Cloning Results

Successfully cloned the PPK1 and PPK2 homolog A genetic constructs.
Successfully created AMP-pSB1C3 and KAN-pSB1C3 constructs from pSB1C3-mRFP1.
Successfully cloned the PPX2 genetic construct.

Cloning PPK1 and PPK2 homolog A into pSB1C3

Before cloning PPK1 and PPK2 homolog A into pSB1C3, we first needed to confirm the restriction digest of the backbone. Since we began with pSB1C3-mRFP1, we first digested backbone with EcoRI and PstI to remove the mRFP1. The following gel confirms that our backbone was successfully digested and ready for assembly. The gel was loaded as follows - Lane #1: Ladder, Lanes #2 - 6: digested pSB1C3-mRFP1

After successfully digesting pSB1C3, we then assembled the first polyphosphate synthesis genetic construct by inserting the PPK1 gene via Gibson Assembly into the backbone. The assembly was transformed into NEB5 alpha cells and grown on chloramphenicol plates. The following image shows colonies grown on our plates.

We conducted a restriction digest screening of the purified plasmid DNA from selected colonies. We used the dual cutter restriction enzyme in the prefix and suffix, NotI. We expect the positive controls of our samples to cut at 2046 bp: unnecessary backbone part and 2378 bp: our promoter-PPK1-terminator insert. We expect the control to cut at 2046 bp: same backbone part, and 1093 bp: part of backbone where our insert should be. The results of the gel suggested to us that our construct was successfully assembled.

We also sent out the purified plasmid DNA from picked colonies for sequencing. Based on the results, we were able to conclude that the His-tagged PPK1 with weak Anderson promoter genetic construct was successfully cloned.

We followed a similar procedure for cloning the PPK2 homolog A gene into pSB1C3 via Gibson Assembly The images below show the colonies after the transformation of the His-tagged PPK2A gene with weak Anderson promoter into NEB5alpha cells on CmR plates The image below suggests that are plasmid transformed into our cell since our pSB1C3 plasmid conferred chloramphenicol resistance.

Like with the PPK1 insert, we again did a restriction digest screening to confirm whether our construct was successfully assembled. We screened PPK2 homolog A with the dual cutter restriction enzyme NotI. Below is the result of our gel restriction screen. The expected banding pattern for the control 2046 bp backbone, 1093 bp mRFP gene) and insert (2046 bp backbone, 1115 bp insert). Lane #1: Ladder, Lanes #2 - 9: plasmid DNA from PPK2A construct. Lane #10: control, pSB1C3-mRFP1

The results from the gel suggest that the PPK2 homolog A construct was successfully cloned since the banding pattern matched the expected banding patterns. To confirm these results, we sent our samples for sequencing and were able to confirm that the sequence matched the sequence of the construct we intended to make.

Creating AMP-pSB1C3 and KAN-pSB1C3 from pSB1C3-mRFP1

Since we did not have pSB1A3 and pSB1K3 available to us and our genetic construct design required us to clone the Pit functional gene groups and the PPX2 homolog gene into an ampicillin-resistant and kanamycin-resistant vector backbone, respectively, another one of our goals was to construct an iGEM ampicillin (AMP-pSB1C3) and iGEM kanamycin backbone (KAN-pSB1C3) from pSB1C3-mRFP.

Constructing the AMP-pSB1C3 and KAN-pSB1C3 vector backbones proved somewhat difficult for us. We spent a great deal of our time troubleshooting errors while creating these backbones. In order to insert Kan-resistant and Amp-resistant genes into pSB1C3, we first needed to digest pSb1C3-mRFP1 with the restriction enzymes EcoRV and AatII. The expected banding pattern for this restriction digest was 2351 bp. The following gel confirms a successful restriction digest of pSB1C3: Lane #1 was the 3 uL of ladder. Lanes #2-6 were 50 uL samples of pSB1C3-mRFP plasmid digested with EcoRV and AatII.

Afterwards, we connected a two-step PCR reaction to amplify the KAN and AMP gene out of pET_28 and pET-18, respectively. In between PCR step #1 and PCR step #2, we ran a restriction digest to confirm that our PCR round #1 product matched the expected KAN and AMP gene sizes. The expected banding patterns were 813 bp and 963 bp for the KAN and AMP gene respectively.

The results of the gel above suggests that KAN and AMP genes were successfully amplified out of their respective vectors. After the second PCR reaction (which , we transformed our PCR round #2 product into NEB5-alpha cells on correct antibiotic-resistant LB plates and screened for colonies. The images below show colonies on both plates, suggesting the plasmid transformed into the E. coli did have the correct antibiotic resistance.

However, when we did a restriction of the purified AMP-pSB1C3 and KAN-pSB1C3 plasmid DNA against the pSB1C3 control, our results did not indicate successful assembly. When cut with NcoI and PstI, the expected banding pattern for each sample was 1415bp, 1284 bp, and 440 bp for control; 1458 bp and 440 bp for AMP-pSB1C3; and 2795 bp and 440 bp for KAN-pSB1C3. The loading was done as follows: Lane #1: KAN-pSB1C3-#1; Lane #2: KAN-pSB1C3-#2; Lane #3: KAN-pSB1C3-#3; Lane #4: Ladder; Lane #5: Ladder with Tiffany’s suggestion; Lane #6: AMP-pSB1C3-#1; Lane #7: AMP-pSB1C3-#2; Lane #8: AMP-pSB1C3-#3; Lane #9: Control (pSB1C3) The results of the gel electrophoresis are indicated below:

Since the banding pattern did not match the expected banding pattern, the data suggests that the construction of the new backbones was not successful. In fact, the sizes of the KAN-pSB1C3 and AMP-pSB1C3 samples were greater than 3000bp, suggesting that we recovered the original pET-28 and pET-18 backbones.

When troubleshooting these results, we realized that a potential source of error was that we picked non-red colonies instead of red colonies. Since the correct antibiotic resistance genes were cloned into a digested pSB1C3-mRFP1 backbone (with intact mRFP), the colonies with correct antibiotic resistance should still express mRFP. While our plates with the AMP-pSB1C3 did have several red colonies, the KAN-pSB1C3 plate only had a singular, small red colony. We still conducted a restriction digest with EcoRV and AatII to determine if the correct backbone was constructed. The gel loading was done as follows: Lane #1: Ladder; Lanes #2- #7: AMP-pSB1C3; Lane #8: KAN-pSB1C3 (only mixed red colony from KAN plate); Lane #9: Control (undigested pSB1C3-mRFP1). The expected banding pattern for AMP-pSB1C3 was 2351 bp backbone, 1009 bp fragment; KAN-pSB1C3 was 2351 bp backbone, 884 bp fragment; and undigested pSB1C3 was 2351 bp backbone, 788 bp fragment.

The results of the gel electrophoresis suggest that samples from the AMP-pSB1C3 were successfully assembled (since the banding patterns matched); however, the KAN-pSB1C3 did not match the expected pattern, suggesting that it was not successfully constructed. As a result, we decided to redo Gibson Assembly with the KAN gene fragment and the pSB1C3-digested with EcoRV and AatII. The image below shows the result of our new KAN-pSB1C3 transformation into NEB5-alpha cells on LB-KAN plates. The KAN transformation yielded many background colonies, but there were a few select colonies that visually expressed RFP.

After picking the colonies visually expressing mRFP, we conducted another restriction digest, where we were able to confirm the correct construction of the KAN-pSB1C3 fragment; however, the concentration yields for both the AMP-pSB1C3 and KAN-pSB1C3 backbones were both low. As a result, we used PCR amplification to amplify the backbone into two parts, one containing the origin and one containing the correct antibiotic resistance gene (AMP or KAN). With this method, we were able to achieve higher concentrations of the KAN- and AMP-pSB1C3 backbones and subsequently utilize them for our other two gene groups.

Cloning PPX2 homolog into KAN-pSB1C3

We were also able to successfully clone our His-tagged PPX2 homolog gene with pBAD promoter into our KAN-pSB1C3 backbone. Like with PPK1 and PPK2, we inserted the PPX2 homolog gene into two part KAN-pSB1C3 backbone (one vector backbone section contained the origin and the other contained the KAN antibiotic resistance gene) via Gibson Assembly into NEB5-alpha cells. The following image shows colonies grown on our plates, suggesting that successful transformation of plasmid with the kanamycin resistance did occur.

Afterwards, we conducted plasmid DNA purification and sent our PPX2 samples for sequencing. Based on the results of our sequencing, we were able to conclude that our genetic construct with PPX2 homolog was successfully cloned.

What Did Not Work For Us

One of the biggest complications of this year's project was major delays due to products being on backorder and out of stock due to the COVID-19 pandemic.
For the PPK2, KAN-pSB1C3 constructs, our concentrations and DNA yields were too low to send out for Sanger sequencing. To work around this we regrew more overnight positive clones from purified plasmids.
We also had a hard time troubleshooting the errors in our Pit function gene group assemblies. Though we assembled the Pit A, Pit B, and Pit C a couple times, we kept seeing unsuccessful restriction digests and sequencing results.

Cloning: Future Plans and Considerations

If we were to continue this project, we would design primers to test different combinations with the Pit, PPK, and PPX genes. Our original plan was to test Pit A alone, Pit C+B, then Pit A+B+C. Due to time limitations and issues with the genes, we were not able to test any of these combinations.
Similarly, we were able to successfully construct the PPK1 and PPK2A genes. We would like to test the PPK1 and PPK2A genes together in one gblock. In this cycle, we were able to test the genes separately rather than together. We also wanted to test PPX, PPX2B, and PPX2C. We were only able to construct and test the PPX gene.
For future plans, we would test and construct PPX2B and PPX2C genes as well.
Additionally, we would like to characterize other plasmid backbones rather than just sequencing them. With the testing and experimentation aspect of our project, we would also like to encapsulate the bacteria as originally planned and measure the escape rate of E.coli from the encapsulated silica bead gel.

Biochemical Assay Results

Objective: From the previous experiments, we had three colonies of bacteria with various genes, PPK1, PPK2, and also a pUC-19 control. After culturing the bacteria with varying amounts of a phosphate source (pH7) in addition to the LB, we sought to determine whether the genes we had induced led to higher intracellular polyP concentrations when compared to the control. The procedure that was followed is from “An improved method for extraction and quantification of polyphosphate granules from microbial cells” by Krishna Ray (2015).

Part 1: Culturing the Bacteria

We cultured each respective bacteria with an antibiotic selection for 26.5 hours, the results of the OD600 readings are seen below. We made 3 samples of each genetically transformed bacteria and had one negative control. For each bacteria, one sample had only LB, the next had a .01/ mg/mL phosphorous source added, and the last had.05 mg/mL source added. We will label these as PXXX-N, PXXX-L, PXXX-H, respectively, for none, low, and high added phosphate. The weights of the cells after centrifugation were also taken.

Part 2: Extraction and Quantification

Then, the bacteria were subject to the extraction procedure, which involved sonification, and 1 hour of boiling in 100C water. Then, toluidine blue was added and the absorbance was measured. Ideally, if polyP is present, the mixture will turn purple instead of its standard blue.

Due to experimental variability, we noticed variability in volume in the samples we test. The OD630 Readings for each sample, along with the proportions tested are tabulated below.

All of our samples did follow the expected shape of absorbance for this kind of test, as shown below.

Then, we compare the differences in absorbance for each sample from the control. Lower absorbance here indicates that more polyphosphate is in the sample, so we took the absolute value of the normal - control.

Interpretation

We can begin to see that there was a reduction in absorbance for all 3 cases, meaning polyP was detected successfully. However, since there was only 1 sample, it is ambiguous to say whether this result was significant or not. It is also ambiguous whether or not there is a phosphate-source dependent absorption of the polyP. If we average all 3 reductions from the values, we find that the PPK2 construct did a much better job of absorbing polyP than the PPK1 construct compared to pUC19. However, neither was a statistically significant reduction.

Achievements and Further Work

Successes:

Phosphorus absorbance was detected via this assay
The PPK2 construct did appear to absorb more phosphorus than the control (p=0.11)

Future Work:

A further test may look at more samples to determine significance. Since this assay is very time-consuming, we were unable to get a clear result as to whether the genetic construct we transformed into the bacteria works for sure. Further work may be tweaking the procedure to see if a more robust decline in absorbance can be detected.

Team:UMaryland/Results