Results
CONSTRUCTION
GOLDEN GATE ASSEMBLY
All Golden Gate Assembly for this project was based on the MoClo Assembly Toolkit from addgene.org. We purchased two kits of parts which allowed us to use the assembly framework outlined in their paper here. The kits are the MoClo Toolkit and the Cyanogate Kit. For reference, Level 0 refers to a basic part, Level 1 refers to a transcriptional unit (Promoter, CDS, and Terminator), and Level 2/T refers to a multi-transcriptional unit expression vector for E. coli / Synechocystis sp. PCC 6803 respectively.
For this project, there were a number of specific constructs we aimed to complete. These are listed below, with
plasmid names according to this scheme:
Level 0: pBLB_ (like BULB) + L0- + id (each CDS has its own number)
Level 1: pBLB_ + L1- + id (such as 1a or 3c, number corresponding to CDS and letter to variant of
promoter or terminator)
Level T: pBLB_ + GLOW- + Δx-id (indicating a change from usual scheme of 1a-2a-3a-4a-5a
construct)
Level 2: pBLB_ + EcoGLOW + Δx-id (indicating a change from usual scheme of 1a-2a-3a-4a-5a construct)
List of Constructs
- pBLB_L0-1
- pBLB_L0-2
- pBLB_L0-3
- pBLB_L0-4
- pBLB_L0-5
- pBLB_L0-6
- pBLB_L0-7
- pBLB_L0-8
- pBLB_L1-1a
- pBLB_L1-1b
- pBLB_L1-1c
- pBLB_L1-2a
- pBLB_L1-3a
- pBLB_L1-4a
- pBLB_L1-5a
- pBLB_L1-6a
- pBLB_L1-7a
- pBLB_L1-8a
- pBLB_GLOW
- pBLB_GLOWΔ1-1c
- pBLB_EcoGLOW
- pBLB_EcoGLOWΔ1-1c
VALIDATION
COLONY PCR ANALYSIS - LEVEL 0
Please note that all agarose electrophoresis gels with a DNA ladder use NEB 1kb Plus, and wells containing ladder DNA do not count towards numbering.
In Figure 1, pBLB_L0-3 and pBLB_L0-4 (H3H, lane 4 and NpgA, land 3 on left) both show a positive result in this colony PCR after our first attempt at assembly and transformation.
Figure 2 shows positive results for transformation with pBLB_L0-1 (gel 1, top; lanes 2-4), pBLB_L0-6 (gel 2, bottom; lanes 1-3), pBLB_L0-7 (gel 3, top; lanes 1-4), and pBLB_L0-8 (gel 3, bottom; lanes 2 and 3).
Figure 3 shows positive results for transformation with pBLB_L0-2 (lane 2), but not pBLB_L0-5 (lane 4). We attained a positive result for pBLB_L0-2 after our third try.
Finally, a gel (Figure 4) that showcases positive results for pBLB_L0-5, the final Level 0 piece to be assembled. Two colonies from a colony PCR conducted on 16 transformant colonies were selected on a whim based on the fact that their results were slightly different from the rest. Plasmids were extracted and digested, getting us Figure 4! In order for us to get this result, we had to undergo at least 9 iterations of the Golden Gate Assembly protocol. Possible reasons for this could be the oligonucleotides present in the gBlocks that IDT sent us (thank you!). Furthermore, the gene was split into 2 fragments with a restriction site to put them together in assembly. Oligonucleotides could have interfered with annealing, as fragments might have taken the binding overhangs. Nevertheless, our approach going forward for assembling gBlocks with plasmid backbones will take into account potential impurities, and involve some sort of purification process or pre-digestion for 2-step assembly.
DIGESTION ANALYSIS - LEVEL 1
pBLB_L1-1a (lane 1), pBLB_L1-6a (lane 3), pBLB_L1-7a (lane 5), and pBLB_L1-8a (lane 7) mini-prepped plasmid DNA samples were digested, validating presence of a correct construct in each of these cultures. This result was attained after the first assembly of the constructs. Would that it were so simple! At least this meant that we were getting better at Golden Gate Assemblies.
In Figure 5, pBLB_L1-1b (lane 1), pBLB_L1-1c (lane 2), pBLB_L1-2a (lane 3), pBLB_L1-3a (lane 4), and pBLB_L1-4a (lane 5) mini-prepped plasmid DNA samples were digested, validating presence of a correct construct in each of these cultures.
PCR ANALYSIS - LEVEL 2/T
This (Figure 6) is where the lack of time kicks in! We were without lab for this section, so we had to borrow a
classroom lab on a week with no lab classes just to get this one result. We ran a colony PCR on a colony of
E. coli which supposedly contained our final Level 2 construct,
pBLB_EcoGLOWΔ1-1c. This result shows a possible successful transformation of our pBLB_EcoGLOWΔ1-1c
plasmid, which is 18kb!
The Wet Lab's next step here would be to perform an SDS-PAGE with a wild-type E. coli in order to see if
any new proteins are being expressed. We would then move on to attempting to fractionate protein extracts
of wild-type and transformed E. coli and running them on SDS-PAGE gels to see if we could see a more
obvious band where we expect to see our target enzymes.
CAFFEIC ACID TRIALS
Our original plan was to clone pBLB_GLOW in E. coli first, to get a high yield of purified plasmid DNA
for transformation into Synechocystis sp. PCC 6803. However, due to time constraints, we decided to use
these transformants to test gene expression of the plasmid first.
As E. coli do not produce caffeic acid, we had to provide it in order to be able to detect activity of
our design. If all enzymes work (excluding CPH) we would expect to see the emission of light from the cells.
To test this theory, we designed an assay with
varying concentrations of caffeic acid to be provided to living E. coli. These E. coli
transformants expressing pBLB_GLOW were combined with caffeic acid in a 96-well plate and placed into a
pitch-black box with a hole for a camera to detect light emission.
To the same effect, we designated additional wells to the addition of E. coli expressing individual
transcriptional units (Level 1 assemblies) in case the metabolic load of all 5 enzymes was too much for our
strain of E. coli DH5α.
Shown by the image above, no luminescence was detected.
FUTURE PLANS
Expression Quantification
An absolutely major limitation for getting results for the duration of this project was always time. As a
first-year team, we spent a lot of time
getting a lab, getting basic materials, ordering E. coli and parts from addgene.org. This took a tremendous amount of time away from working on
constructs and testing them thoroughly.
Furthermore, near the end of the season and as school started again, we lost the ability to work in the lab.
This was because the lab space we were given was turned into
a classroom for the semester. This left us with a couple weeks where we couldn't do much work, and any work we
did do was in a different lab!
Despite this hardship, we believe that this project still has potential. This season, we were not able to make any organism glow using fungal bioluminescent genes, but we can still see the path forward!
First, we would like to undertake more rigorous validation of the Level 2 and Level T constructs, pBLB_EcoGLOW and pBLB_Glow, respectively. This would involve:
- Expression of pBLB_EcoGLOW and variants in a strain of E. coli like BL21 to express high amounts of protein
- Protein Extraction and Fractionation by Column Chromatography
- SDS-PAGE Analysis of Fractions
- Further Fractionation and Purification
- Substrate Assays
- Individual Expression and Purification of Enzymes
1. Expressing our constructs in a strain like E. coli BL21 instead of DH5alpha would facilitate the extraction of our target proteins. This would allow us to better quantify the expression of our protein and leave us with some flexibility with yield.
2. We were not able to procure any materials dedicated to extract proteins for analysis. For this reason, we would like to dedicate budget to purchase them. We would like to extract our protein from our cultures so that we can fractionate them using a method such as His-tagging. This way, we would be able to be more precise with quantifying expression of our genes of interest.
3. Next, we would use an SDS-PAGE to visualize the presence of our target protein in comparison to a wild-type negative control. If a band appears in our transformed which does not appear in the wild-type, it is likely that it is our target protein. We can then fractionate the fraction it was located in to get a pure sample.
4. After identifying which fraction our protein belongs to, we could either use a different column chromatography method or move to HP-LC analysis to purify our target protein.
5. After purification, we would be able to use our pure protein samples to guide an assay. This assay would be dependant on our primary substrate: caffeic acid. After introducing pathway proteins to the substrate, we can analyse the conversion of substrate into product using LCMS-MS analysis on the resulting solution.
6. To assist the characterization of the activity of enzymes, we can express each enzyme separately using our pBLB_L1 transcriptional unit variants in E. coli BL21 and purify them to get each enzyme on their own. Then, we can do assays such as the following:
- Conversion of Caffeic Acid into Hispidin by HispS enzyme with presence or absence of the NpgA enzyme.
- Conversion of Hispidin into 3-Hydroxyhispidin by H3H enzyme, using substrate purified from above reaction products.
- Luminescence of the nnLuz and variants or psLuz enzymes by catalyzing conversion of 3-Hydroxyhispidin into Fungal Oxyluciferin
- Cyclic conversion of Caffeic Acid → Hispidin → 3-Hydroxyhispidin → Oxyluciferin → Caffeic Acid using all enzymes in the cycle.
- With pure caffeic acid as a substrate, characterize the whole bioluminescent cycle by introducing one enzyme at a time.
- With proper time, quantification of quantum yield and overall light emission using specialized instruments and software.
- Comparison of nnLuz, nnLuz variants, and psLuz's quantum yields in vitro and in vivo (E. coli)
Then, with credence to the whole project's purpose, or even at the same time with a big enough lab, we will pioneer the expression of the fungal bioluminescent biosynthesis pathway in Synechocystis sp. PCC 6803, as well as take measurements of quantum yield and overall light emission in vivo. Below is a picture of some fun we had with the calibration plate, post-calibration.
For the Caffeic Acid Trials, we would like to attempt this again using E. coli BL21 transformants of pBLB_GLOW or pBLB_EcoGLOW, as well as Synechocystis sp. PCC 6803 transformants of pBLB_GLOW. We strongly believe that it will shed some light!
INTERLAB CONTRIBUTION
CALIBRATION
Our team this year decided to attempt every protocol of this year's Interlab Study. However, due to extremely transformation efficiency for unknown reasons and a severe lack of time, we were not able to complete any protocol other than the calibration measurements.
