Team:Freiburg Cloning Strategy

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Introduction

To form different types of synthetic receptor constructs a principle of a building set was used. There were two different parts for an extracellular binding protein (scFv anti NIP, Lipocalin) and four different components of an intracellular signal transduction reporter protein, Cerulean CFP, Venus YFP, β-Lactamase and Luciferase. These reporters were used as Split-proteins to achieve that there is only detection when two receptors come together and form a cluster. With this strategy, a signal is only given by two binding-molecules that are next to each other as it is realized in the DNA-origami structure or the NIP and Fluorescein-coupled BSA.
The whole receptor construct consists out of (i) a signal peptide, (ii) an extracellular receptor domain, (iii) a GGGS-linker followed by (iv) a transmembrane region and (v) an intracellular split-reporter-protein. The signal peptide sequence according to the EGF-receptor erbb1 ensures the transport of the protein construct to the cytoplasma membrane. A GGGS-linker is necessary to create a distance between membrane and the recognition-site to overcome cell surface structures as glycoproteins and glycolipids. The sequence of the transmembrane region is taken from the EGF-receptor erbb1 as well. The C-terminal split-parts of Venus-YFP and Cerulean CFP were not directly fused to the transmembrane region. To get a little bit more flexibility in binding to the N-terminal part, a split fluorophor-linker was assembled in between.

Almost all parts were ordered by gene synthesis in a pMA-vector system that is adapted for cloning BioBrick parts. The constructs were designed according to BioBrick 3.0 standard with the modification for fusion-proteins proposed by the iGEM Freiburg Team 2007 ([http://parts.mit.edu/igem07/index.php/Freiburg07/report_fusion_parts FreiGEM07_report_fusion_part]). Keeping the standard iGEM prefix and suffix, the restriction sites NgoMIV and AgeI were added to create functional fusions without stop-codons.
All possible variations of the receptor constructs were fused together in the pMA-vector and afterwards inserted into a transfection vector. The transfection vector is a BioBrick 2006 part from the Ljubljana, Slovenia team ([http://partsregistry.org/Part:BBa_J52017 BBa_J52017]) and includes a kanamycin and ampicillin resistance cassette. There is a pMB1 Ori and the multiple cloning site consist of the BioBrick Prefix and Suffix restriction enzymes (Prefix: EcoRI, NotI, XbaI/ Suffix: SpeI, NotI, PstI) followed by an eukaryotic terminator sequence. To achieve a strong expression, a constitutive promoter construct was needed. Because of this, the Cytomegalovirus [CMV] promotor was amplified by PCR using the BioBrick part [http://partsregistry.org/Part:BBa_J52038 BBa_J52038 ]from the 2006 Ljubljana, Slovenia team as well and primers mentioned below. The forward primer bound in the iGEM-prefix, while the reverse primer bound at the end of the CMV-coding region. The reverse primer also contained an overhang to get the iGEM-suffix directly to the end of the promoter sequence which later allowed the cloning into the transfection vector. The CMV promoter was cloned into the vector by using the EcoRI and PstI restriction sites. To test the expression activity of the vector with the CMV promoter, the gene of the yellow fluorescent protein [YFP] was cloned downstream of the promoter region by using XbaI, PstI restriction enzymes for the YFP insert and SpeI, PstI for the vector to open the Biobrick suffix. The transfection of the resulting plasmid into 293T cells shows full functionality.
An overview about the cloning is given in table 1



Figure 1_cloning strategy . A shows the Biobrick part BBa_J52017. The transfection vector for eukaryotic cell systems has an ampicillin and a kanamycin resistance cassette. The multiple cloning site contains the Biobrick standard restriction sites EcoRI, NotI, XbaI, SpeI, NotI, PstI followed by a eukaryotic terminator sequence. B The CMV promotor fragment was obtained by PCR from the Biobrick BBa-J52038 template. C The PCR product was cloned into the transfection vector by EcoRI and PstI to get a final eukaryotic transfection system. D To test the efficiency of expression, a gene fragment coding for the yellow fluorescent protein was cloned into the vector downstream of the CMV Promotor.

Figure 2_cloning strategy . A figure 2 A gives an overview about the cloning constructs. The N-terminal signal peptide ensures protein transport to the cytoplasma membrane. Lipocalin and the scFv-anti-NIP are the extracytoplasmatic parts of the construct to mediate signal transduction into the cell. The GGGS-Linker keeps a distance to the transmembrane region to overcome surface structures of the cell and to avoid a total inflexibility. The split fluorophor linker is only necessary for the C-terminal split parts of Cerulean-CFP and Venus-YFP.  The split enzymes β-Lactamase and Luciferase and the split-fluorophors CFP and YFP are the cytoplasmatic parts of the constructs. If there is a clustering of this synthetic receptor-system caused by the corresponding binding parts of Lipocalin and scFv-anti-NIP, the split parts come together to create a functional protein, which allows detection.
B The different constructions described in figure 2.A were cloned into the transfection vector system by using the restriction sites XbaI and PstI to ensure a functional ATG-start codon which is part of the XbaI recognition-sequence in the iGEM-prefix.

Table1_cloning strategy overview about the cloning steps to create the different types of synthetic receptors. To get further information about the composite parts see

https://2008.igem.org/Team:Freiburg/Parts

Methods

The cloning was started with a preparative digestion of the DNA-Plasmids. To clone fusion parts the vector constructs were digested with AgeI and PstI to open the Biobrick suffix. The inserts were digested with NgoMIV and PstI. For cloning into the transfection-vector the enzymes SpeI and PstI were used for vector and XbaI, PstI for insert to keep up the ATG-start codon in the XbaI restriction site of the biobrick suffix. All restriction-enzymes were ordered from New England Biolabs. After digestion the DNA-fragments were separated on a 1% agarose gel. The DNA-band of interest was isolated and purified with the QIAGEN QIAquick Gel Extraction Kit. For the ligation a 3 molar excess of the insert was put together with the vector-fragment and ligated with a Quick ligase (New England Biolabs). After half an our at room temperature the DNA was transformed to chemical competent E.coli strain XL1 cells, plated on 2YT-agar-plates and incubated at 37°C over night. After picking clones and growing in 5ml LB-medium, the plasmid DNA was isolated by QIAGEN QIAprep Spin Miniprep Kit. A test digestion was prepared with about 0,5µg Plasmid DNA and NotI restriction enzyme to isolate the fusion-protein from the vector and to control if the expected bands were obtained. After a positive result the clones were sent to GATC-Biotech for sequencing. The GGGS-Linker was produced by Klenow -fill-in-PCR. Two primers were designed align to each other at 60°C and filled to a complete dobble-strand by Klenow Polymerase fragment.

Digestion Protocol

- about 2µg Plasmid-Prep in 20µl
- 2 µl NEB Buffer 10x
- 1 µl NEB enzyme 1 (NgoMIV, AgeI, XbaI, EcoRI)
- 1 µl NEB enzyme 2 (PstI, SpeI)
- 0.2µl BSA 100x

Ligation

- 10µl volume of vector and insert DNA (about 50ng vector-DNA)
- 1 µl DNA Quick Ligase (New England Biolabs)
- 10 µl Quick Ligase Buffer

Analytic digestion

- about 0.5 µg Plasmid-DNA in 5µl
- 5µl H2O
- 0.5 µl NotI
- 1µl NEB-Buffer
- 0.1 µl BSA

Transformation

- Competent cells (100µl) werde defrosted on ice
- 10µl of the ligation was added
- DNA and cells werde mixed softly
- Incubation on ice for 20-30 min
- Heat shock at 42°C for 40 sek
- cells were cooled down on ice for 5-10 min
- 900µl sterile 2YT Medium was added
- Incubation at 37°C for 60-70 min (shaker)
- cells werde plated on 2YT-agar-plates with antibiotics

Klenow fill in reaction

- 25pmol forward primer
- 25pmol reverse primer
- 0.5 µl Klenow-fragment without exonuclease activity (Fermentas)
- 2µl Klenow Buffer
- 1µl dNTPs
- Add H2O to a volume of 20µl
program: 94°C for 3min, cool down to 37°C, adition of klenow enzyme, 37°C for 1 hour

Results

All composite parts were succesfully cloned and controlled by sequencing and transfection as well (Freiburg Transfection and Synthetic Receptor)

Biobrick fusion protein

The present BioBrick prefix and suffix rules are not compatible with modular protein design. Thus as in 2007, we proposed an extension of the present standard for fusion proteins in which two restriction sites are added in frame adjacent to the coding sequence. The basic parts and as well all composite parts follow this strategy. To get further information see [http://parts.mit.edu/igem07/index.php/Freiburg07/report_fusion_parts FreiGEM07_report_fusion_part]

Cloned parts

The cloning parts scFv-anti-NIP, lipocalin-FluA, Split-Venus-N-YFP, Split-Venus-C-YFP, Cerulean-N-CFP and Cerulean-C-CFP were obtained by genesynthesis and ordered by the GENEART company. The β-Lactamase fragments were adopted from genesynthesis GENEART order in 2007. The erbb1-transmembrane, Split-Fluorophor-Linker and the erbb1-signalpeptide were synthesized by ATG:biosynthetics. The CMV promoter cloning part was produced by PCR with the template J52038 using the CMV_RLuc_Forward-primer and CMV_RLuc_Reverse-primer. To synthesize the GGGS-linker a Klenow-fill-in-reaction was performed by using Klenow-fragment without exonuclease activity and GGGS-forward and reverse primer.

CMV-Promotor-PCR [http://partsregistry.org/wiki/index.php?title=Part:BBa_K157040 BBa_K157040]
CMV_RLuc_Forward-primer: TCGCTAAGGA TGATTTCTGG AA
CMV_RLuc_Reverse-primer: AACGATCCTG CAGCGGCCGC TACTAGTAAT TTCGATAAGC CAGTAAGCAG

GGGS-Linker  (primer-fill-in)
AA sequence:
GGGSGGGSGG GSGGG
gene sequence:
GGAGGAGGTG GTTCAGGTGG TGGTGGTAGT GGAGGAGGAG GATCC

GGGS_Forward-primer: ATTATATTGA ATTCGCGGCC GCTTCTAGAt gGCCGGCGGA GGAGGTGGTT CAGGTGGTGG TGGTAGTGGA
GGGS_Reverse-primer: TTATATTTCT GCAGCGGCCG CTACTAGTAT TAACCGGTGG ATCCTCCTCC TCCACTACCA CCACCACCT

GGGS-linker (pMA) [http://partsregistry.org/wiki/index.php?title=Part:BBa_K157013 BBa_K157013]
AA sequence:
GGGSGGGSGG GSGGG
gene sequence:
GGTGGAGGAG GTTCTGGAGG CGGTGGAAGT GGTGGCGGAG GTAGC

Erbb1-transmembraneregion [http://partsregistry.org/wiki/index.php?title=Part:BBa_K157002 BBa_K157002]
IAA sequence:
ATGMVGALLL LLVVALGIGL FM
gene sequence:
ATAGCTACCG GAATGGTGGG TGCACTTTTG CTCCTTTTGG TCGTTGCCCT GGGGATAGGA CTCTTTATG

Signalpeptide [http://partsregistry.org/wiki/index.php?title=Part:BBa_K157001 BBa_K157001]
AA sequence:
MRPSGTAGAA LLALLAALCP ASRA
gene sequence:
ATGAGACCAT CTGGTACTGC TGGAGCCGCA TTGCTGGCAC TTTTGGCTGC GCTGTGCCCT GCAAGCAGAG CA

Split-Fluo-Linker [http://partsregistry.org/wiki/index.php?title=Part:BBa_K157009 BBa_K157009]
AA sequence:
RPACKIPNDL KQKVMNH
gene sequence:
CGACCAGCCT GTAAGATTCC AAATGACCTG AAGCAGAAAG TTATGAATCA C

Sc-Fv anti NIP [http://partsregistry.org/wiki/index.php?title=Part:BBa_K157003 BBa_K157003]
AA sequence:
QVQLQQPGAE LVKPGASVKL SCKASGYTFT SYWMHWVKQR PGRGLEWIGR IDPNSGGTKY NEKFKSKATL TVDKPSSTAY MQLSSLTSED SAVYYCARYD YYGGSYFDYW GQGTTVTVSS GGGGSGGGGS GGGGSQAVVT QESALTTSPG ETVTLTCRSS TGAVTTSNYA NWVQEKPDHL FTGLIGGTNN RAPGVPARFS GSLIGDKAAL TITGAQTEDE AIYFCALWYS NHWVFGGGTK LTVLG
gene sequence:
CAGGTGCAGC TCCAGCAGCC CGGAGCCGAA CTGGTGAAGC CAGGCGCCAG CGTGAAGCTG TCCTGCAAGG CCAGCGGCTA CACCTTCACC AGCTACTGGA TGCACTGGGT GAAACAGAGG CCCGGCAGAG GCCTGGAATG GATCGGCCGG ATCGACCCCA ACAGCGGCGG CACCAAGTAC AACGAGAAGT TCAAGAGCAA GGCCACCCTG ACCGTGGACA AGCCCAGCAG CACCGCCTAC ATGCAGCTGT CCAGCCTGAC CAGCGAGGAC AGCGCCGTGT ACTACTGCGC CAGATACGAC TACTACGGCG GCAGCTACTT CGACTACTGG GGCCAGGGCA CCACCGTGAC CGTGTCCTCT GGGGGAGGGG GCTCAGGAGG AGGAGGAAGC GGGGGAGGGG GCAGCCAGGC CGTGGTGACC CAGGAAAGCG CCCTGACCAC TCCCCTGGCG GACAGTGACC TGACCTGCCG TCCTCTACAG GCGCCGTGAC CACAAGCAAC TACGCCAACT GGGTGCAGGA AAAGCCCGAC CACCTGTTCA CCGGCCTGAT CGGCGGCACA AACAACAGAG CCCCTGGCGT GCCCGCTAGA TTCAGCGGCA GCCTGATCGG GGACAAGGCC GCCCTGACAA TCACAGGCGC CCAGACCGAG GACGAGGCCA TCTACTTTTG CGCCCTGTGG TACAGCAACC ACTGGGTGTT CGGCGGAGGG ACCAAGCTGA CCGTGCTGGG C

Split-Venus-N-YFP [http://partsregistry.org/wiki/index.php?title=Part:BBa_K157008 BBa_K157008]
AA sequence:
MVSKGEELFT GVVPILVELD GDVNGHKFSV SGEGEGDATY GKLTLKLICT TGKLPVPWPT LVTTLYLQCF ARYPDHMKQH DFFKSAMPEG YVQERTIFFK DDGNYKTRAE VKFEGDTLVN RIELKGIDFK EDGNILGHKL EYNYNSHNVY ITADKQ
gene sequence:
ATGGTGTCCA AGGGCGAGGA ACTGTTCACC GGCGTGGTGC CCATCCTGGT GGAGCTGGAC GGCGACGTGA ACGGCCACAA GTTCAGCGTG TCCGGCGAGG GCGAAGGCGA CGCCACCTAC GGCAAGCTGA CCCTGAAGCT GATCTGCACC ACCGGCAAGC TGCCCGTGCC CTGGCCCACC CTGGTGACCA CCCTGTACCT CCAGTGCTTC GCCAGATACC CCGACCACAT GAAGCAGCAC GATTTCTTCA AGAGCGCCAT GCCCGAGGGC TACGTGCAGG AACGGACCAT CTTCTTCAAG GACGACGGCA ACTACAAGAC CAGAGCCGAA GTGAAGTTCG AGGGCGACAC ACTGGTGAAC CGGATCGAGC TGAAGGGCAT CGACTTCAAA GAGGACGGCA ATATCCTGGG CCACAAGCTG GAATACAACT ACAACAGCCA CAACGTGTAC ATCACCGCCG ACAAGCAG

Split-Venus-C-YFP [http://partsregistry.org/wiki/index.php?title=Part:BBa_K157007 BBa_K157007]
AA sequence:
KNGIKANFKI RHNIEDGGVQ LADHYQQNTP IGDGPVLLPD NHYLSYQSKL SKDPNEKRDH MVLLEFVTAA GITHGMDELY K
gene sequence:
AAGAACGGCA TCAAGGCCAA CTTCAAGATC CGGCACAACA TCGAGGACGG CGGCGTGCAG CTGGCCGACC ACTACCAGCA GAACACCCCC ATCGGCGACG GCCCCGTGCT GCTGCCCGAC AACCACTACC TGAGCTACCA GAGCAAGCTG TCCAAGGACC CCAACGAGAA GCGGGACCAC ATGGTGCTGC TGGAATTTGT GACAGCCGCC GGAATCACCC ACGGCATGGA CGAGCTGTAC AAG

Cerulean-N-CFP [http://partsregistry.org/wiki/index.php?title=Part:BBa_K157006 BBa_K157006]
AA sequence:
MSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTL VTTLGVQCFA RYPDHMKRHD FFKSAMPEGY VQERTIFFKD DGNYKTRAEV KFEGDTLVNR IELKGIDFKE DGNILGHKLE YNAISDNVYI TADKQ
gene sequence:
ATGAGCAAGG GCGAGGAACT GTTCACCGGC GTGGTGCCCA TCCTGGTGGA GCTGGACGGC GACGTGAACG GCCACAAGTT CAGCGTGTCC GGCGAGGGCG AAGGCGACGC CACCTACGGC AAGCTGACCC TGAAGTTCAT CTGCACCACC GGCAAGCTGC CCGTGCCCTG GCCCACCCTG GTGACCACCC TGGGCGTGCA GTGCTTCGCC AGATACCCCG ACCACATGAA GCGGCACGAT TTCTTCAAGA GCGCCATGCC CGAGGGCTAC GTGCAGGAAC GGACCATCTT CTTCAAGGAC GACGGCAACT ACAAGACCAG AGCCGAAGTG AAGTTCGAGG GCGACACACT GGTGAACCGG ATCGAGCTGA AGGGCATCGA CTTCAAAGAG GACGGCAATA TCCTGGGCCA CAAGCTGGAA TACAACGCCA TCAGCGACAA CGTGTACATC ACCGCCGACA GCAG

Cerulean-C-CFP [http://partsregistry.org/wiki/index.php?title=Part:BBa_K157005 BBa_K157005]
AA sequence:
KNGIKANFKI RHNIEDGSVQ LADHYQQNTP IGDGPVLLPD NHYLSTQSKL SKDPNEKRDH
gene sequence:
MVLLEFVTAA GITLGMDELY KAAGAACGGC ATCAAGGCCA ACTTCAAGAT CCGGCACAAC ATCGAGGATG GCAGCGTGCA GCTGGCCGAT CACTACCAGC AGAACACCCC CATCGGCGAC GGCCCCGTGC TGCTGCCCGA CAACCACTAC CTGAGCACCC AGAGCAAGCT GTCCAAGGAC CCCAACGAGA AGCGGGACCA CATGGTGCTG CTGGAATTTG TGACAGCCGC CGGAATCACC CTGGGCATGG ACGAGCTGTA CAAG

Split beta-Lactamase 1/2 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K157018 BBa_K157018]
AA sequence:
MAGIATGMVG ALLLLLVVAL GIGLFMTGHP ETLAKVKDAE DQLGARVGYI ELDLNSGKIL ESFRPEERFP MMSTFKVLLC GAVLSRIDAGQ EQLGRRIHYS QNDLVEYSPV TEKHLTDGM TVGELCSAAI TMSDNTAANL LLTTIGGPKE LTAFLRNMGDH VTRLDRWEPE LNEAIPNDE RDTTTPVAMAT TLRKLLTGEL LGTG*
gene sequence:
ATGGCCGGCA TAGCTACCGG AATGGTGGGT GCACTTTTGC TCCTTTTGGT CGTTGCCCTG GGGATAGGAC TCTTTATGAC CGGCCACCCG GAAACCCTGG CCAAAGTGAA AGATGCGGAA GATCAGCTGG GTGCGCGTGT GGGCTATATT GAACTGGATC TGAACAGCGG CAAAATTCTG GAATCTTTTC GTCCGGAAGA ACGTTTTCCG ATGATGAGCA CCTTTAAAGT GCTGCTGTGC GGTGCGGTTC TGAGCCGTAT TGATGCGGGC CAGGAACAGC TGGGCCGTCG TATTCATTAT AGCCAGAACG ATCTGGTGGA ATATAGCCCG GTGACCGAAA AACATCTGAC CGATGGCATG ACCGTGGGCG AACTGTGCAG CGCGGCGATT ACCATGAGCG ATAACACCGC GGCGAACCTG CTGCTGACCA CCATTGGCGG TCCGAAAGAA CTGACCGCGT TTCTGCGTAA CATGGGCGAT CATGTGACCC GTCTGGATCG TTGGGAACCG GAACTGAACG AAGCGATTCC GAACGATGAA CGTGATACCA CCACCCCGGT GGCCATGGCG ACCACCCTGC GTAAACTGCT GACCGGCGAA CTGCTGGGCA CCGGTTAA

Split beta-Lactamase 2/2 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K157019 BBa_K157019]
AA sequence:
MAGIATGMVG ALLLLLVVAL GIGLFMTGGT PASRQQLMDW MKADKVAGPL LRSVLPAGWF IADKSGAGER GSRGIIAALG PDGKPSRIVV IYTTGSQATM DELNRQIAEI GASLIKHWTG *
gene sequence:
ATGGCCGGCA TAGCTACCGG AATGGTGGGT GCACTTTTGC TCCTTTTGGT CGTTGCCCTG GGGATAGGAC TCTTTATGAC CGGCGGTACT CCGGCTTCCC GGCAACAATT AATGGACTGG ATGAAAGCGG ATAAAGTTGC AGGACCACTT CTGCGCTCGG TGCTTCCGGC TGGCTGGTTT ATTGCTGATA AATCTGGAGC CGGTGAGCGT GGGTCTCGCG GTATCATTGC AGCACTGGGG CCAGATGGTA AGCCCTCCCG TATCGTAGTT ATCTACACGA CGGGGAGTCA GGCAACTATG GATGAACTGA ATCGTCAGAT CGCTGAGATA GGTGCCTCAC TGATTAAGCA TTGGACCGGT TAA

Split Luciferase 58 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K157020 BBa_K157020]
AA sequence:
EAIVDIPEIP GFKDLEPMEQ FIAQVDLCVD CTTGCLKGLA NVQCSDLLKK WLPQRCATFA SKIQGQVDKI KGAGGD
gene sequence:
GAAGCGATTG TGGATATTCC GGAAATTCCG GGCTTTAAAG ATCTGGAACC GATGGAACAG TTTATTGCGC AGGTGGATCT GTGCGTGGAT TGCACCACCG GCTGCCTGAA AGGCCTGGCC AACGTGCAGT GCAGCGATCT GCTGAAAAAA TGGCTGCCGC AGCGTTGCGC GACCTTTGCG AGCAAAATTC AGGGCCAGGT GGATAAAATT AAAGGCGCGG GTGGCGAT

Split Luciferase 57 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K157021 BBa_K157021]
AA sequence:
KPTENNEDFN IVAVASNFAT TDLDADRGKL PGKKLPLEVL KEMEANARKA GCTRGCLICL SHIKCTPKMK KFIPGRCHTY EGDKESAQGG IG
gene sequence:
AAACCGACCG AAAACAACGA AGATTTTAAC ATTGTGGCGG TGGCGAGCAA CTTTGCGACC ACCGATCTGG ATGCGGATCG TGGCAAACTG CCGGGCAAAA AACTGCCGCT GGAAGTGCTG AAAGAAATGG AAGCGAACGC GCGTAAAGCC GGTTGCACCC GTGGCTGCCT GATTTGCCTG AGCCATATTA AATGCACCCC GAAAATGAAA AAATTTATCC CGGGTCGTTG CCATACCTAT GAAGGCGATA AAGAAAGCGC GCAGGGCGGC ATTGGC