Part:BBa_K5261015
FLO11, an anchoring protein for cell adhesion
The FLO11 gene is an important gene in yeast and is mainly associated with cell adhesion, community behaviour and biofilm formation. It plays a key role in regulating cell-cell interactions and cell-substrate attachment. The FLO11 gene encodes a serine - and threonine-rich GPI-anchored glycoprotein that enhances cell-to-cell and cell-to-substrate carrier interactions in the presence of Ca(Ⅱ) in solution
Result
We tried to obtain FLO11 gene by PCR. The results of agarose gel electrophoresis showed (Figure 1) that the FLO11 gene (theoretical band 4129 bp) could be successfully obtained. However, its electrophoretic band brightness was low and the PCR product DNA concentration was extremely low. Therefore, we continued to purify and recover the PCR product of FLO11 by 8 fold and obtained 8×FLO11 with increased DNA concentration.
Figure 1. Electrophoresis results of FLO11 gene and 8×FLO11 gene
The pUMRI-HO plasmid vector skeleton and 8×FLO11 gene were assembled by Gibson Assembly. The ligation product was transformed into competent E. coli DH5α, and positive clones were screened on Kan plate. Six E. coli clones were selected for colony PCR verification of plasmid construction (theoretical band of 297 bp), and electrophoresis results (Figure 2) showed non-specific bands.
Figure 2. Electrophoresis results of colony PCR of pUMRI-HO-FLO11 plasmid
For further verification, we took the above E. coli monoclonal amplification, extracted the plasmid and sequenced it. The sequencing results (Figure 5) showed that the construction of pUMRI-HO-FLO11 plasmid failed.
Figure 3. The sequencing results of pUMRI-HO-FLO11 plasmid showed that the construction failed.
After that, we conducted two rounds of repeated experiments and still failed to construct.
Conclusion
The FLO11 gene mediates the encoding of a GPI-anchored glycoprotein rich in serine and threonine, and its length is also diverse between parents and offspring. The results of electrophoresis experiments showed that no matter we changed the PCR DNA polymerase or changed the primer design, it was difficult to obtain a single FLO11 gene band, and it was often prone to bright primer dimer bands. After 8×FLO11 concentration, the brightness of the band was still low, and it was difficult to obtain correctly assembled positive clones after Gibson Assembly.
We presume that the reason is that the FLO11 gene is longer (full length of 4104 bp) and the CDS region contains a large number of serine and threonine repeat sequences. On the one hand, it is prone to gene self-pairing during PCR amplification. On the other hand, it is also prone to mutations such as displacement during replication, which makes the PCR gene bands impure and trailing.
Reference
[1] Zara G, Zara S, Pinna C, Marceddu S, Budroni M. FLO11 gene length and transcriptional level affect biofilm-forming ability of wild flor strains of Saccharomyces cerevisiae. Microbiology (Reading). 2009 Dec;155(Pt 12):3838-3846.
[2] Watari, Junji, et al. Molecular cloning and analysis of the yeast flocculation gene FLO1. Yeast. 1994, 10(2): 211-225.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 75
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Illegal PstI site found at 3989 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 75
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Illegal PstI site found at 3989 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 88
Illegal XhoI site found at 4020 - 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 75
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Illegal PstI site found at 3989 - 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 75
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Illegal PstI site found at 3989 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 1016
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