Difference between revisions of "Part:BBa K4181020"

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	CotE as an anchor protein can be used to bind melanin to spores. To test whether it is expressed, a His tag is used for verification, followed by a linker to link the subsequent cotB protein.		CotE as an anchor protein can be used to bind melanin to spores. To test whether it is expressed, a His tag is used for verification, followed by a linker to link the subsequent cotB protein.
−	<!-- Add more about the biology of this part here	+	===Plasmid profiles===
−	===Usage and Biology===	+	[[File:2022-SJTU-Biox-Shanghai-11.png|500px|center]]<br>
		+	<p align="center">'''Figure 1.'''Pht01.<br></p>
		+	===Biology===
		+	1. It is a shuttle plasmid with dual-resistance. In E.coli, it mainly expresses Amp resistance, while in B.subtils, it expresses Cm resistance.
		+	2. Tyrosinase-linker-cotE gene complex were introduced. Tyrosinase is used for catalyzing exogenous L-DOPA into melanin; cotE is used for anchoring tyrosinase on the spore surface and linker joint these two together.
		+	3. 4B4-cotB gene complex displays the melanin-binding peptide gene on the spore surface so that it can capture the melanin right after it is produced.
		+	4. At the end is our Dsup protein gene, it expresses Dsup protein which can bind to DNA and form a shield.
		+	5. Lactose promoter is used for all the three genes so that it needs IPTG to induce expressions.
		+	6. His-tag is linked to Dsup and tyrosinase module, for further detection by Western blot and flow cytometry.
		+	7. Flag-tag is linked to melanin-binding-peptide 4B4 for demonstrsting its expression by flow cytometry.</p>
		+	<p>The melanin-binding-peptide 4B4 is an oligopeptide with only 10 amino acids, and according to the procedures mentioned above, we had added the flag-tag to it for verification. As another peptide on the spore surface, we chose the immunofluorescence assay similar to the ones done on tyrosinase. Anti-flag antibody coupled with PE-CY7 was used and we observe the results both from phase contract/ fluorescecnce microscopy and the flow cytometer. The excitation light is at 562nm and the emitted light is 770nm, so theoritically, red dots could be recognized if there were 4B4 expression on the surface. The microscope we used only got the excitation wave length for RFP, which is 532nm. Given that there must be a error band for the real wave length, we determined to have a try using 532nm light as the excitation light of CY7. The results of immunofluorescence is as below.</p>
		+
		+	[[File:2022-SJTU-Biox-Shanghai-1.png|500px|center]]<br>
		+	'''Figure 1.'''  Electrophoresis results after PCR of cotE and the first PCR electrophoresis results of cotB. The picture indicates a positive result.<br>
		+	<p>Next, PCR the cotB gene sequence again, and add 4B4 melanin-binding peptide and the stop codon to the 3' end of this sequence by designing your primers for this PCR.</p>
		+	<p style="text-align: center;">
		+	[[File:2022-SJTU-Biox-Shanghai-2.png|500px|center]]<br>
		+	'''Figure 2.''' Electrophoresis results of cotB after the second PCR and Dsup-encoded gene after PCR. The picture indicates a positive result.<br>
		+	===Characterization===
		+	cotE and cotB proteins are closely related to tyrosinase activity.Tyrosinase and the melanin-binding-peptide were the next to be demonstrated because of their display on the spore surface, and the process of sporulation has been mentioned above. Because there was his-tag linking with our tyrosinase protein, we decided to use immunofluorescence to detect it. The following is the result of our immunofluorescence test:</p>
		+	<p style="text-align: center;">
		+	[[File:2022-SJTU-Biox-Shanghai-3.png|500px|center]]<br>
		+	'''Figure 3.'''  Immunofluorescence assay on tyrosinase anchoring on the spore surface. Groups: Samples containing plasmid with IPTG induced; Samples containing plasmid without IPTG induced; WB800N wildtype. <br>
		+	.
		+
		+	===Flow Cytometry===
		+	FCM was also conducted by similar procedures and the results are as below:</p>
		+	[[File:2022-SJTU-Biox-Shanghai-4.png|500px|center]]<br>
		+	<p align="center">'''Figure 4.'''  Results of flow cytometry for the detection of tyrosinase. <br></p>
		+	<p>From the results, compared with the control group, samples with IPTG-induced plasmids showed more positive signals, whilst nearly no signals had been detected in the negative controls, suggesting a similar results as the immunofluoresecnce results. However, the peak of positive signals is just by the side of the negative peak, indicating a weak fluorescence intensity. From the result of FCM, it is more obvious that the total expression proportion of the target protein is relatively low. After discussion, we thought this was caused by several reasons. Firstly, during the growth and propagation of B.subtilis, the plasmid may be lost and thus no proteins can be generated. Secondly, as we could observe a large amount of spores under the phase-contract microscopy, it reminded us that we added IPTG for induction at the time when the OD of becteria was ranging from 0.6-0.8. This concentration was before the log-growth phase so that the bacteria would still propogate after IPTG induction. With the mother cells absorbing IPTG, less IPTG is left for daughter cells so that the bacteria produced by binary fissions later could not exposed to suffient amount of IPTG. Of course, as it can be indicated by fig20, B.subtilis would go through a quick propagation after transferred into sporulation medium, the concentration of ITPG at that stage of experiment could went even lower since no extra IPTG was added given its negative effects on the bacteria. Therefore, the concentration might be too low to induce the later produced bacteria generating proteins. Thirdly, we thought this might be caused by competitions between endogeneous cot proteins and the ones we introduced by exogeneous plasmid. As it is mentioned in our design, we cloned cotB and cotE proteins directly form the bacterial genome. So, there’re intrinsic prioteins to express under the regulation of sporulation signals naturally and thus compete with our exogeneous proteins to occupy the space on the spores’ outer surface. According to our results from Western blot, the expression level of our target priteins was not so much. In this regard, an unclear competitive advantage of exogeneous introduced proteins over the endogeneous ones might lead to the low anchoring rate of target proteins displayed in the outer surface. Generally, these results suggested we had successfully induced the expression and anchoring of tyrosinase on the spore surface. And there was notable expression disparity between the bacteria with IPTG-induced plasmid and the bacteria with no IPTG-induced plasmid and the wildtype.</p>
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Revision as of 10:44, 12 October 2022

cotE→His tag→linker

CotE as an anchor protein can be used to bind melanin to spores. To test whether it is expressed, a His tag is used for verification, followed by a linker to link the subsequent cotB protein.

Plasmid profiles

Figure 1.Pht01.

Biology

1. It is a shuttle plasmid with dual-resistance. In E.coli, it mainly expresses Amp resistance, while in B.subtils, it expresses Cm resistance. 2. Tyrosinase-linker-cotE gene complex were introduced. Tyrosinase is used for catalyzing exogenous L-DOPA into melanin; cotE is used for anchoring tyrosinase on the spore surface and linker joint these two together. 3. 4B4-cotB gene complex displays the melanin-binding peptide gene on the spore surface so that it can capture the melanin right after it is produced. 4. At the end is our Dsup protein gene, it expresses Dsup protein which can bind to DNA and form a shield. 5. Lactose promoter is used for all the three genes so that it needs IPTG to induce expressions. 6. His-tag is linked to Dsup and tyrosinase module, for further detection by Western blot and flow cytometry. 7. Flag-tag is linked to melanin-binding-peptide 4B4 for demonstrsting its expression by flow cytometry.</p>

The melanin-binding-peptide 4B4 is an oligopeptide with only 10 amino acids, and according to the procedures mentioned above, we had added the flag-tag to it for verification. As another peptide on the spore surface, we chose the immunofluorescence assay similar to the ones done on tyrosinase. Anti-flag antibody coupled with PE-CY7 was used and we observe the results both from phase contract/ fluorescecnce microscopy and the flow cytometer. The excitation light is at 562nm and the emitted light is 770nm, so theoritically, red dots could be recognized if there were 4B4 expression on the surface. The microscope we used only got the excitation wave length for RFP, which is 532nm. Given that there must be a error band for the real wave length, we determined to have a try using 532nm light as the excitation light of CY7. The results of immunofluorescence is as below.

Figure 1. Electrophoresis results after PCR of cotE and the first PCR electrophoresis results of cotB. The picture indicates a positive result.

Next, PCR the cotB gene sequence again, and add 4B4 melanin-binding peptide and the stop codon to the 3' end of this sequence by designing your primers for this PCR.

Figure 2. Electrophoresis results of cotB after the second PCR and Dsup-encoded gene after PCR. The picture indicates a positive result.

Characterization

cotE and cotB proteins are closely related to tyrosinase activity.Tyrosinase and the melanin-binding-peptide were the next to be demonstrated because of their display on the spore surface, and the process of sporulation has been mentioned above. Because there was his-tag linking with our tyrosinase protein, we decided to use immunofluorescence to detect it. The following is the result of our immunofluorescence test:

Figure 3. Immunofluorescence assay on tyrosinase anchoring on the spore surface. Groups: Samples containing plasmid with IPTG induced; Samples containing plasmid without IPTG induced; WB800N wildtype.
.

Flow Cytometry

FCM was also conducted by similar procedures and the results are as below:

Figure 4. Results of flow cytometry for the detection of tyrosinase.

From the results, compared with the control group, samples with IPTG-induced plasmids showed more positive signals, whilst nearly no signals had been detected in the negative controls, suggesting a similar results as the immunofluoresecnce results. However, the peak of positive signals is just by the side of the negative peak, indicating a weak fluorescence intensity. From the result of FCM, it is more obvious that the total expression proportion of the target protein is relatively low. After discussion, we thought this was caused by several reasons. Firstly, during the growth and propagation of B.subtilis, the plasmid may be lost and thus no proteins can be generated. Secondly, as we could observe a large amount of spores under the phase-contract microscopy, it reminded us that we added IPTG for induction at the time when the OD of becteria was ranging from 0.6-0.8. This concentration was before the log-growth phase so that the bacteria would still propogate after IPTG induction. With the mother cells absorbing IPTG, less IPTG is left for daughter cells so that the bacteria produced by binary fissions later could not exposed to suffient amount of IPTG. Of course, as it can be indicated by fig20, B.subtilis would go through a quick propagation after transferred into sporulation medium, the concentration of ITPG at that stage of experiment could went even lower since no extra IPTG was added given its negative effects on the bacteria. Therefore, the concentration might be too low to induce the later produced bacteria generating proteins. Thirdly, we thought this might be caused by competitions between endogeneous cot proteins and the ones we introduced by exogeneous plasmid. As it is mentioned in our design, we cloned cotB and cotE proteins directly form the bacterial genome. So, there’re intrinsic prioteins to express under the regulation of sporulation signals naturally and thus compete with our exogeneous proteins to occupy the space on the spores’ outer surface. According to our results from Western blot, the expression level of our target priteins was not so much. In this regard, an unclear competitive advantage of exogeneous introduced proteins over the endogeneous ones might lead to the low anchoring rate of target proteins displayed in the outer surface. Generally, these results suggested we had successfully induced the expression and anchoring of tyrosinase on the spore surface. And there was notable expression disparity between the bacteria with IPTG-induced plasmid and the bacteria with no IPTG-induced plasmid and the wildtype.

Sequence and Features