Difference between revisions of "Part:BBa K3796203"

Revision as of 17:16, 11 October 2021 (view source) Chouuken (Talk | contribs) (→‎Design Page) ← Older edit		Latest revision as of 03:06, 21 October 2021 (view source) Yang Feiyue (Talk | contribs)
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−	<!-- Add more about the biology of this part here
	===Usage and Biology===		===Usage and Biology===
		+	MazF is an mRNA interferase which cleaves mRNAs at a specific site, and <i>ndoA</i>, also called <i>ydcE</i> or <i>mazF-bs</i> shows a similar activity according to references given below. <i>ndoA</i> belongs to a type Ⅱ toxin-antitoxin system in <i>Bacillus subtilis</i>. There is great possibility that no antitoxin exists in <i>C. glutamicum</i> to inhibit EndoA encoded by <i>ndoA</i>. So it can be used to kill engineered <i>C. glutamicum</i> at a proper time.
−	<!-- -->	+	===Characterization===
		+	<p>We aimed to test if the over-expression of the toxin gene <i>ndoA</i> from <i>Bacillus subtilis</i> can kill <i>Corynebacterium glutamicum</i>  effectively as well.</p>
		+	[[image:T--CAU China--ndoA_1.svg|600px|thumb|center|]]
		+	<p style="text-align: center;"><b>Fig.1 Genetic circuit for <i>ndoA</i> verification</b></p>
		+
		+	<p>To characterize its killing effect, <i>ndoA</i> is inserted into the plasmid pXMJ19 to construct a circuit with tac promotor, lac operator, <i>ndoA</i> and terminator rrnB using the ClonExpress II one-step cloning kit (Vazyme Biotech, China). The expression vector is transformed into <i>E. coli</i> DH5&alpha; first and then into <i>C. glutamicum</i> by electroporation.</p>
		+
		+	<p>To design a quick quanlitative test, 'divided plate' assay was carried out at the beginning. We divided a plate with LB medium into four parts and use the spread plate method to see if <i>ndoA</i> can kill <i>C. glutamicum</i> and this killing effect is not caused by the toxicity of IPTG. Bacteria carrying empty vector was added into 1 Ep tube and bacteria carrying the gene circuit was added into 2 Ep tubes with 1.8 mL LB liquid containing 10 μg/mL chloramphenicol respectively. After incubating the culture in a shaker at 30 °C, 220 rpm until OD<sub>600</sub> reached 0.6, we prepared LB plates that were separated into four quarters marked A,B,C,D. We spread the diluted bacteria solution on the quarters respectively, and all plates were incubated at 30 °C for a certain time.</p>
		+
		+	[[image:T--CAU China--ndoA_2.png|600px|thumb|center|]]
		+	<p style="text-align: center;">
		+	<b>Figure 34 Divided Plate Assay</b><br>
		+	Quarter A: <i>C. glutamicum</i> carrying empty vector and 0.8mM IPTG was added; Quarter B: <i>C. glutamicum</i> carrying empty vector without adding 0.8mM IPTG; Quarter C: <i>C. glutamicum</i> carrying the vector pXMJ19-<i>ndoA</i> without adding 0.8mM IPTG; Quarter D: <i>C. glutamicum</i> carrying the vector pXMJ19-<i>ndoA</i> and 0.8mM IPTG was added.
		+	</p>
		+
		+	<p>Comparing Quarter A and Quarter B, we can see that the growth condition of the two is very similar, which means that the toxicity of 0.8mM IPTG is very low and it hardly kills <i>C. glutamicum</i>. Comparing Quarter C and Quarter D, we can see that there is apparently less bacteria survived in Quarter C, and the diameter of the colonies in Quarter C is rather small as well. As for the comparison between Quarter B and Quarter D, we assume that there is serious leaky expression of <i>ndoA</i> controlled by tac promoter in Quarter D, which deteriorates its growth condition. We repeated this experiments for 5 times and got nearly the same result. Hence, we can get the preliminary conclusion that <i>ndoA</i> does have a killing effect on <i>C. glutamicum</i>.</p>
		+
		+	<p>To give a further quantitative test, we carried out CFU assay to characterize the killing effect of <i>ndoA</i> instead of determining OD<sub>600</sub> in order to get rid of dead bacterial cells. CFU was quantified by counting the colonies on one plate and normalizing the number to volume of 1 mL culture. We added 0.8mM IPTG as OD<sub>600</sub> reached 0.6, and estimate CFU by spreading 25&mu;L of the culture on 3 LB solid medium every hour after the induction with 0.8mM IPTG, and our results are as follows.</p>
		+
		+	[[image:T--CAU China--ndoA_3_2.png|600px|thumb|center|]]
		+	<p style="text-align: center;"><b>Figure 35 Results of the CFU Assay, plotted against induction </b></p>
		+
		+	<p>It is visually discovered that the number of colonies carrying pXMJ19-<i>ndoA</i> fell off in the presence of IPTG, while the same bacteria grew well without induction within the first four hours and then its CFU decrease probably due to the leaky expression. We can also see that the growth condition of the bacteria carrying the empty vector hasn’t been affected by 0.8mM IPTG, since its CFU curve increases as normal. We repeated this experiments for 3 times and all the curves show similar trends.</p>
		+	<p>Therefore, we can finally come to the conclusion that the <i>ndoA</i> does have a killing effect on <i>C. glutamicum</i>, and can be used in our project as the toxin gene.</p>
		+
		+	<!-- --><br><br><br>
	<span class='h3bb'>Sequence and Features</span>		<span class='h3bb'>Sequence and Features</span>
	<partinfo>BBa_K3796203 SequenceAndFeatures</partinfo>		<partinfo>BBa_K3796203 SequenceAndFeatures</partinfo>
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	<partinfo>BBa_K3796203 parameters</partinfo>		<partinfo>BBa_K3796203 parameters</partinfo>
	<!-- -->		<!-- -->
−
−	===Usage and Biology===
−	MazF is an mRNA interferase which cleaves mRNAs at a specific site, and <i>ndoA</i>, also called <i>ydcE</i> or <i>mazF-bs</i> shows a similar activity according to references given below. ndoA belongs to a type Ⅱ toxin-antitoxin system in Bacillus subtilis. There is great possibility that no antitoxin exists in C. glutamicum to inhibit EndoA encoded by ndoA. So it can be used to kill engineered <i>C. glutamicum</i> at a proper time.
−	===Characterization===
−	This year, our team designed a kill switch in <i>C. glutamicum</i> using <i>ndoA</i> from <i>Bacillus subtilis</i> subsp. subtilis str. 168 under the control of PgsiB (alkali inducible promoter) and Patp2 (alkali inducible promoter). We aimed to test if the over-expression of <i>ndoA</i> in <i>C. glutamicum</i> can kill the engineered bacteria effectively, and documented our works and failures in iGEM Parts to inspire future iGEM teams for kill switch design.
−	To verify that <i>ndoA</i> has function of killing <i>C. Glutamicum</i>, <i>ndoA</i> was inserted into PXMJ19 to construct a simple circuit with tac promotor, lac operon, <i>ndoA</i> and terminator rrnB.<br><hr>
−	CFU assay was carried out to determine the effect of <i>ndoA</i>. Three groups of plates, empty vector (with IPTG), Ptac/<i>lacIq-ndoA</i> (non-induction) and Ptac/<i>lacIq-ndoA</i> (induction) were used. CFU of three groups above is determined every one hour to catch the killing process. At first, LB solid medium was used in each plate for these three groups. However, there was no significant difference between the last two group. We assume that there must be leakiness of Ptac/lac operon in <i>C. glutamicum</i>.
−	So, we used LBG medium to replace LB medium of Ptac/<i>lacIq-ndoA</i> (without induction) in order to repress the expression of <i>ndoA</i> when no IPTG exists.
−	It was discovered that the number of colonies carrying lac operon and <i>ndoA</i> fell at presence of IPTG, while the same bacteria without IPTG grew well at first and died due to leakiness after 4 hours.<br>
−	//image
−	In the assay, we noticed that toxin gene <i>ndoA</i> resulted in a very slow colony growth, which was indirect evidence. <br>
−	What’s more, <i>ndoA</i> couldn’t be detected by the method of PCR as time went in the CFU assay, which cut the contrast.<br><hr>
−	To give a sharp view of toxin <i>ndoA</i> and avoid leakiness, another assay was also carried out by dividing a plate into four parts equally and used LBG medium in one part, LB in the others. Empty vector (with IPTG), empty vector (without IPTG) and Ptac/<i>lacIq-ndoA</i> (induction) were grown respectively on LB parts, Ptac/<i>lacIq-ndoA</i> (non-induction) was grown on the LBG part. <br>
−	//image
−	As you can see, the number of colonies of <i>C. glutamicum</i> carrying <i>ndoA</i> will can be much smaller because of the presence of IPTG.
−
−	===Design Page===
−
−	<h2>Design</h2><br>
−	In the very beginning, <i>ndoA</i> from <i>B. Subtilis</i>, <i>mazF</i> from <i>E. Coli</i> and <i>ndoA</i> from <i>C. Glutamicum</i> were considered to be the toxin gene in our kill switch. However, <i>ndoA</i> stood out for two reasons. On one hand, its annotation score is 5 on the Uniprot website. On the other hand, <i>B. Subtilis</i> is Gram-positive bacteria just like <i>C. Glutamicum</i>.<br>
−	<h3>build</h3>
−	We have only constructed a simple genetic circuit for <i>ndoA</i> test so far, which is composed with Ptac, <i>ndoA</i> and rrnB T1&T2 using ClonExpress II one-step cloning kit (Vazyme Biotech, China). This expression clone is transformed into DH5α first and then into <i>C. glutamicum</i> by Electroporation.
−	//image 饿

---

Latest revision as of 03:06, 21 October 2021

ndoA (endoribonuclease toxin in Bacillus subtilis)

This coding region ndoA(also mazF, ydcE) encodes endoribonuclease EndoA, a toxic component of a type II toxin-antitoxin (TA) system. It encodes endoribonuclease toxin in Bacillus subtilis that is a UACAU‐specific mRNA interferase, cleavaging after the first U. Previous research has proved that its overexpression is toxic for cell growth and it functions as normal in Escherichia coli.

Usage and Biology

MazF is an mRNA interferase which cleaves mRNAs at a specific site, and ndoA, also called ydcE or mazF-bs shows a similar activity according to references given below. ndoA belongs to a type Ⅱ toxin-antitoxin system in Bacillus subtilis. There is great possibility that no antitoxin exists in C. glutamicum to inhibit EndoA encoded by ndoA. So it can be used to kill engineered C. glutamicum at a proper time.

Characterization

We aimed to test if the over-expression of the toxin gene ndoA from Bacillus subtilis can kill Corynebacterium glutamicum effectively as well.

Fig.1 Genetic circuit for ndoA verification

To characterize its killing effect, ndoA is inserted into the plasmid pXMJ19 to construct a circuit with tac promotor, lac operator, ndoA and terminator rrnB using the ClonExpress II one-step cloning kit (Vazyme Biotech, China). The expression vector is transformed into E. coli DH5α first and then into C. glutamicum by electroporation.

To design a quick quanlitative test, 'divided plate' assay was carried out at the beginning. We divided a plate with LB medium into four parts and use the spread plate method to see if ndoA can kill C. glutamicum and this killing effect is not caused by the toxicity of IPTG. Bacteria carrying empty vector was added into 1 Ep tube and bacteria carrying the gene circuit was added into 2 Ep tubes with 1.8 mL LB liquid containing 10 μg/mL chloramphenicol respectively. After incubating the culture in a shaker at 30 °C, 220 rpm until OD₆₀₀ reached 0.6, we prepared LB plates that were separated into four quarters marked A,B,C,D. We spread the diluted bacteria solution on the quarters respectively, and all plates were incubated at 30 °C for a certain time.

Figure 34 Divided Plate Assay
Quarter A: C. glutamicum carrying empty vector and 0.8mM IPTG was added; Quarter B: C. glutamicum carrying empty vector without adding 0.8mM IPTG; Quarter C: C. glutamicum carrying the vector pXMJ19-ndoA without adding 0.8mM IPTG; Quarter D: C. glutamicum carrying the vector pXMJ19-ndoA and 0.8mM IPTG was added.

Comparing Quarter A and Quarter B, we can see that the growth condition of the two is very similar, which means that the toxicity of 0.8mM IPTG is very low and it hardly kills C. glutamicum. Comparing Quarter C and Quarter D, we can see that there is apparently less bacteria survived in Quarter C, and the diameter of the colonies in Quarter C is rather small as well. As for the comparison between Quarter B and Quarter D, we assume that there is serious leaky expression of ndoA controlled by tac promoter in Quarter D, which deteriorates its growth condition. We repeated this experiments for 5 times and got nearly the same result. Hence, we can get the preliminary conclusion that ndoA does have a killing effect on C. glutamicum.

To give a further quantitative test, we carried out CFU assay to characterize the killing effect of ndoA instead of determining OD₆₀₀ in order to get rid of dead bacterial cells. CFU was quantified by counting the colonies on one plate and normalizing the number to volume of 1 mL culture. We added 0.8mM IPTG as OD₆₀₀ reached 0.6, and estimate CFU by spreading 25μL of the culture on 3 LB solid medium every hour after the induction with 0.8mM IPTG, and our results are as follows.

Figure 35 Results of the CFU Assay, plotted against induction

It is visually discovered that the number of colonies carrying pXMJ19-ndoA fell off in the presence of IPTG, while the same bacteria grew well without induction within the first four hours and then its CFU decrease probably due to the leaky expression. We can also see that the growth condition of the bacteria carrying the empty vector hasn’t been affected by 0.8mM IPTG, since its CFU curve increases as normal. We repeated this experiments for 3 times and all the curves show similar trends.

Therefore, we can finally come to the conclusion that the ndoA does have a killing effect on C. glutamicum, and can be used in our project as the toxin gene.

Sequence and Features