Part:BBa_K1468008
pT7 + gene encoding blue chromoprotein* (BBa_K864401). Codon usage optimized for Pseudomonas
BL21 is an Escherichia coli strain that bears the T7 phage polymerase gene into its genome. Therefore, it is used to express proteins that are controlled by T7 phage promoters, as is the case of three of our Biobricks (Biobricks 8, 9 & 10). However, when we transformed the strain with the Biobricks (blue and yellow chromoproteins), we could not measure any signal. After many tests, we hypothesized that the T7 phage polymerase gene was unstable and did not resist stressful situations such as competence or transformation protocols.
We performed a PCR screening to find out whether the T7 gene was present. We used colonies of BL21 transformed with Biobrick 8 (blue chromoprotein) and grew them on LBA medium. We performed a colony-PCR with a couple of T7 specific oligos designed by ourselves targeting a 1.5 kb region of the T7 polymerase coding sequence. On a 0.8% agarose gel electrophoresis, we saw that the bacteria forming the colonies did not have the gene that codes for the polymerase.
We then decided to check whether our glycerol stock of bacteria contained the gene. Maybe during the process of competence the culture was contaminated. To our surprise, the gene was present in every single colony we analyzed. Therefore, we decided to do a final colony PCR using the bacteria from the glycerol stock as positive controls, and using colonies from the non-transformed competent stock.
Our results reveal that BL21 loses the T7 polymerase gene at some point during the process of making the cells competent, as only three out of the ten colonies analyzed did result in (a rather faint) amplification of the gene.
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% BL21 colonies with T7 polymerase gene |
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Glycerol Stock |
100 |
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Competent cells (non transformed) |
30 |
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Transformed cells |
0 |
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |