Part:BBa_K883162:Design

CCMV Delta 26 coat protein with N-terminal K-coil

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 172
  
• 1000
  COMPATIBLE WITH RFC[1000]

First approach: 4 step PCR making a delta 25 mutant with a k-coil attached

The approach of making this construct was by deleting the first 25 amino acids, and adding the K-coil with consecutive PCR steps. The PCR strategy is a 4 step N-terminal addition of the linker, K-coil and prefix (Figure 1). This construct might expose the K-coil on the outside of the CCMV VLP.

Figure 1: The PCR steps involved in getting an K-coil delta 25 CCMV

Frameshift occured:

The full construct of the delta 25 mutant was amplified and cloned. After sequencing and conformation of intended modifications, we found out that one of the many primers contained a minor mistake having major implications: a frameshift in the CCMV coat protein gene. The primer that was supposed to add the flexible linker to the coat protein contained a minor mistake: One ‘G’ was not taken up in the primer, causing a frameshift. After all the subsequent steps were undertaken, we found that the whole coil was on the coat protein as intended. However, due to the mistake, the open-reading frame of the coat protein was a mess, creating an early STOP-codon.

Frame shift in delta 25 mutant primers
After receiving the sequencing results, we saw that there was a frame shift in the sequence, leading to the faulty translation of the [http://2012.igem.org/Team:Wageningen_UR/ModifyingtheCCMV| CCMV] delta 25 coat protein, with an early stop codon(figure 2).

	Faulty	Intended
BP sequence	GTTTCTTCGAATTCGCGGCCGCTTCTAGATGAAGATAGCGG CGTTGAAGGAGAAAATCGCAGCACTAAAAGAAAAGATAGCG GCGTTGAAGGAGCTTGGTGGTGGTTCTGGTGGTGGTGGTTC TGCTGCTGCTGTGTGGTCCAACCTGTTATTGTAGAACCCAT CGCTTCAGGCCAAGGCAAGGCTATTAAAGCATGGACCGGTT ACAGCGTATCGAAGTGGACCGCCTCTTGTGCGGCTGCCGAA GCTAAAGTAACCTCGGCTATAACTATCTCTCTCCCTAATGA GCTATCGTCCGAAAGGAACAAGCAGCTCAAGGTAGGTAGAG TTTTATTATGGCTTGGGTTGCTTCCCAGTGTTAGTGGCACA GTGAAATCCTGTGTTACAGAGACGCAGACTACTGCTGCTGC CTCCTTTCAGGTGGCATTAGCTGTGGCCGACAACTCGAAAG ATGTTGTCGCTGCTATGTACCCCGAGGCGTTTAAGGGTATA ACCCTTGAACAACTCACCGCGGATTTAACGATCTACTTGTA CAGCAGTGCGGCTCTCACTGAGGGCGACGTCATCGTGCATT TGGAGGTTGAGCATGTCAGACCTACGTTTGACGACTCTTTC ACTCCGGTGTAT	GTTTCTTCGAATTCGCGGCCGCTTCTAGATGAAGATAGCGG CGTTGAAGGAGAAAATCGCAGCACTAAAAGAAAAGATAGCG GCGTTGAAGGAGCTTGGTGGTGGTTCTGGTGGTGGTGGTTC TGCTGCTGCTGGTGTGGTCCAACCTGTTATTGTAGAACCCA TCGCTTCAGGCCAAGGCAAGGCTATTAAAGCATGGACCGGT TACAGCGTATCGAAGTGGACCGCCTCTTGTGCGGCTGCCGA AGCTAAAGTAACCTCGGCTATAACTATCTCTCTCCCTAATG AGCTATCGTCCGAAAGGAACAAGCAGCTCAAGGTAGGTAGA GTTTTATTATGGCTTGGGTTGCTTCCCAGTGTTAGTGGCAC AGTGAAATCCTGTGTTACAGAGACGCAGACTACTGCTGCTG CCTCCTTTCAGGTGGCATTAGCTGTGGCCGACAACTCGAAA GATGTTGTCGCTGCTATGTACCCCGAGGCGTTTAAGGGTAT AACCCTTGAACAACTCACCGCGGATTTAACGATCTACTTGT ACAGCAGTGCGGCTCTCACTGAGGGCGACGTCATCGTGCAT TTGGAGGTTGAGCATGTCAGACCTACGTTTGACGACTCTTT CACTCCGGTGTAT
Primer FW1 FL-Delta25	TGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTGCTGC TGTGTGGTCCAACCTGTTATTGTAG	TGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTGCTGC TGGTGTGGTCCAACCTGTTATTGTAG

Figure 2: translation of the sequenced part, where the start codon (green) K-coil (red) and the flexible linker (light blue) are translated as intended. Between the linker and the start of the delta 25 CCMV coat protein CDS (dark blue) a frame shift occured, which leads into wrong AA sequence from that point, and an early stop codon

Fixing the frameshift with a Mutagenesis PCR, removing 2 basepairs

The frameshift was due to a single mistake in one of our primers. One approach of fixing this brick was by performing an mutagenisis PCR step, deleting 2 basepairs where the frameshift occurs, resulting in the right ORF. Mutagenesis depends on the priciple of whole plasmid PCR and Dpn1 digestion of methylated and hemimethylated DNA.An overview is given in figure 3.

Figure 3: Schematic overview of the Mutagenesis frameshift repair

Special primers were designed to remove 2 basepares from the plamsid construct (step 1). The primers used in this reaction are:
Coil_del204-205 5'-ggttctgctgctgctgtggtccaacctgtt-3'
Coil_del204-205-antisense 5'-aacaggttggaccacagcagcagcagaacc-3'

These primers are used in a PCR programme, as discribed in the [http://2012.igem.org/Team:Wageningen_UR/Protocol/Mutagenesis| QuikChange® Lightning Site-Directed Mutagenesis Kit protocol] (step 2). The newly created plasmids in the PCR are not methylated. The restriction enzyme Dpn1 only digests methylated DNA, so this enzyme can be used to digest and remove the parental plasmid (step 3). In the end we yield a plasmid where the 2 basepares are removed, and so yields a delta 26 K-coil CCMV coat protein coding sequence, with a correct ORF. This plasmid was used to transform Top10 cells (Step 4).

Source

We recieved the CCMV coat protein coding sequence on a pET28 plasmid from Dr. Kormelink from the Virology group Wageningen.

===References===