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Team:Glasgow/Project/Mobility Proteins
From 2014.igem.org
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<h2 id="subheading">Motility Genes: MotA and MotB</h2> | <h2 id="subheading">Motility Genes: MotA and MotB</h2> | ||
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We were investigating motility genes which would allow bacteria to swim when the switch was off. Our main working strains DS941, DH5α and TOP10 had motA knock-out variants and, thus, we decided to incorporate motA into our switch on a different plasmid and rescue the gene. | We were investigating motility genes which would allow bacteria to swim when the switch was off. Our main working strains DS941, DH5α and TOP10 had motA knock-out variants and, thus, we decided to incorporate motA into our switch on a different plasmid and rescue the gene. | ||
Wild-type motA was obtained from XB3.1 strain genomic gDNA, as the one already present in the registry didn't have the right beginning (based on Colibri E.coli genome database). Primers were designed to change the starting codon of the gene from GTG to ATG to match registry requirements and stop codon was changed from TGA to a stronger TAA; ribosome binding site was also added. <br><br> | Wild-type motA was obtained from XB3.1 strain genomic gDNA, as the one already present in the registry didn't have the right beginning (based on Colibri E.coli genome database). Primers were designed to change the starting codon of the gene from GTG to ATG to match registry requirements and stop codon was changed from TGA to a stronger TAA; ribosome binding site was also added. <br><br> | ||
MotA was then ligated into the pSB1C3 and plasmid J61002 which had a J23100 promoter. Those plasmids were then transformed into strains DH5α and TOP10. First transformation was only successful with the pSB1C3 plasmid whereas transformation of promoter plasmid had to be repeated. Sequencing data shown that pSB1C3 had motA insert without any mutations whereas the only two colonies of J23100 had mutations; in one variant in the ribosome binding site and in another variant in there was a 5 base depletion in the 5' end of the gene. This suggested that promoter J23100 was too strong for motA expression and was possibly toxic to the cells. We decided to use a variant with mutated RBS for our further experiments.<br> | MotA was then ligated into the pSB1C3 and plasmid J61002 which had a J23100 promoter. Those plasmids were then transformed into strains DH5α and TOP10. First transformation was only successful with the pSB1C3 plasmid whereas transformation of promoter plasmid had to be repeated. Sequencing data shown that pSB1C3 had motA insert without any mutations whereas the only two colonies of J23100 had mutations; in one variant in the ribosome binding site and in another variant in there was a 5 base depletion in the 5' end of the gene. This suggested that promoter J23100 was too strong for motA expression and was possibly toxic to the cells. We decided to use a variant with mutated RBS for our further experiments.<br> | ||
| - | Swarm assays (semi-solid agar motility test) were developed to investigate whether inserted plasmid expressing motA would rescue swimming and also to check whether our knock-outs did not swim. Strain DS941 ΔmotA (with motA knock-out) was transformed with pSB1C3 motA and J23100 motA and then carried out swarm experiments. Strain MG1655-z1 was used as a positive swimming control <strong>(Figure 1)</strong>. | + | Swarm assays (semi-solid agar motility test) were developed to investigate whether inserted plasmid expressing motA would rescue swimming and also to check whether our knock-outs did not swim. Strain DS941 ΔmotA (with motA knock-out) was transformed with pSB1C3 motA and J23100 motA and then carried out swarm experiments. Strain MG1655-z1 was used as a positive swimming control <strong>(Figure 1)</strong>.<br><br> |
| + | As the first swarm assay didn't show good gene rescuing, we decided to use weaker promoters:</p> | ||
| + | <ul> | ||
| + | <li>J23106 (½ the strength of J23100)</li> | ||
| + | <li>J23116 (¼ the strength of J23100)</li> | ||
| + | <li>J23103 (very weak promoter)</li> | ||
| + | <li>J23112 (weakest promoter we could find in the registry, barely no expression)</li> | ||
| + | </ul> | ||
| + | <p>(Strength measured with RFP: <a href="http://parts.igem.org/Part:BBa_J23100" target="_window">Part BBa_J23100</a> | ||
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</p> | </p> | ||
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| + | <div id="figure1"><img id="swarm1" class="allimage" src="https://static.igem.org/mediawiki/2014/4/45/GU_Gintare_illustration_1.png"/><p class="figuretext">Figure 1: Swarm assay. 5µ drop of overnight culture was added on a soft-agar plate and left incubated overnight at 37°C.</p></div> | ||
Revision as of 22:28, 10 October 2014
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Motility Genes: MotA and MotB
We were investigating motility genes which would allow bacteria to swim when the switch was off. Our main working strains DS941, DH5α and TOP10 had motA knock-out variants and, thus, we decided to incorporate motA into our switch on a different plasmid and rescue the gene.
Wild-type motA was obtained from XB3.1 strain genomic gDNA, as the one already present in the registry didn't have the right beginning (based on Colibri E.coli genome database). Primers were designed to change the starting codon of the gene from GTG to ATG to match registry requirements and stop codon was changed from TGA to a stronger TAA; ribosome binding site was also added.
MotA was then ligated into the pSB1C3 and plasmid J61002 which had a J23100 promoter. Those plasmids were then transformed into strains DH5α and TOP10. First transformation was only successful with the pSB1C3 plasmid whereas transformation of promoter plasmid had to be repeated. Sequencing data shown that pSB1C3 had motA insert without any mutations whereas the only two colonies of J23100 had mutations; in one variant in the ribosome binding site and in another variant in there was a 5 base depletion in the 5' end of the gene. This suggested that promoter J23100 was too strong for motA expression and was possibly toxic to the cells. We decided to use a variant with mutated RBS for our further experiments.
Swarm assays (semi-solid agar motility test) were developed to investigate whether inserted plasmid expressing motA would rescue swimming and also to check whether our knock-outs did not swim. Strain DS941 ΔmotA (with motA knock-out) was transformed with pSB1C3 motA and J23100 motA and then carried out swarm experiments. Strain MG1655-z1 was used as a positive swimming control (Figure 1).
As the first swarm assay didn't show good gene rescuing, we decided to use weaker promoters:
- J23106 (½ the strength of J23100)
- J23116 (¼ the strength of J23100)
- J23103 (very weak promoter)
- J23112 (weakest promoter we could find in the registry, barely no expression)
(Strength measured with RFP: Part BBa_J23100
Figure 1: Swarm assay. 5µ drop of overnight culture was added on a soft-agar plate and left incubated overnight at 37°C.
