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Team:Kyoto/Project/Goal C
From 2010.igem.org
Contents |
Discussion
SΔTMD1 cannot inhibit Lysis cassette completly
These results showed that SΔTMD1 cannot inhibit completely Lysis cassette. This results can't supported a report on lysis (Ref.1) which says that SΔTMD1 without TMD1 dominant-negatively inhibit lysis cassette. Why did we get the result against that report? Probably, there were three reasons below, we thought.
- SΔTMD1 in the plasmid did not worked correctly.
- S&DeltaTMD1 is weaker anti-killer gene than we expected.
- less SΔTMD1 proteins or more lysis cassette proteins was expressed than we expected.
Reason1:SΔTMD1 in the plasmid did not worked correctly.
To make sure the reason, we sequenced BBa_K358021 we used. It’s result showed that this construct is inserted into the plasmid correctly, which demonstrated that SΔTMD1 worked correctly. So, first reason was not right.
Reason2:S&DeltaTMD1 is weaker anti-killer gene than we expected.
As anti-killer gene against lysis cassette, there are two genes, S107 and SΔTMD1(Learn more). S107 is anti-killer gene, but because it has essential TMD1 of S for function of this killer gene, it’s function as anti-killer gene is weak compared with SΔTMD1, which does not have TMD1 of （Learn more). So, we thought that SΔTMD1 is a strong anti-killer gene, but from result of experiment 1 we doubted SΔTMD1 was a strong anti-killer gene. However, I could not make sure the reason.
Reason3:less SΔTMD1 proteins or more lysis cassette proteins was expressed than we expected.
As a report (Ref.2) says, it is necessary for the inhibition of lysis cassette that there are at least 4.73 times more S107, anti-killer gene than lysis cassette proteins. S gene encodes not only S105 but also S107. S107 is anti-killer gene, but because it has essential TMD1 of S for function of this killer gene, it’s function as anti-killer gene is weak compared with SΔTMD1, which does not have TMD1 of S [1]. So SΔTMD1 should have inhibited lysis cassette if at least 4.73 times more SΔTMD1 than lysis cassette were expressed. Actually, we selected <partinfo>R0011</partinfo> and <partinfo>J23105</partinfo> as each promoter of lysis cassette and SΔTMD1 so that the activity of the promoter of SΔTMD1 is at least five times higher than that of lysis cassette. However, S&Delta TMD1; couldn’t inhibit lysis cassette. Then, in order to solve this problem we changed the promoter of SΔTMD1 to stronger one, and now, are checking the function of SΔTMD1. Surely, this result will help us discuss why SΔTMD1 couldn’t function as we expected. You should expect it!!
