Team:Rotterdam HR/Results
Results
Results
General
Our plan was to create multiple composite parts, which included CooA.
To be pure the DNA needed to have a A260/280 value between 1,80-2,00 and a A260/230 value between 2,00 and 2,20.
These composite parts would have different
promoters and output genes, so we can measure carbon monoxide in multiple ways.
For further steps we isolated the DNA. The DNA had to be pure for digestions. To be sure the DNA was pure, requirements were set. Hereby there would be looked at the curve of the line also. If the line wasn’t smooth, the DNA was most likely not pure. A pure sample has a curve like shown in figure 1.
To produce enough DNA of BBa_J04450 (an plasmid containing a functional spacer) we performed multiple mini preps (DNA isolations), which gave us the following results:
| BBa_J04450 | pSB1C3 1 | 270,71 ng/µL |
| BBa_J04450 | pSB1C3 2 | 119,84 ng/µL |
| BBa_J04450 | pSB1C3 1 | 451,17 ng/µL |
| BBa_J04450 | pSB1C3 2 | 319,01 ng/µL |
| BBa_J04450 | pSB1C3 1 | 136,98 ng/µL |
| BBa_J04450 | pSB1C3 2 | 80,90 ng/µL |
Successful isolated DNA could be digested and ligated with another biobrick for testing. Early named biobricks were paired with each other, where BBa_K133071 and BBa_K173003 functioned as vectors and BBa_I134353 and BBa_J23100 as inserts. We used the backbone from the vectors, so we only needed to insert 1 biobrick into another biobrick. To know if the ligation had worked the DNA was transformed into the E.coli strain: NEB10Bèta. Colonies that grow were tested by digesting again. The digested DNA was put on gel, and the length was determined.
CooA production
When we started our project our first order of business was to reliably produce CooA, the carbon monoxide dependent transcription factor. We knew we had to constitutively produce CooA, and had to produce as much as we could to elicit a high enough response, but we also knew that our cells wouldn’t be able to produce it indefinitely. To this end we decided to combine different constitutive promoters (BBa’s J23100, J23105 and J23113), and combine them with different ribosomal binding sites (BBa’s B0030, B0031 and B0032). This could then be combined with CooA (BBa_K352001) and a double terminator (BBa_B0015).
At first we tried to insert our ribosomal binding site into the plasmids with the different promoters, though because the backbones of the promoters were different from the standard registry backbones we had some difficulty with the cloning, so we decided to turn our plan around and tried to insert the promoters into the plasmids containing the different ribosomal binding sites. The next problem we had with our cloning however was the size of the promoters themselves. As they are only around 15 base pairs long, and the ribosomal binding sites are around 35 base pairs long, when combined the total change in plasmid size would only be around 50 to 60 base pairs, which we couldn’t visualize on our electrophoresis gels.
After this conundrum we had decided to follow the standard iGEM 3A assembly method, using BBa_J04450 (pSB1C3, pSB1K3 and pSB1A3 versions of this biobrick) as a quick screening method.
After a few attempts we seemed to have successfully cloned BBa_J23105
with BBa_B0032 and BBa_K352001
with BBa_B0015.
This has then been cloned together to make the full CooA production plasmid. Of the resulting colonies we made 10
different colony PCR’s, and put these on gel, of which one seemed to be the expected size.
Sadly when we sequenced the CooA part in pSB1A3, the sequence conformed to the sequence of BBa_J04450, which is about the same length as the expected CooA product from the PCR.
Gas production
For our gas production system we started with transformations of the biobricks:
-BBa_J23100: Constitutive promoter.
-BBa_K133071: UreB.
-BBa_K173003: Pyruvate decarboxylase + ethanol dehydrogenase.
-BBa_I13453: Arabinose Promoter.
Things went wrong, so after a time we had transformed the biobrick combinations BBa_J23100 + BBa_K133071, BBa_J23100 + BBa_K173003, BBa_I13453 + BBa_K133071 and BBa_I13453 + BBa_K173003 in the E.coli strain NEB10Bèta.
After the transformations in NEB10bèta we started testing with our self invented gas production test tubes.
Due to us not having any experience with our setup as seen in these last two images, when we placed the smaller tube into a 15 mL tube there was some gas present. Though when comparing before and after pictures there is undoubtedly more gas present in our tubes.
| Bacteria | LB | LB | Urea | Urea | Pyruvate | Pyruvate |
|---|---|---|---|---|---|---|
| NEB10beta | + | +++ | +++ | +++ | + | ++/+++ |
| BL21 (DE3) | - | ++ | - | - | - | - |
| BL21 | - | - | - | - | - | - |
| DH5a | +++ | +/- | - | - | +/- | + |
| HB101 | - | - | - | - | + | - |
| JM109 | - | - | - | - | - | - |
-
-: No/ minimal gas production,
+: Little bit of gas production
++: More than a little bit of gas production
+++: Most gas production
The strains are tested with different substrates in Lysogeny Broth (LB) culture medium (See table 2). HB101 and JM109
can be used best as a negative control for our gas production tests.
For easier use we gave our combinations abbreviations. This can be seen in the following tabel:
| Number | Promotor | Gene | Backbone |
|---|---|---|---|
| A | K352002 (CooF) | K173013 | pSB1K3 |
| B | K352002 (CooF) | K173003 | pSB1K3 |
| C | K352002 (CooF) | K133071 | pSB1K3 |
| D | K352003 (CooM) | K173003 | pSB1K3 |
| E | K352003 (CooM) | K173013 | pSB1K3 |
| F | K352003 (CooM) | K173013 | pSB1K3 |
| H | I13453 | K173013 | pSB1K3 |
| I | I13453 | K173003 | pSB1K3 |
| J | I13453 | K133071 | pSB1K3 |
When looking at figures 6 and 7 we can conclude that most biobricks did not give the expected results, because they were cut twice. H1, D2, I2 and J2 were digested once,so we expect they include the right biobricks and BBa_K352002 + BBa_K133116 and BBa_K352003 + BBa_K133116 were dropped because those lanes are empty.
Only I1, J1 and J2 gave usable results so we sequenced all parts that we couldn't say were definitely wrong.
Sequencing and shipping
We sequenced the previously mentioned gas production parts and CooA 8 (refer to figure: 2). When comparing the sequencing results to the expected sequences we concluded that B1, B6, D1, I1 and J1 were as we expected. These BioBricks were then digested and ligated into pSB1C3, after which we transformed them, mini prepped them, and dried the DNA onto a 96 wells plate for submission.
Hardware
For information about our hardware results, please navigate to the Hardware page.
To the hardware pageFuture plans
If we want to make a fully usable product firstly we would have to finish our CooA production plasmid. Secondly we would like to look at a cell-free system, probably by altering the CooA structure, to avoid regulations regarding GMOs outside of a laboratory setting. This would also mean a relatively simpler hardware design, cutting down costs and the amount of work. If a cell-free system is not feasible we could also give more thought to safety measures, adding a working kill switch to our system, and also pursuing possible hardware contingency plans (like releasing toxic substances in our system when part of it breaks).
Replicating the experiments
If we would do our experiments again, we would start the change by performing our digestions in a smaller end volume, so we can get a more concentrated digest product, enabling us to use more nanograms of DNA when performing our ligation. Secondly we would like to sequence our intermediate constructs, as most parts we wanted to use are fairly small, and thus they don’t make a significant enough change in construct length to easily and reliably detect on an electrophoresis gel. So as to be sure that our intermediate constructs are as we want them to, sequencing would be the most reliable method.