Team:UM Macau/Results
Results
I. Plasmid Construction
Pet11a-OPHT1/OPHT2/OPH3/OPHM/OPHA
PCR and gel running results
Figure A-1: Gel result of the PCR amplification for clone identity checking for OPHT1 and OPHT2
We amplified OPHT1 DNA fragment from plasmids of colonies #1, #2, #3 and OPHT2 DNA fragment from plasmids of colonies #1, #2 by using PCR. The OPHT1 fragment bands appear slightly below 1500bp size and OPHT2 fragment bands are at around 2000bp; which indicated the pET11a-OPHT1 and pET11a-OPHT2 plasmid have been successfully constructed.
Figure A-2: Gel result of the PCR amplification for the clone identity checking for OPHT3 and OPHM
We amplified OPHT3 DNA fragment of colonies #1, #2 and OPHTM DNA fragment of colonies #1, #2 by using PCR. The OPHT3 fragment bands appear slightly below 1500bp size and OPHTM fragment bands are at the range between 1500~2000bp; which indicated that pET11a-OPHT3 and pET11a-OPHTM plasmid have been successfully constructed.
Figure A-3: Gel result which shows the amplified PCR product which pertains to OPHA.
We amplified OPHA DNA fragment from plasmids of colonies #1, #2 through PCR. The OPHA fragment bands appear slightly below 1500bp size; which indicated that pET11a- OPHA plasmid is successfully constructed.
OPHT1 plasmid’s sequencing result
In all the plasmid sequencing results figures below, each white gap in between primer pairs of each colony indicates a mismatched pair.
Figure A-4: Sequencing result for the OPHT1 variant of the Surface nanoparticle capturing protein (SNCP).
From the plasmid sequencing results for OPHT1 colony 1,2 and 3, we can see that there are very few mismatched pairs in each forward and reverse primer pairs of each colony.
OPHT2 plasmid’s sequencing result
Figure A-5: Sequencing result for the OPHT2 variant of the Surface nanoparticle capturing protein (SNCP).
From the plasmid sequencing results for OPHT2 colony 2 and 5, we can see that there are very few mismatched pairs in each forward and reverse primer pairs of each colony.
OPHT3 plasmid’s sequencing result
Figure A-6: Sequencing result for the OPHT3 variant of the Surface nanoparticle capturing protein (SNCP).
From the plasmid sequencing results for OPHT3 colony 2 and 4, we can see that there are very few mismatched pairs in each forward and reverse primer pairs of each colony. On the other hand, there are a lot of mismatch pairs in colony 3, therefore the bacteria culture from this colony is not appropriate to be used.
OPHM plasmid’s sequencing result
Figure A-7: Sequencing result for the OPHM variant of the Surface nanoparticle capturing protein (SNCP).
From the plasmid sequencing results for OPHM colony 2,3 and 4, we can see that there are very few mismatched pairs in each forward and reverse primer pairs of each colony.
OPHA plasmid’s sequencing result
Figure A-8: Sequencing result for the OPHA variant of the Surface nanoparticle capturing protein (SNCP).
From the plasmid sequencing results for OPHA colony 1 and 2, we can see that there are very few mismatched pairs in each forward and reverse primer pairs of each colony. There are more mismatch pairs in colony 3 compared to colony1 and 2.
II. Protein Expression Check Results
Western blotting results: Surface nanoparticle capturing protein variants
Figure A-9: This western blotting result shows the band signals for all the variants for our surface nanoparticle capturing protein (SNCP)
Immunofluorescence: Surface nanoparticle capturing protein variants
Figure A-10: Immunofluorescence imaging result of our bacteria transformed with our Surface nanoparticle capturing protein (SNCP)
We want to examine whether our target protein is being expressed in our 5 different surface NPs capturing protein: OPHT1, OPHT2, OPHT3, OPHM, and OPHA. From Figure A-9, we can observe that our OPHT1, OPHT2, and OPHA show the band on the correct size. OPHT1 and OPHA1 show bands at around 50kDa, while OPHT2 which tagged with GFP shows the band at around 75kDa; which are at the estimated protein band size. However, unfortunately, OPHT3 and OPHM did not show any protein band. Although there are no positive results from western blot in OPHT3 and OPHM, the green light from the anti-mouse secondary antibody can be seen in immunofluorescence (IF) assay which shows that the all adhesion constructive system express on the surface of our bacteria (Figure A-10).
III. Functional Test
Yellow fluorescent nanospheres from Spherotech catalog number FP-00552-2 with and excitation wavelength of 440 nm and emission wavelength of 480 nm; with the size of 40 nm to 90 nm were used in all of the test. Refer to our protocol file for the details of the assay: Click here!
Figure A-11: RFU of 5 variants and wild-type bacteria after 6-minute treatment
Figure A-12: RFU of 5 variants and wild-type bacteria after 12-minute treatment
Figure A-13: RFU of 5 variants and wild-type bacteria after 24 minute treatment
Figure A-14: Figure A-14: RFU of 5 variants and wild-type bacteria after 48 minute treatment
Based on the successful capturing protein expression results, we want to test the nanoparticle binding capability for future potential application. We incubated our construct variant bacteria, which are expressing NPs capturing protein, with nanoparticles and measured the fluorescence signal to quantify NPs (nanospheres with fluorescence) signals in the supernatant after centrifugation. The fluorescence delta change is determined by individually subtracting the absorbance readings of the negative control and construct variants, which both incubated with nanoparticle, to the positive control reading, which contains the same type and concentration of nanoparticle in phosphate buffer. The greater the delta change value is, the more efficient our capturing protein function is. In conclusion, OPHM and OPHT have consistently high Relative Fluorescence Unit (RFU) throughout 6, 12, 24 and 48 minutes incubation time from Figure A-11 to A-14. In terms of incubation time, 6 minutes is considered as the best incubation time length for bacteria to capture the most amount of nanoparticles from Figure A-11. The figures above show that compared to the wild-type bacteria, consistent in different time points, our bacteria with the surface nanoparticle capturing protein performs better.
Figure A-15: Average standard curve for our yellow fluorescence nanoparticle
We made an average standard curve for the yellow fluorescence nanoparticle to convert Relative Fluorescence Unit (RFU) to concentration in parts per million unit (mg/L) for more straightforward data display. This was done to put the RFU into a more relatable for industrial application in the future.
Figure A-16 to A-25: Contrast of nanoparticle concentration (ppm) between in pellet and in supernatant collected from 5 construct variants with different incubation time length
By utilizing the same raw data of the capturing function, the relationship between the various incubation time points and capturing function were analyzed. After collecting the pellet of each bacteria culture, the trendlines show that all five bacteria construct included OPHT1, OPHT2, OPHT3, OPHM and OPHA have a significant increased capturing ability compared to the wild-type bacteria which may contain unspecific bindings. (Figure A-16, A-18, A-20, A-22 and A-24). The concentration of nanoparticle in each pellet of bacteria constructs has a significant increase which indicates that the capturing protein on the bacteria enhances its ability to capture more nanoparticles. The concentration of nanoparticle in the supernatant of all five bacteria constructs have more significant and drastic decrease compared to wild-type bacteria (Figure A-17, A-19, A-21, A-23, A-25). As the concentration of nanoparticles which are unbound to the capturing protein of the bacteria will remain in supernatant after centrifugation, thus the lesser nanoparticles left in the supernatant implies that there are more nanoparticles bound onto the capturing protein on the bacteria cell surface. These declining trendlines show that the observed increase in the concentration of nanoparticles in the pellet as shown in Figures A-16, A-18, A-20, A-22 and A-24 is reflected clearly as a decrease of the nanoparticle concentration in the supernatant as shown in Figures A-17, A-19, A-21, A-23, A-25. All the evidence mentioned above shows that all five capturing protein is successfully secreted and the capturing system is performing well. Thus, ouur bacteria SANCE is working well.
Reference
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I. Plasmid Construction
PCR and Gel Running Results
Figure B-1: Size of Fragment NSP4-tyrosinase=1022bp
When have received our fragments from IDT. We digested the vector pBAD24 in order to directly ligate the received fragment. After gibson assembly, we have transformed the plasmid into the Stbl3 strain of bacteria and then picked 6 colonies and extracted the plasmid. After that, we amplified the NSP4-Tyrosinase DNA fragment of colonies 1, 2, 3, 4, 5 and 6 through PCR. From the gel run results above, we can see that all the bands’ size of NSP4-tyrosinase DNA fragments from all the colonies are at around 1000bp which is close to 1022bp which is what we expected. This result proves that we have successfully constructed pBAD24-NSP4.
NSP4-tyrosinase plasmid’s sequencing result
Figure B-2: This is the aligned plasmid sequencing result. Two primers were used to check our gene of interest
Two flanking primers were used to overlap the gene of interest NSP4-Tyrosinase. From the plasmid sequencing results for NSP4-Tyrosinase colony 3 and 6, we can see that there are very few mismatched pairs in each forward and reverse primer pairs of each colony. But there are so many mismatch pairs in colony 5, therefore we didn’t use the plasmids from this colony to finish the further experiment. Colony 6 plasmid was chosen for the subsequent experiments.
II. Protein Expression Test
Western blotting results: NSP4-Tyrosinase induced and uninduced pellets
Figure B-3: This Western Blot result shows our pellet samples signal after lysis of the bacteria
We cultured NSP4-tyrosinase transformed bacteria, saved some volume of the culture as uninduced bacteria sample and induced the rest volume of bacteria culture with 0.2% arabinose for the next 6~8 hours. Next, we centrifuged both induced and uninduced samples, we saved both pellet and supernatant for target protein expression check using western blotting. We lysed the cells using a lysis buffer. However, in this first western blotting result as shown in Figure B-3, we were only able to clearly show that our target protein is expressed in the pellet sample. Tyrosinase which has the size of 34kDa has a strong detection signal compared to the uninduced samples. Although we got the right protein size, we want our protein to be secreted onto the supernatant. A possible explanation for this was that our protein concentration was not high enough in the supernatant sample thus we couldn’t see a signal for it.
Western blotting results: Nickel pull-down of NSP4 supernatant
Figure B-4: A. NSP4 arabinose induced (before pull-down) B. NSP4 arabinose induced (after pull-down) C. bacteria transformed by vector without the insert(before pull-down). D. bacteria transformed by vector without the insert(after pull-down).
The results of Figure B-3 shows that there is not enough tyrosinase in the supernatant therefore we have performed Nickel pull-down. Figure B-4 above shows our results after pulling down the supernatant samples. It makes sense that our target protein is not detectable in either before (C sample) or after pull-down (D sample) in the supernatant from bacteria transformed with vector without tyrosinase fragment insert after centrifugation. By concentrating our protein of interest from the supernatant using pull-down assay (B sample), we were able to see, as shown in Figure B-4, the band of our target protein Tyrosinase, which is 34kDa in size. Indicating that it was pulled-down from the supernatant of induced samples while our target protein bands don’t show in the supernatant from bacteria transformed with tyrosinase gene without pull down. This result indicates that our protein is secreted in the extracellular space due to the NSP4-tyrosinase inserted in the pBAD24 vector backbone as it does not express in the bacteria sample which is only transformed with pBAD24 vector backbone without the insert.
III. Functional Test
Figure B-5: The bar chart above shows three information, arabinose concentration, Specific activity and time of incubation.
In order to investigate the best condition for activating the tyrosinase secretion system, we induced the E.coli with a series of concentrations of arabinose ranging from 0 to 133000μM as well as two different incubation times: 4 and 8 hours under each concentration. In Figure B-5, the tyrosinase activity increases while being induced by a higher concentration of arabinose, it reaches the highest tyrosinase activity when the concentration of arabinose added is the highest. However, longer incubation time would not contribute to a higher tyrosinase activity as the activity obviously does not increase much with 8 hour incubation even with very high arabinose concentration. Based on this functional test result, we can deem 133000μM arabinose induction with 4 hours incubation time with the induction chemical arabinose to be the best condition to collect the supernatant of bacteria culture and be used for further tests. With a higher concentration of arabinose induction, the expression of tyrosinase will increase in an appropriate incubation time length and would be able to show better enzyme function. The results that we have obtained shows that we are successful in secreting a functional tyrosinase enzyme out of our bacteria.
Reference
- Valipour, E., & Arikan, B. (2016). Increased production of tyrosinase from Bacillus megaterium strain M36 by the response surface method. Archives of Biological Sciences Arhiv Za Bioloske Nauke, 68(3), 659–668. doi: 10.2298/abs151002058v
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- Xu, D.-Y., Chen, J.-Y., & Yang, Z. (2012). Use of cross-linked tyrosinase aggregates as catalyst for synthesis of l-DOPA. Biochemical Engineering Journal, 63, 88–94. doi: 10.1016/j.bej.2011.11.009
I. Plasmid Construction
PCR and Gel Running Results
Figure C-1: Gel running result for plasmid identity confirmation. Expected size of the FtnA fragment 574bp.
After performing gibson assembly of the FtnA fragment into the pBAD24 plasmid backbone we picked 3 colonies and then we extracted their plasmid and then we used PCR to amplify the FtnA DNA fragment. From the gel run results above, we can see that all the band size of FtnA DNA fragment from all the colonies are at around the expected size of 574 bp. This result shows that we have successfully constructed pBAD24-FtnA.
FtnA plasmids sequencing result
Figure C-2: Sequencing result for the pBAD24_FtnA plasmid
After we have built the plasmid we sent them for sequencing. The sequencing results for FtnA colony 1, 2 and 3 are as shown in Figure C-2. We can see that there are very few mismatched pairs in the reverse primer of each colony. Therefore, all of them are usable. However, for our further experiments, we have used the plasmids from Colony #1 only.
II. Protein Expression Test
Western Blotting Results: Checking of FtnA Expression
Figure C-3: Western Blot result containing our protein bands pertaining to the FtnA size
Our FtnA gene was tagged with FLAG tag at the C-terminal. To confirm whether our FtnA protein is successfully expressed in our bacteria, we ran western blot with 7 protein samples which respectively extracted from 6 different bacteria colonies transformed with FtnA gene fragment inserted plasmid shown as above (colonies 1.1, 1.2, 1.3, 2.1, 2.2 and 2.3) and from bacteria without FtnA-plasmid transformation as our negative control in the western blot analysis (Figure C-3). We have used an anti-Flag primary antibody with HRP tag and used chemiluminescence to produce detectable signals. We can see that FtnA protein is successfully expressed in all 6 bacteria colonies compared to the negative control. Although there is variation in the amount of protein shown, this result tells us that after successful transformation we can be confident that the protein would be expressed regardless of which colony we choose.
Western blotting results: FtnA expression level induced with different arabinose concentrations
Figure C-4: Western Blot result containing our protein bands which we later analyzed
Figure C-5: Bar chart to represent the relative expression level and arabinose concentration
To investigate whether concentrations of arabinose will affect FtnA protein expression level, we ran western blot with bacteria lysis sample from BL-21 (DE3) bacteria incubated with 6 different concentrations of arabinose: 0-13300uM (Figure C-4). Band signal’s intensity from the membrane were analyzed using Image Studio. Normalized with internal control rpoB (which the protein size is 150kDa), we can see that FtnA protein expression level rises when the arabinose concentrations are increased (Figure C-5). Therefore, it indicates that the concentrations of arabinose can affect the FtnA protein expression level in the same bacteria strain. We have used both of these results for the characterization of the part number BBa_K1679029 by OUC China 2015. This result would be a valuable characterization contribution to the said part for future teams who wishes to use the part and express the protein under the araBAD promoter.
Western blotting results: FtnA expression level in different bacteria strains
Figure C-6: The figure shows the membrane containing our protein signal bands which we analyzed later
Figure C-7: The bar chart represents the different strains and their relative protein expression levels
Different E.coli strain types can influence FtnA protein expression levels. To investigate which E.coli strain is the best strain for Ftna protein expression, we ran western blot with the extracted protein samples from 6 different kinds of bacteria strains (Figure C-6). We used rpoB (DNA-directed RNA 9polymerase subunit beta) as an internal control for our western blot analysis. Band signal’s intensity from the membrane were analyzed using Image Studio. After being normalized against the internal control (rpoB), we can see that different protein expression levels could be observed in different bacteria strains and all BL-21 derivatives strains which includes Rosetta, Star, pLys and DE3 showed a much higher level of protein expression compared to wild-type BL-21 strain (WT) as well as Stbl3 and DH5α strains (Figure C-7). Therefore, we can conclude that BL-21 derivatives: Rosetta, Star, pLys and DE3 are suitable strains in expressing FtnA protein.
III. Functional Test
Figure C-8: This shows the magnetization test setup for checking the function of ftna protein.
After testing the protein expression level, we also want to know whether those expressed proteins are functional. A functional FtnA protein would increase the magnetization efficiency; we expect that after 1 hr, there would be significant migration of the bacteria toward the magnet. Therefore, we did a magnetization test: The pBAD24-FtnA construct was transformed respectively into the six different strains which are BL21 (Rosetta), BL-21 (Star), Stbl3, BL-21 (pLys), DH5 alpha and BL-21(DE3). Our negative control is the wild type BL-21 DE3 which is without the construct. Overnight cultures of 5 mL were made and after overnight culture they were all induced by 0.02% arabinose and cultured overnight. The next day, 1ml of 100mM ferric ammonium citrate for 24 hours was added. After incubation, we centrifuged the bacteria culture, resuspended it with autoclaved water and poured 2ml into the small dish. A round shape magnet was set under each small dish during the 1hour magnetization. In figure C-8, we can see that the strains, which includes BL-21 Rosetta, BL-21 Star, BL-21 pLys and BL-21 DE3 showed a clear bacteria migration towards the shape of the magnet after 1 hour. Meanwhile, no significant change can be observed in the wild type BL-21 strain (DE3) as well as Stbl3 and DH5α strains. This functional test results shows consistent results with our protein expression test result in Figure 4, and it also can demonstrate that our FtnA overexpression system can work in these strains that we would recommend using in the future.
Reference
- Lu, Y.-J., & Sun, M. (2014). A simple method for constructing magnetic Escherichia coli. doi: 10.1101/010249
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Discussion
Initially, we planned to combine all these three functional systems in our engineered E.coli. However, we were not able to achieve it due to insufficient time to perform the combination tests. We selected pET11a plasmid which has ribosome binding site (RBS) as vector backbone of nanoparticle capturing system, pBAD24 plasmid with NSP4 as vector backbone for tyrosinase secretion system and FtnA overexpression system respectively. pET11a and pBAD24 plasmid are both Ampicillin resistant as their origins of replication are compatible, we will not be able to select colonies which possess construction of all three features. Although we are able to do checking by using PCR technique whether the picked colony has three constructs, it will be very time consuming and laborious. Moreover, the incompatibility of plasmids is a statistical phenomenon, when one of the plasmids divides faster than others. They compete with each other for the replication machinery, thus we are not able to guarantee the ratio of our three final plasmid features as one might dominate over another. In addition, there will be only one plasmid would be passed down to the next generation after a couple of divisions, this can lead to heterogeneous population of bacteria in our culture. In order to maintain the homogeneous population during sub-culture, we must apply combined antibiotic for selection. Therefore, possible solutions are to reconstruct the system with compatible plasmids or to combine all three features into single plasmid by utilizing Golden Gate assembly. In this case, this plasmid may finally contains three different cloning sites and promoters for three features we suggested.