Difference between revisions of "Part:BBa K4491135"

Latest revision as of 02:14, 14 October 2022

StayGold GFP characterisation (ID42)

The aim of constructing this plasmid is to characterise StayGold GFP with RBS B0033

Usage

As we are using a low copy number plasmid in our main project, we characterise a new fluorescent protein StayGold GFP which has a considerably better excitation coefficient, brightness and quantum yield compared to other GFPs.

Designing plasmid for characterisation

To characterise StayGold GFP, we have decided to use a low-copy number Level 1 acceptor plasmid (pJUMP27-1A) as a backbone where we will make use of both the main cloning site and the downstream cloning site.

Our circuit consists of 1 transcription unit in pJUMP27-1A backbone, consisting of PTet*, RBS, GFP and DT5. For GFP, in order to compare StayGold GFP with other GFPs available (Figure 1), we look through the Open Reporters in the Distribution Kit Plate 2 to find the 2 GFPs (afraGFP, efasGFP) that has the closest excitation and emission wavelength compared to StayGold GFP to allow better comparison. For the RBS, we have decided to characterise StayGold GFP with both B0033 (weak), B0032 (medium) and B0034 (strong) to see the effect. Quantifying the effect of the strength of RBS is important because fluorescent protein form aggregates when existing in high amounts. DT5 is an ultrastrong double terminator.

Figure 1. Design of the transcript unit to characterise StayGold GFP with different RBS strengths

Figure 2. Design of the transcript unit to characterise StayGold GFP with different GFPs with a similar emission and excitation wavelength

The backbone chosen for the characterisation of StayGold GFP is pJUMP27-1A because it is a low copy number plasmid. As discussed in the our Wiki, a lower copy number plasmid both reduce metabolic burden and noise.

Build

We perform JUMP assembly to clone the transcription unit into pJUMP27-1A using the enzyme BsaI to clone the transcription unit into the main cloning site. We perform JUMP assembly to clone the transcription unit into pJUMP27-1A using the enzyme BsaI to clone the transcription unit into the main cloning site.

Characterisation

Experiment 1

To test out how the concentration of inducers will affect the expression of StayGold GFP, we have designed our plate reader assay with different amounts of aTc inducers on several of our different constructs. We have chosen the range of aTc inducers used based on the characterisation done in Meyer (2019) on the Ptet* promoter. In the experiment, we have put in 2 negative controls (no_cells, Marionette_LVL1sinI) and Marionette_sfGFP as positive control as superfolder GFP is known to fluoresce. The test plasmids we have decided to use is StayGold with B0033 (ID42) and StayGold with B0032 (ID41) which is a transcription unit consisting of B0032 and B0033 with StayGold GFP respectively. The aim of the experiment is to compare the fluorescent intensity of StayGold GFP with superfolder GFP and to compare the fluorescent intensity of StayGold GFP with different strengths of ribosome binding sites.

Results of Experiment 1

Looking at the numerical data of the plate reader experiment. We can see that the fluorescent intensity of the superfolder GFP is a lot higher than that of the constructs containing StayGold GFP. To confirm this, a graph of the fluorescent intensity against time is plotted which is shown in Figure 3.

Figure 3. Result from Experiment 1 showing sfGFP has a much higher fluorescence than StayGold GFP

Experiment 2

During Experiment 1, we did the experiment pipetting by hand which we have learnt is an error prone process. Moreover, as the first experiment shows that StayGold is not fluorescing, we have decided to repeat the experiment to troubleshoot the results. To do this, we have decided to conduct the experiment using both DH5a and the Marionette strain with all the constructs we have for StayGold, efasGFP and afraGFP with Ptet* with no inducer (aTc) added or with a concentration (2μM) leading to maximal response added. We have also included a construct with the Pbad wt promoter with StayGold GFP to confirm that it is not the promoter that is not working with and without arabinose. Therefore, for this second iteration of the experiment, we have 14 different culture conditions altogether which makes pipetting manually difficult and error prone. As a result, we have decided to make use of automation. We perform the experiment using OT-2 robot which allows automated pipetting with a Python script invented by Camillo Moschner, one of the instructors of the Cambridge iGEM Team.

Results of Experiment 2

The results again show that StayGold GFP does not fluoresce as expected. The following graphs are plotted as a comparison. We first plotted efasGFP, afraGFP and StayGold GFP (all RBS strengths (ID42: B0032, ID43: B0033, ID44: B0034) on the same plots under the same promoters (Ptet*) for both the DH5a strain and the "Marionette" strain. The result shows that inside both Marionette strain (with aTc added) or DH5a, although efasGFP (ID45) is fluorescing strongly and is saturating the plate reader at this gain, both afraGFP (ID45) and StayGold GFP did not have a high fluorescence intensity.

Figure 4. Result from Experiment 2 showing efasGFP has a lot more fluorescence than StayGold GFP and afraGFP in DH5a

Figure 5. Result from Experiment 2 showing efasGFP has a lot more fluorescence than StayGold GFP and afraGFP "Marionette" strain

Sequence and Features