to a final concentration of 4% and stirred gently for 30 min. The solution was then centrifuged at 15,000 rpm for 30 min at 4°C. This pulls down the smaller bits of DNA that were not removed previously. The supernatant was again saved and added to a beaker. We then

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Viability of Flp Site-Specific Recombinase in DNA Recombination

slowly add (NH4)2SO4 while stirring and then stirred for an additional 30 min. The final concentration of ammonium sulfate was ~.258g/ml of solution. The solution was then centrifuged at 12,000 rpm for 10 min at 4°C. This pulls down the protein. The supernatant was removed and the pellet gently respuspended in 3ml of Buffer A0.2. The solution was then further diluted to 50ml in A0.2. 5. The solution was then run for 8 times sample volume over a Sephadex G-25 column and then began collecting .5ml fractions and, according to ultraviolet absorption readings, the Flp moved through the column when the NaCl concentration was .6M. To determine which fractions had the highest Flp concentration a protein electrophoresis was performed (see “Protein Electrophoresis”). We then exchanged the .6M NaCl buffer with A0.1 Buffer, which was 50% glycerol. The sample was then divided into several tubes, flash frozen in liquid N2 and stored at -70°C.

Protein Purification: His Tag 1. We prepared a 2ml Ni-NTA column by running a 50% Ni slush through the column filters and allowing the Ni to settle. We then prepared our washing buffer (SB) and our elution buffer (EB). They contained equal concentrations of NaCl and PO4, but SB was 5mM Imidazole and EB was 250mM Imidazole. We then primed our Ni-NTA column with SB. 2. We then performed the same steps 1 and 2 as in “Protein Purification: No His Tag”. Then we spun down the solution at 25,000 rpm for 30 min at 4°C. The supernatant was saved, diluted to 50ml and added to a beaker. 3. The sample was run for 8 times sample volume over the Ni-NTA column and then collected in .5ml fractions when run with the elution buffer. Then, based of ultraviolet absorption readings, we tested for highest concentration of Flp by protein electrophoresis (see “Protein Electrophoresis”). We then exchanged Buffer EB for A0.1 Buffer, which was 50% glycerol. The sample was then divided into several tubes, flash frozen in liquid N2 and stored at -70°C.

Protein Electrophoresis 1. We first prepared separation gel. The gel consists of H2O, 40% Bis/Acrylamide, 1.5M Tris (pH 8.8), 10% SDS, 10% APS (ammonium persulfate), and TEMED to harden (for amounts, see Appendix D). We then prepared our stacking gel which consists of all the previous items, but in smaller amounts. We added a few microliters of the fractions which appeared to have Flp, based on ultraviolet absorption, which had been heat shocked at 90°C for 5min with a running dye into slots made by a “comb” in the stacking gel. We then ran the gel at 25mAmps for 90min in a 1x SDS buffer. 2. After the gel had run, we cut the stacking buffer and placed the separation buffer in a stain that contained Coomasse Blue, methanol, H2O, and acetic acid (for amounts, see Appendix E). The gel was stained for approximately 30min and then placed in an acetic acid solution for destaining. The results are shown in Appendix F and the arrow indicates the line which contains Flp. Results show that fractions 34-38 contain the highest concentrations of Flp.

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Viability of Flp Site-Specific Recombinase in DNA Recombination

Competent Cell Preparation 1. We prepared a 10ml overnight culture of E. coli using 25µg/ml kanamycin in LB. We added 1ml of overnight culture to 100ml prewarmed LB broth containing 25µg/ml kanamycin in a 250-ml flask and shook at 37°C until an OD of .5 was reached. The culture was cooled on ice for 5min and transferred to a sterile, round-bottom centrifuge tube. 2. The cells were centrifuged at 4,000 rpm for 5min at 4°C. We removed the supernatant while keeping the cells on ice. The cells were resuspended in 4ml of TFB2 buffer and then prepared into aliquots of 200µl in centrifuge tubes, flash frozen in liquid nitrogen, and store at -70°C.

In Vivo Recombination Assay 1. Plates were prepared for this assay containing LB, agar, kanamycin, ampicillin and X-gal. The double antibiotics ensure that for any cells to grow, they must have both the plasmid and Flp expression protein. 2. We first added .5µl of diluted plasmid (pBU1 or pBU11) per 10µl of competent cells which had been thawed on ice. We then incubated the cells for 30min on ice. The cells were then heat shocked at 42°C for 45 seconds and cooled on ice for 1min and 15 seconds. 200µl of LB was added and the cells were incubated at 37°C for 2 minutes. 3. Flp (either wild-type or K82M) production was induced by adding 2.2µl of 10% arabinose and the cells were incubated at 37°C for 2.5 hours. The tubes were placed in the rotary to ensure even mixing, but tubes could have been inverted every 15 minutes for the same effect. 4. The cells were then plated in 1µl, 10µl, and 100µl concentrations. Only those that had both the plasmid (either pBU1 or pBU11, Ampr) and the Flp expression (p4BFlp, Kmr) could grow on the plate. Once the cells grew, recombination could be determined by colony color. If recombination had taken place the cells would be white because the lacZα coding site would be gone. If recombination hadn’t taken place, the colony would be blue. Control plates were added to show that the cells could not grow if Flp was not introduced. Results are shown in Appendix G.

In Vitro Recombination Assay #1 1. A cocktail consisting of pIG243 (control plasmid), recombination buffer (to be diluted 10 times), 50% PEG and 50% glycerol was made (for all amounts in this assay, see Appendix H-1). This cocktail was to be used to be used in tubes 1-4. A second cocktail, consisting of the same substances, was prepared and added to tubes 5-8. We then added TE and wild-type His tagged Flp in various amounts to each tube. The tubes were then incubated at 30°C for 30min. 2. At the end of the incubation period we added 1% SDS to a final concentration of .1% (see Appendix H-2). We then added equal amounts of Protenase K. The tubes were then incubated at 37°C for 30min. 3. We then began phenol purification by diluting the solution to 200µl with TE and add 200µl of phenol. The tubes were vortexed until white and foamy and then spun down at maximum speed in the microcentrifuge for 5min. 195µl of the supernatant was removed from each tube and placed in clean tubes. Then 25µl of 1M NaCl was added

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Viability of Flp Site-Specific Recombinase in DNA Recombination

and the solution was diluted to 1ml with 100% ethanol. The tubes were then flash frozen in liquid nitrogen and stored at -70°C for 1 hour. 4. The tubes were thawed at the end of one hour and spun down at maximum speed for 15min. The supernatant was removed and the pellets washed with 80% ethanol. The tubes were then spun down for 5min and washed again. We then dried the tubes in the vacuum centrifuge. 5. We then resuspended the pellets in a cocktail of TE, BSA (10X diluted), NEB2 (10X diluted) and Xho1 (see Appendix H-3). Then a control was made that contained pIG243, TE, BSA (10X diluted), NEB2 (10X diluted), and Xho1. The solutions were then allowed to digest for 2 hours at 37°C. 6. The tubes were removed from digestion and a few microliters of dye were added to each tube. We then ran a DNA electrophoresis using the 8 samples, the control, and a marker. Results showed that recombination had occurred and are posted in Appendix I.

In Vitro Recombination Assay #2 1. A cocktail was made consisting of 10x recombination buffer, 50% PEG and 50% glycerol (for amounts used in this assay, see Appendix J-1). The cocktail was then added in various amounts to each tube, which were numbered 1-12. Tubes 1-3 and 10 contained plasmid pIG243, 4-6 and 11 contained plasmid pBU1, and 7-9 and 12 contained plasmid pBU11. Then His tagged Flp (K82M), TE, and 1M NaCl were added in various amounts to each tube. Tubes10-12 were then incubated for 30min. 4 incubation periods were assigned to tubes 1-9: A for 30min, B for 60min, C for 90min and D for 120min. All tubes were incubated at 30°C. 2. At the end of the incubation period (A, B, C, or D), 30µl was removed from each tube and added to a fresh tube (1A-9A, 1B-9B, etc.). 4µl of 1% SDS and 4µl of Protenase K were then added to get a final concentration of .1%. The tubes were incubated at 37°C for 30min. Then phenol purification was performed exactly as stated in “In Vitro Recombination Assay #1”, steps 3 and 4. 3. The pellets were then resuspended in a cocktail consisting of TE, 10x BSA, and 10x NEB2 (see Appendix J-2). Then, dependent on the tube, either Xho1 or HindIII was added. Control tubes 13, 14, 15 were then made using pIG243, pBU1, and pBU11, respectively, with TE, 10x BSA, 10x NEB2 and, dependent on the tube, either Xho1 or HindIII. The tubes were then digested for 2 hours at 37°C. 4. The tubes were removed from digestion and a few microliters of dye added to each tube. We then ran 6 gels consisting of the samples, control tubes, and marker. Results showed no recombination and are some are listed in Appendix K.

In Vitro Recombination Assay #3 1. A cocktail was made consisting of 10x recombination buffer, 50% PEG and 50% glycerol and distributed to each tube (for amounts in this assay, see Appendix L-1). The tubes were labeled 1-5 and either pBU11 or pIG243 was added dependent on the tube. Then, dependent on the tube, either His tagged K82M Flp of non-His tagged K82M Flp was added to the tube with either TE or 1M NaCl. The tubes were then incubated for 30min at 30°C.

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Viability of Flp Site-Specific Recombinase in DNA Recombination

2. At the end of the incubation 4µl 1% SDS and 4µl Protenase K were added to a final concentration of .1%. The tubes were then incubated for 30min at 37°C. Then phenol purification was performed as described in “In Vitro Recombination Assay #1”, steps 3 and 4. 3. We then resuspended the pellets in a cocktail of TE, 10x BSA, and 10x NEB2. We then add either Xho1 or HindIII, dependent on the tube. Then we made two control tubes consisting one of pBU11 and the other pIG243, TE, 10x BSA, 10x NEB2 and for pBU11, HindIII, and for pIG243, Xho1 (see Appendix L-2). The solutions were then digested for 2 hours at 37°C. 4. After digestion, a few microliters of dye were added to each tube. We then ran a DNA electrophoresis using our samples, our controls, and a marker. Results showed that recombination took place and are posted in Appendix M.

DNA Electrophoresis 1. We first prepared a gel made of agarose and ethidium bromide, which we placed slots into using a “comb”. While the gel hardened, we took a few microliters of each sample for one of the in vitro assays and added a 6x dye (diluted 6 times). The dye consisted of 70µl Bromophenol Blue, .5ml 100% glycerol, and 470µl H2O. 2. We then placed the gel into a 1x TAE buffer and began to slowly add the samples into the slots along with a marker. The gel was then run at either 50 or 100 Volts until the dye was approximately ¾ of the way down the gel. The gel was then removed from the buffer and placed under ultraviolet light, which made visible the ethidium bromide stained bands. These results are shown under Appendixes I, K, and M.

Conclusions Based on the results of the in vivo assay, it can be inferred that the wild-type Flp has little or no attraction to the mutant mFRT11 contained in pBU11, and near 100% attraction to the natural FRT in pBU1. It can also be inferred that K82M Flp has an almost equal attraction for both the natural FRT site and the mutant mFRT11 site. This shows that K82M Flp has a broader site-specificity than wild-type Flp. The in vitro assays show that both wild-type and non-His tagged K82M Flp are viable Flp strands. The negative results of in vitro assay #2 are most likely due to reduced reactivity by the His tag and the use of excess tubes which lengthened the process out long enough for trace nucleases to eat away the protein. Finally, the results that show that mutant Flp is viable and has a broader sitespecificity may lead to new methods in biotechnology and genome engineering. Continued mutation of FRT sites and recombinases can lead to individually designed recombinases with either broad or narrow site-specificity, as is required. As further process is made, Flp protein, and recombinases like it, may one day aid in the fight against genetic diseases, including cystic fibrosis.

Works Cited 1. Voziyanov, Yuri; Stewart, Francis; Jayaram, Makkuni; Nucleic Acids Research, 2002, Vol. 30, No. 7

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Viability of Flp Site-Specific Recombinase in DNA Recombination

2. Saxena, Pratibha; Whang, Ilson; Voziyanov, Yuri; Harkey, Cecil; Argos, Patrick; Jayaram, Makkuni; Dandekar, Thomas; Biochemica et Biophysica Acta 1340 (1997)

Acknowledgements My most sincere gratitude goes out to the Welch Foundation, Dr. J. J. Lagowski, and all those who have made the Welch Summer Scholar Program possible with their ongoing support. I would like to thank my chemistry teacher, Mr. Teyneyck, who nominated me for the program and taught me most of what I know about chemistry. I would also like to thank my family for their support. I am most appreciative to Dr. Makkuni Jayaram, who allowed me to enter his lab and take part in his research, and to the student I worked, Jay Konieczka, for his guidance and information provided on the research and how the recombination process takes place. Finally I would like to thank the other “Summer Scholars” and all those who helped me in this project.

Appendix A

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Viability of Flp Site-Specific Recombinase in DNA Recombination

Appendix B Amino Acids Alanine Arginine Asparagine Aspartic Acid Cysteine Glutamine Glutamic Acid Glycine Histidine Isoleucene Leucene Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine

Abbreviations Ala/a Arg/r Asn/n Asp/d Cys/c Gln/q Glu/e Gly/g His/h Ile/i Leu/l Lys/k Met/m Phe/f Pro/p Ser/s Thr/t Trp/w Tyr/y Val/v

Appendix C mpqfgilckt ppkvlvrqfv erferpsgek lcwmithngt aikratfmsy ntiisnslsf divnkslqfk yktqkatile eftiipyygq khqsditdiv sslqlqfess eeadkgnshs kkmlkallse ilnsfeytsr ftktktlyqf lflatfincg rfsdiknvdp ksfklvqnky etktsvsrhi yffsargrid plvyldeflr nsepvlkrvn rtgnsssnkq rsynkalkkn apysifaikn gpkshigrhl mtsflsmkgl teltnvvgnw ttythqitai pdhyfalvsr yyaydpiske mialkdetnp ieewqhieql pawngiisqe vldylssyin rri

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ialcaaelty aslkklipaw gesiweitek lgviiqclvt eyqllkdnlv sdkrasavar kgsaegsiry

Viability of Flp Site-Specific Recombinase in DNA Recombination

Appendix D Separation Gel 4.3 ml 3 ml 2.5 ml 100 µl 100 µl 10 µl

ddH2O 40% Bis/Acrylamide 1.5M Tris (pH 8.8) 10% SDS 10% APS TEMED

Stacking Gel 2.87 ml .5 ml .5 ml 40 µl 40 µl 8 µl

Appendix E Components Coomasse Blue Methanol ddH2O Acetic Acid

Amounts .75 g 135 ml 135 ml 30 ml

Appendix F M = Marker, S = Sample, F = Flowthrough

Flp protein bands

M

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F

33 34 35

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Viability of Flp Site-Specific Recombinase in DNA Recombination

Appendix G

Flp/pBU1 Plate

K82M/pBU1 Plate

Flp/pBU11 Plate

K82M/pBU11 Plate

Appendix H-1 Tube 1 2 3 4 5 6 7 8

pIG243 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl

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10x Rec. Buffer 3 µl 3 µl 3 µl 3 µl 4 µl 4 µl 4 µl 4 µl

50% PEG 6 µl 6 µl 6 µl 6 µl 8 µl 8 µl 8 µl 8 µl

UT3-11

50% glycerol 6 µl 6 µl 6 µl 6 µl 8 µl 8 µl 8 µl 8 µl

Flp 1 µl 2 µl 5 µl 10 µl 5 µl 7 µl 10 µl 15 µl

TE 9 µl 8 µl 5 µl – 10 µl 8 µl 5 µl –

Viability of Flp Site-Specific Recombinase in DNA Recombination

Appendix H-2 Tube 1-4 5-8

1% SDS 4 µl 5 µl

Protenase K 4 µl 5 µl

Appendix H-3 Tube 1-8 Control

pIG243 – 5 µl

TE 22 µl 17 µl

10x BSA 3 µl 3 µl

10x NEB2 3 µl 3 µl

Xho1 2 µl 2 µl

Appendix I M = Marker, C = Control

Recombination bands

M C

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2

3

4

M

5

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6

7

8

Viability of Flp Site-Specific Recombinase in DNA Recombination

Appendix J-1 Tube 1 2 3 4 5 6 7 8 9 10 11 12

Amt. Plasmid 20 µl 20 µl 20 µl 20 µl 20 µl 20 µl 20 µl 20 µl 20 µl 5 µl 5 µl 5 µl

10x Buffer 12 µl 12 µl 12 µl 12 µl 12 µl 12 µl 12 µl 12 µl 12 µl 3 µl 3 µl 3 µl

50% PEG 24 µl 24 µl 24 µl 24 µl 24 µl 24 µl 24 µl 24 µl 24 µl 6 µl 6 µl 6 µl

50% glycerol 24 µl 24 µl 24 µl 24 µl 24 µl 24 µl 24 µl 24 µl 24 µl 6 µl 6 µl 6 µl

Flp

TE

NaCl

4 µl 8 µl 20 µl 4 µl 8 µl 20 µl 4 µl 8 µl 20 µl 2 µl 2 µl 2 µl

25.2 µl 22.4 µl 18 µl 25.2 µl 22.4 µl 18 µl 25.2 µl 22.4 µl 18 µl 5 µl 5 µl 5 µl

10.8 µl 9.6 µl 6 µl 10.8 µl 9.6 µl 6 µl 10.8 µl 9.6 µl 6 µl 3 µl 3 µl 3 µl

10x NEB2 2 µl 2 µl 2 µl 2 µl 2 µl

HindIII – 2 µl – 2 µl 2 µl

Appendix J-2 Tube Amt. Plasmid 1-3, 10 – 4-9, 11, 12 – 13 5 µl 14 5 µl 15 5 µl

TE 14 µl 14 µl 9 µl 9 µl 9 µl

10x BSA 2 µl 2 µl 2 µl 2 µl 2 µl

Appendix K M = Marker

M 10 1A 2A 3A 1B 2B 3B 13

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M 12 7A 8A 9A 7B 8B 9B 15

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Xho1 2 µl – 2 µl – –

Viability of Flp Site-Specific Recombinase in DNA Recombination

Appendix L-1 Tube

Plasmid

1)K82M/pBU11/.1M NaCl 2)K82M/pIG243/.1 M NaCl 3)K82M/pBU11/.6M NaCl 4)K82M/pIG243/.6 M NaCl 5)His-Flp/pIG243

50% PEG 6 µl

50% glycerol 6 µl

Flp

TE

NaCl

5 µl

10x Buffer 3 µl

7.5 µl

–

2.5 µl

5 µl

3 µl

6 µl

6 µl

7.5 µl

–

2.5 µl

5 µl

3 µl

6 µl

6 µl

7.5 µl

2.5 µl

–

5 µl

3 µl

6 µl

6 µl

7.5 µl

2.5 µl

–

5 µl

3 µl

6 µl

6 µl

7.5 µl

–

2.5 µl

Appendix L-2 Tube 1 2 3 4 5 6)pBU11 7)pIG243

Plasmid – – – – – 5 µl 5 µl

TE 22 µl 22 µl 22 µl 22 µl 22 µl 17 µl 17 µl

10x BSA 3 µl 3 µl 3 µl 3 µl 3 µl 3 µl 3 µl

Appendix M M = Marker

M

1

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3

4

5

6

7

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10x NEB2 3 µl 3 µl 3 µl 3 µl 3 µl 3 µl 3 µl

Xho1 – 2 µl – 2 µl 2 µl – 2 µl

HindIII 2 µl – 2 µl – – 2 µl –

Viability of Flp Site-Specific Recombinase in DNA Recombination

Appendix N

UV absorption peaks

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