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Team:Ciencias-UNAM/Project/WetLab/Characterization
From 2013.igem.org
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We also worked with another team's part:<br> | We also worked with another team's part:<br> | ||
<br> | <br> | ||
| - | + | BBa_K875009<br> | |
| + | Given Trieste Team couldn't characterise this part properly, we analysed the problems with theirs characterisation to express this protein properly. | ||
They used Lac promoter to express this peptide, hoping to inhibit cell growth. They did not have positive results, as the control experiment growth the same way as the one with LL-37 expression with IPTG. We approached this problem with two solutions. The first problem we saw in Trieste's characterisation was that Lac promoter has a very high basal level of transcription. Given pSB1C3 has a high copy number, it is transcribing a high amount of LL-37, still with no IPTG in the media. This could mean that there is selection pressure on E. coli to loose the plasmid, which could be one of the reasons the did not see changes between the control cells and the cells under IPTG induction. We addressed this problem by changing the promoter, constructing device BBa_K1230007. This device is under the pBad promoter, which works with arabinose. This promoter has a basal level of almost 0, so we are sure that it is not transcribing any LL-37 that could be making the cells to loose the plasmid. The second problem we addressed was the change in the codon usage they used. By changing the codons to produce more peptide in E. coli, the peptide may not have sufficient time to fold properly, altering it's function. We solved this problem by using the normal codon usage of this peptide, allowing it to fold properly into it's final conformation. | They used Lac promoter to express this peptide, hoping to inhibit cell growth. They did not have positive results, as the control experiment growth the same way as the one with LL-37 expression with IPTG. We approached this problem with two solutions. The first problem we saw in Trieste's characterisation was that Lac promoter has a very high basal level of transcription. Given pSB1C3 has a high copy number, it is transcribing a high amount of LL-37, still with no IPTG in the media. This could mean that there is selection pressure on E. coli to loose the plasmid, which could be one of the reasons the did not see changes between the control cells and the cells under IPTG induction. We addressed this problem by changing the promoter, constructing device BBa_K1230007. This device is under the pBad promoter, which works with arabinose. This promoter has a basal level of almost 0, so we are sure that it is not transcribing any LL-37 that could be making the cells to loose the plasmid. The second problem we addressed was the change in the codon usage they used. By changing the codons to produce more peptide in E. coli, the peptide may not have sufficient time to fold properly, altering it's function. We solved this problem by using the normal codon usage of this peptide, allowing it to fold properly into it's final conformation. | ||
For the characterization of the LL-37 antimicrobial peptide from Trieste, with another promoter, we inoculated 12.5 ml of LB of the strain without the plasmid, and the strain with the plasmid (we made pressure selection with chloramphenicol to make sure our plasmid was still there). We did the inoculation at 0.05 of OD at 600A and incubating at 37°C until it got to 4.0 OD at 600A. Then we separated each of the two tubes in 5 15ml falcon tubes, 2 ml of the inoculums. We induced with arabinose as we mention in the following table. From each tube we did 8 decimal dilutions in 1.5 Eppendorf tubes with LB. These are the results. | For the characterization of the LL-37 antimicrobial peptide from Trieste, with another promoter, we inoculated 12.5 ml of LB of the strain without the plasmid, and the strain with the plasmid (we made pressure selection with chloramphenicol to make sure our plasmid was still there). We did the inoculation at 0.05 of OD at 600A and incubating at 37°C until it got to 4.0 OD at 600A. Then we separated each of the two tubes in 5 15ml falcon tubes, 2 ml of the inoculums. We induced with arabinose as we mention in the following table. From each tube we did 8 decimal dilutions in 1.5 Eppendorf tubes with LB. These are the results. | ||
Latest revision as of 04:01, 28 September 2013
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Check our parts in Results and Biobricks sections.
We also worked with another team's part:
BBa_K875009
Given Trieste Team couldn't characterise this part properly, we analysed the problems with theirs characterisation to express this protein properly.
They used Lac promoter to express this peptide, hoping to inhibit cell growth. They did not have positive results, as the control experiment growth the same way as the one with LL-37 expression with IPTG. We approached this problem with two solutions. The first problem we saw in Trieste's characterisation was that Lac promoter has a very high basal level of transcription. Given pSB1C3 has a high copy number, it is transcribing a high amount of LL-37, still with no IPTG in the media. This could mean that there is selection pressure on E. coli to loose the plasmid, which could be one of the reasons the did not see changes between the control cells and the cells under IPTG induction. We addressed this problem by changing the promoter, constructing device BBa_K1230007. This device is under the pBad promoter, which works with arabinose. This promoter has a basal level of almost 0, so we are sure that it is not transcribing any LL-37 that could be making the cells to loose the plasmid. The second problem we addressed was the change in the codon usage they used. By changing the codons to produce more peptide in E. coli, the peptide may not have sufficient time to fold properly, altering it's function. We solved this problem by using the normal codon usage of this peptide, allowing it to fold properly into it's final conformation.
For the characterization of the LL-37 antimicrobial peptide from Trieste, with another promoter, we inoculated 12.5 ml of LB of the strain without the plasmid, and the strain with the plasmid (we made pressure selection with chloramphenicol to make sure our plasmid was still there). We did the inoculation at 0.05 of OD at 600A and incubating at 37°C until it got to 4.0 OD at 600A. Then we separated each of the two tubes in 5 15ml falcon tubes, 2 ml of the inoculums. We induced with arabinose as we mention in the following table. From each tube we did 8 decimal dilutions in 1.5 Eppendorf tubes with LB. These are the results.
For more information on the characterisation, visit our notebook on our wiki https://2013.igem.org/Team:Ciencias-UNAM/Project/WetLab/Protocols As we can see here, there is no basal level transcription on this experiment. So the problem in theirs characterisation may be due to the codon usage they used.
