Part:BBa_K4335003

ChlamyHgR

Berthold et al. (2002)([1]) found that the aminoglycoside phosphotransferase gene (aphVII) from Streptomyces hygroscopicus works as a selectable marker for C. reinhardtii establishing a hygromycin resistance.

BBa_K4335003 is a resistance gene optimized for Chlamydomonas reinhardtii based on the original gene. We inserted a 145bp intron into the original gene to facilitate its expression in Chlamydomonas reinhardtii.

Introns are ubiquitous in eukaryotes and are an important feature that distinguishes them from prokaryotes. In higher organisms, introns have been reported to regulate expression at multiple levels. The main function of introns is to generate different exon combinations by alternative splicing and then translate different proteins, which improves the complexity of the proteome. [2]

Validation

We found from the registry page that BBa_K2984012 from iGEM19_Humboldt_Berlin's team was very similar, so we compared BBa_K4335003 with BBa_K2984012 using snapgene (Figure 1)

Figure 1 Comparison of BBa_K4335003 and BBa_K2984012 DNA sequences

The sequence difference between the two is only two bases. And the sequence of the protein expressed is exactly the same.

Usage

We inserted ChlamyHgR into plasmids pTX2038 and pTX2040 as marker genes for transformation screening.

Result

Plasmid construction

To verify our successful assembly, we designed two primers, Hgy-F and Hgy-R, for PCR verification. The primer location, primer sequence and gel map are shown in the following figure:

Figure 2.The primers：Hgy-F and Hgy-R

Figure 3.The length of the amplified sequence was 606bp；M is DNA Marker.

The determination of optimal screening concentration

We introduced plasmids pTX2038 and pTX2040 containing APHVII ChlamyHgR gene into Chlamydomonas reinhardtii by electrical transformation.

According to the specificity of the codon of Chlamydomonas reinharditii, our team optimized and designed a new part for the ChlamyHgR (Part:BBa_K4335003) based on the existing part, and the new part acted as the selection standard for transforming positive clones. However, the inhibition effect of hygromycin varies with species. Therefore, we designed and performed experiments to determine the minimum inhibitory concentration of Hyg on the growth of Chlamydomonas reinhardtii in solid medium.

Figure 4. Sequence alignment of Hyg resistance gene.
M:2000bp DNA marker; WT:wild type; Plasmid: Linear plasmid corresponding to the vector.

Figure 4. shows that the DNA sequence in the single algal colony was successfully amplified by PCR from pTX2038 and pTX2040. From this electrophoretogram, we can see the brightness of Hyg resistance gene fragment is consistent with the DNA bands of the positive control group, and match with the DNA Marker marking position, which indicates that the PCR products of the fragments transferred into the vector are in a high concentration and normal expression state.

Reference

Sequence and Features

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 NgoMIV site found at 782
  Illegal NgoMIV site found at 890
  
• 1000
  COMPATIBLE WITH RFC[1000]