Chlamydomonas adapted Green Fluorescent Protein (CrGFP)

Transcription

1 Chlamydomonas adapted Green Fluorescent Protein (CrGFP) Plasmid pfcrgfp for fusion proteins Sequence of the CrGFP In the sequence below, all amino acids which have been altered from the wildtype GFP from Aequorea victoria are printed in bold face. 1 ATG GCC AAG GGC GAG GAG CTG TTC ACC GGT GTG GTC CCC ATC CTG GTG 48 M A K G E E L F T G V V P I L V 49 GAG CTG GAC GGC GAC GTG AAC GGC CAC AAG TTC TCC GTC TCC GGC GAG 96 E L D G D V N G H K F S V S G E 97 GGT GAG GGT GAC GCC ACC TAC GGC AAG CTG ACC CTG AAG TTC ATC TGC 144 G E G D A T Y G K L T L K F I C 145 ACC ACC GGC AAG CTG CCC GTG CCC TGG CCC ACC CTG GTC ACC ACC CTG 192 T T G K L P V P W P T L V T T L 193 ACC TAC GGT GTG CAG TGC TTC TCC CGC TAC CCC GAC CAC ATG AAG CAG 240 T Y G V Q C F S R Y P D H M K Q 241 CAC GAC TTC TTC AAG TCC GCC ATG CCC GAG GGC TAC GTG CAG GAG CGC 288 H D F F K S A M P E G Y V Q E R 289 ACC ATC TTC TTC AAG GAC GAC GGC AAC TAC AAG ACC CGC GCC GAG GTC 336 T I F F K D D G N Y K T R A E V 337 AAG TTC GAG GGC GAC ACC CTG GTG AAC CGC ATC GAG CTG AAG GGC ATC 384 K F E G D T L V N R I E L K G I 385 GAC TTC AAG GAG GAC GGC AAC ATC CTG GGC CAC AAG CTG GAG TAC AAC 432 D F K E D G N I L G H K L E Y N 433 TAC AAC TCC CAC AAC GTG TAC ATC ATG GCC GAC AAG CAG AAG AAC GGC 480 Y N S H N V Y I M A D K Q K N G 481 ATC AAG GTG AAC TTC AAG ATC CGC CAC AAC ATC GAG GAC GGC TCC GTG 528 I K V N F K I R H N I E D G S V 529 CAG CTG GCC GAC CAC TAC CAG CAG AAC ACC CCC ATC GGC GAT GGC CCC 576 Q L A D H Y Q Q N T P I G D G P 577 GTG CTG CTG CCC GAC AAC CAC TAC CTG TCC ATC CAG TCC GCC CTG TCC 624 V L L P D N H Y L S I Q S A L S 625 AAG GAC CCC AAC GAG AAG CGC GAC CAC ATG GTC CTG CTG GAG TTC GTC 672 K D P N E K R D H M V L L E F V 673 ACC GCT GCC GGC ATC ACC CAC GGC ATG GAC GAG CTG TAC AAG TAA 717 T A A G I T H G M D E L Y K * Base composition of the coding region: A 23,01 % GC 61,51 % C 34,31 % AT 38,49 % G 27,20 % T 15,48 % Amino acid exchanges from the wildtype gene from Aequorea victoria Two amino acids have been exchanged in the gene, F64L and S65T. The exchanges have effects three properties of the protein: The fluorescence intensity at an excitation wavelength of 480 nm increased 30fold; the formation of the chromophore is accelerated 10 to 20fold; and the protein shows only one excitation maximum at 489 nm versus two maxima (at 395 nm and 470 nm) in the wildtype. The reduction to one maximum suites the protein for confocal microscopy, because most laser-scanning confocal microscops (LSCM) work with lasers of 488 nm emission wavelength

2 The plasmid pfcrgfp The algal CrGFP is carried by the plasmid pcrgfp. This plasmid is based on the vector pksii -. The CrGFP has been inserted into the MCS of pksii - by using the restriction sites XhoI and BamHI. The restriction sites have been conserved. The CrGFP is not in frame with the lacz α gene β -galactosidase. Therefore, transformation of E.coli does not lead to functional expression, there is no GFP fluorescence. By inserting another gene in between the lac promoter and the GFP and correction of the frameshift, you can achieve functional expression of the fusion protein of the new gene and the GFP. The GFP fluorescence indicates then the successful construction of the fusion protein. Map of restriction sites for the vector Enzymes that do not cut in pfcrgfp: AatII AflII AscI AvrII BaeI BaeI BbsI BclI BglII Bpu10I Bpu1102I BsaBI BsmI BspEI BspMI BstAPI BstZ17I Bsu36I ClaI Eco47III EcoRI EcoRV FseI HindIII HpaI MluI MunI NarI NcoI NdeI NheI NruI NsiI NspV PacI PmeI PmlI PshAI PstI RsrII SalI SanDI SbfI SexAI SfiI SgfI SmaI SmiI SnaBI SphI SrfI Sse8647I StuI SunI Tth111I UbaEI XcmI XmaI Enzymes that cut, with position of restriction site: AgeI 1 AhdI 1 AlwNI 1 ApaI 1 BamHI 1 BcgI 1 BcgI 1 BplI 1 BsaI 1 BsbI 1 BseRI 1 BsmBI 1 BspGI 1BspLU11I 1 BstEII 1 DrdII 1 EagI 1 EcoNI 1 HgiEII 1 KpnI 1 MscI 1 NotI 1 Pfl1108I 1 RleAI 1 SacI 1 SacII 1 SapI 1 ScaI 1 SgrAI 1 SpeI 1 UbaDI 1 XbaI 1 XhoI 1 XmnI 1 AclI 2 ApaLI 2 BciVI 2 BfiI 2 BglI 2 BsrDI 2 BsrGI 2 BssHII 2 BssSI 2 BstXI 2 DraIII 2-2 -

3 FspI 2 NgoAIV 2 PflMI 2 PvuI 2 RcaI 2 SspI 2 BpmI 3 Bsp24I 3 Bsp24I 3 DraI 3 DrdI 3 EarI 3 PvuII 3 TaqII 3 VspI 3 Bce83I 4 BsaXI 4 BsgI 4 Eco57I 4 TaqII 4 BsrBI 5 EciI 6 AceIII 7-3 -

4 The multiple cloning site of pfcrgfp The CrGFP is not in frame with the lacz α gene of β -Galactosidase that is under control of the lac promoter. The frameshift is indicated by a hyphen in the MCS (after the XhoI restriction site). By using the restriction sites preceeding the GFP you can insert another gene and correct the frameshift. Please check that the insert has the correct number of nucleotides. The inserted gene must not contain a stop codon. The resulting fusion protein can be translated and shows the GFP fluroescence, indicating the correct construct

5 Transformation of E. coli with pfcrgfp To transform E. coli with pfcrgfp in its original or a modified version, you need competent E. coli cells. You find detailed instructions to prepare such cells for electrical or chemical transformation in "Current Protocols in Molecular Biology" (Ausubel et al., 1990) or in "Molecular Biology: A Laboratory Manual" (Sambrook et al. 1989). Map of the restriction sites of the CrGFP gene The CrGFP gene starts in the plasmid at position 2223 and ends at position The position of the restriction sites in this paragraph is given relative to the first nucleotide of the CrGFP, which has been asigned the number 1. You get the absolute position in the plasmid therefore by adding 2222 to the specified positions below. Enzymes that do not cut in CrGFP: AatII AclI AflII AhdI AlwNI ApaI ApaLI AscI AvrII BaeI BaeI BamHI BbsI Bce83I BcgI BcgI BciVI BclI BfiI BglI BglII Bpu10I Bpu1102I BsaI BsaBI BsbI BsmI BspEI BspLU11I BspMI BsrBI BsrDI BssHII BssSI BstAPI BstZ17I Bsu36I ClaI DraI DrdII EagI EarI Eco47III EcoRI EcoRV FseI FspI HgiEII HindIII HpaI KpnI MluI MunI NarI NcoI NdeI NheI NotI NruI NsiI NspV PacI Pfl1108I PmeI PmlI PshAI PstI PvuI RcaI RsrII SacI SacII SalI SanDI SapI SbfI ScaI SexAI SfiI SgfI SmaI SmiI SnaBI SpeI SphI SrfI Sse8647I SspI StuI SunI TaqII Tth111I UbaDI UbaEI VspI XbaI XcmI XhoI XmaI XmnI Enzymes that cut in CrGFP, with position of restriction site: AgeI 1 BplI 1 BseRI 1 BsmBI 1 Bsp24I 1 Bsp24I 1 BspGI 1 BstEII 1 BstXI 1 DraIII 1 DrdI 1 EcoNI 1 MscI 1 NgoAIV 1 PvuII 1 RleAI 1 SgrAI 1 TaqII 1 BpmI 2 BsaXI 2 BsrGI 2 Eco57I 2 PflMI 2 EciI 3 AceIII 4 BsgI 4-5 -

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