Difference between revisions of "Part:BBa K515005"
 
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a translational unit of superfolder GFP
 
a translational unit of superfolder GFP
  
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===Characterization for Universal Golden Gate Cloning (Alma College)===
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===Usage and Biology===
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We used this BioBrick to characterize a new approach to Golden Gate assembly - one inspired by a paper by [https://www.ncbi.nlm.nih.gov/pubmed/25524097 Bryksin et al]. In this approach, universal PCR primers add BsaI sites and overhang sequences to any BioBrick, thanks to a specialized mismatch priming. This eliminates the need to order separate primers for every BioBrick you want to assembly using Golden Gate. We designed forward primers with overhangs sequences AATT, AAGG, ACTC, AGGT, and reverse primers with overhangs AAGG, ACTC, AGGT, and TGCA. For more information, refer to the diagram to the right or visit the contribution page on our Wiki.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K515005 SequenceAndFeatures</partinfo>
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Using this (and other) BioBricks and the above primers, we carried out PCR reactions to determine if this approach was feasible. The reactions were generally setup as follows: 0.5uL of purified plasmid DNA for BioBrick BBa_K515005 was mixed with 9.5uL ddH20, 1.25uL of each primer (@ a 10uM concentration), and finally with 12.5uL of a 2X Mix for [https://www.neb.com/products/m0492-q5-high-fidelity-2x-master-mix Q5 DNA Polymerase]. Reaction conditions were setup as suggested by NEB for Q5 polymerase, typically with an annealing temperature between 51 and 55C. In some trials, 9uL of ddH20 and 0.5ul of DMSO were added.  
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<p><b>This BioBrick has been sequence verified.</p></b>
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<h2>Thermostability</h2>
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<p>This test is to show the thermostability of sfGFP, by finding the temperature at which the protein denatures. Stock solutions of sfGFP were prepared by extracting the protein from cell lysate, and then 50 μl aliquots of the solution were heated in a PCR thermocycler along a temperature gradient.</p>
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<p>After two hours, 30 μl was removed from each aliquot and diluted with 170 μl of 20 mM Tris buffer to give 200 μl samples. The samples were then measured by fluorescence on a 96-well plate. The corresponding curve was plotted on a graph and the curve was used to calculate the denaturation temperature.</p>
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<p><img class="border" src="https://static.igem.org/mediawiki/2011/thumb/7/75/Curve.png/800px-Curve.png" width="600px" /></div>
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A depiction of the primer binding, and a representative gel, is below.
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<p><i>Fig. 4: Results of the heat denaturation experiment. The temperature at which half of the protein is denatured measured by looking at its fluorescence (PTm50) mRFP1: 82.2°C; GFPmut3b: 61.6°C; Dendra2: 89.1°C; sfGFP: 75.0°C.</i></p>
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<h2>Soil survivability and plasmid retainment testing</h2>
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<p style="float:right;">
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[[File:T--Alma--universalprimers2019_2.png|400px]]
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<a href="http://2011.igem.org/Team:Imperial_College_London/Protocols_Switch"><img src="https://static.igem.org/mediawiki/2011/5/58/ICL_ProtocolIconDark.png" width="180px" /></a></p>
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<p>To test for the survivability of <i>E. coli</i> in soil, we set up an experiment. We initially transformed chemically competent DH5alpha cells with superfolder GFP. These cells were inoculated on small (about 0.5 cm diameter) filter discs, which were placed in autoclaved and non-autoclaved soil. We periodically grew up cultures from these filter discs over the course of six weeks.</p>
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<p>After six weeks, we were able to recover fluorescent bacteria from sterilised soil. Colonies appearing to be <E. coli</i> on the plates from non-sterile soil had lost fluorescence (Fig. 2).
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<p><img class="border" src="https://static.igem.org/mediawiki/2011/b/b6/Fluorescent_soil_plates.png" width="500" />
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PCR products from some reaction combinations were selected and sequenced from the middle towards the end - this way, we can confirm if the overhang and BsaI site were added correctly. VF2 and VR primers were used as controls in the event that some primer pairs gave no or incorrect products.  
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<p><i>Figure 2. Colonies recovered from filter discs and grown on LB plates containing selective antibiotics imaged using a LAS-3000 gel imager. a) Sample taken from non-sterilised soil b) Sample taken from sterilised soil  (Data by Imperial College London iGEM 2011).</i></p>
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As you can see in the table below, we had mixed results - some of these PCRs required optimization, and were sensitive to annealing temperature and the speed at which the temperature shifted (evident by comparing the reaction in an old thermocycler versus a new mini8 PCR machine). However, we were able to show that this approach can be viable and does indeed lead to products that should work in a Golden Gate assembly reaction.
  
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<p>As is visible from these plates, fluorescence was present in bacteria recovered from sterile but not from non-sterile soil. The control plate showed that there was no contamination with other fluorescent lab bacteria. In order to investigate whether the fluorescence observed was due to the presence of the original sfGFP construct and whether the <i>E. coli</i>-like colonies from the non-sterile sample had retained a plasmid we extracted plasmid DNA using a miniprep kit and did a digest with EcoRI and PstI and with EcoRI on its own to check for presence of the original insert and size of the unfolded vector, respectively (Fig. 3).</p>
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[[File:T--Alma--universalprimers2019_3.png|400px]]
  
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<img class="border" src="https://static.igem.org/mediawiki/parts/9/9c/Soil_digest_gel.png" width="250" />
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Legend: green indicates a successfully confirmed reaction (with the bold cell representing the result corresponding to the representative chromatogram above), light green indicates a sequencing analysis with partial success/confirmation/matching, orange-pink (EtBr color) indicates a PCR reaction that gave the correct product, and purple indicates a reaction that did not work. Empty cells represent untested combinations. *These reactions did not work initially, but were successful at least once (required further optimization).
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<p><i>Figure 3. Gel digests of bacteria displaying colony morphology typical of </i>E. coli<i> recovered from non-sterilised and sterilised soil. These bacteria exhibited colony morphologies typical of </i>E. coli.<i> (Data by Imperial College iGEM team 2011)</i></p>
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== '''Team iBowu-China 2021 Contribution'''==
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<b>Group: iBowu-China 2021 </b>
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<b>Author: Rachel Chen</b>
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<b>Introduction:</b>
  
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<p>The insert is very clearly visible at just below 2 kb. This confirms the presence of superfolder GFP in both cultures. Sequencing of the GFP insert revealed that a single frameshift mutation had taken place in the colonies grown up from non-sterile soil. No mutations were observed in the superfolder GFP gene contained in the bacteria inoculated in sterile soil. This explains the absence and presence of fluorescence in the respective colonies. However, the bacteria were still resistant to the antibiotics and contained the plasmid. We will be replicating these results with other samples to ensure this is representative.
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<p>In addition, small colonies appeared on the non-sterile plate that had very different colony morphology. We grew this colony up in LB medium containing selective antibiotic and subsequently performed a separate miniprep. No DNA was yielded in this miniprep. It is therefore likely that the plasmid was not transferred to these bacteria but that they either possess natural antibiotic resistance or were able to survive on plates that whose antibiotics had already been depleted by the presence of resistant engineered bacteria.
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We used this part as a positive comparison for control group in the measurement of green fluorescence. Carried on a pSB1C3 plasmid, this part can effectively produce green fluorescence, together with a constitutive promoter. Our contribution includes
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# We transformed both DH5a and BL21(DE3) and produced green fluorescence without any addition of induction reagents such as IPTG.
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# In both cases, the light intensity is much stronger than the background by about 100 fold.  
  
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<h2>Plant uptake of <i>E. coli</i></h2>
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<p>As described by Paungfoo-Lonhienne et al. (2010)<sup>[1]</sup>, plants are able to actively take up microbes. We replicated these findings using <i>E. coli</i> DH5alpha cells expressing sfGFP. Bacteria were grown into exponential phase, spun down and resuspended in 5mM MES to OD 30. 2, 4, and 8 ml of these bacteria were added to 100ml half-MS cultures containing three-week old <i>Arabidopsis thaliana</i> Columbia strain wild type plants.</p>
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<p>Subsequently, the roots were washed in PBS to avoid imaging of any false positives - bacteria on the outside of rather than inside the roots. The sfGFP allowed us to very clearly identify bacteria inside the roots (Figure 3).
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<b>Protocol:</b>
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The plasmid was obtained from BNDS-China 2021 team. It was then expressed both in E. coli DH5a and E. coli BL21(DE3) with Cm resistance. The bacteria culture was incubated without addition of any iPTG in both cases overnight at 37 degree Celsius. GFP was measured with a microplate reader and OD600 was taken for normalization of the concentration. A control group was also measured for background light intensity where the E. coli BL21(DE3) without this plasmid was cultured. A picture was taken to show the effect of green fluorescence protein expression.
  
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<p><img class="border" src="https://static.igem.org/mediawiki/2011/b/b7/Awesome_bac_in_roots_16bit.png" width="450px;" />
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<p><i>Figure 3. </i>Escherichia coli<i> cells expressing superfolder GFP (sfGFP) can be seen inside an </i>Arabidopsis thaliana<i> root using confocal microscopy after overnight incubation of the plants with bacteria. Roots were washed in PBS prior to imaging to avoid "false positives" of bacteria adhering to the outside of the root (data and imaging by Imperial College iGEM 2011).</i></p>
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<b>Results:</b>
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<p><b>References:</b>
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<p>[1] Paungfoo-Lonhienne, C. et al. (2010) Turning the table: plants consume microbes as a source of nutrients. <i>PLoS One</i> <b>5(7):</b>, e11915.
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[[File: T--iBowu-China--2021gfp1.png |800px|thumb|center| Figure 1. Green fluorescence protein expressed by <i>E. coli</i> DH5a and BL21(DE3) under visible light and their measured intensity normalized by their concentration (OD600). The third column was the control group where the E. coli BL21(DE3) without this plasmid was used. ]]
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</html>
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<br>
  
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The results show this part can produce green fluorescence protein highly effectively. In BL21(DE3), the expression is stronger than in DH5a. The intensity of the GFP light in BL21(DE3) is about 2x the intensity in DH5a. In both cases, the light intensity is much stronger than the background by about 100 fold.
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===Functional Parameters===
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<partinfo>BBa_K515005 parameters</partinfo>
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<b>Summary:</b>
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This part can be used for effective expression of green fluorescence protein. It is an ideal choice for control group, and it can also be engineered to express other sequences.
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</p>
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Latest revision as of 11:36, 11 October 2022

superfolder GFP (sfGFP)

a translational unit of superfolder GFP

Characterization for Universal Golden Gate Cloning (Alma College)

We used this BioBrick to characterize a new approach to Golden Gate assembly - one inspired by a paper by Bryksin et al. In this approach, universal PCR primers add BsaI sites and overhang sequences to any BioBrick, thanks to a specialized mismatch priming. This eliminates the need to order separate primers for every BioBrick you want to assembly using Golden Gate. We designed forward primers with overhangs sequences AATT, AAGG, ACTC, AGGT, and reverse primers with overhangs AAGG, ACTC, AGGT, and TGCA. For more information, refer to the diagram to the right or visit the contribution page on our Wiki.

Using this (and other) BioBricks and the above primers, we carried out PCR reactions to determine if this approach was feasible. The reactions were generally setup as follows: 0.5uL of purified plasmid DNA for BioBrick BBa_K515005 was mixed with 9.5uL ddH20, 1.25uL of each primer (@ a 10uM concentration), and finally with 12.5uL of a 2X Mix for Q5 DNA Polymerase. Reaction conditions were setup as suggested by NEB for Q5 polymerase, typically with an annealing temperature between 51 and 55C. In some trials, 9uL of ddH20 and 0.5ul of DMSO were added.

A depiction of the primer binding, and a representative gel, is below.

T--Alma--universalprimers2019 2.png

PCR products from some reaction combinations were selected and sequenced from the middle towards the end - this way, we can confirm if the overhang and BsaI site were added correctly. VF2 and VR primers were used as controls in the event that some primer pairs gave no or incorrect products.

As you can see in the table below, we had mixed results - some of these PCRs required optimization, and were sensitive to annealing temperature and the speed at which the temperature shifted (evident by comparing the reaction in an old thermocycler versus a new mini8 PCR machine). However, we were able to show that this approach can be viable and does indeed lead to products that should work in a Golden Gate assembly reaction.

T--Alma--universalprimers2019 3.png

Legend: green indicates a successfully confirmed reaction (with the bold cell representing the result corresponding to the representative chromatogram above), light green indicates a sequencing analysis with partial success/confirmation/matching, orange-pink (EtBr color) indicates a PCR reaction that gave the correct product, and purple indicates a reaction that did not work. Empty cells represent untested combinations. *These reactions did not work initially, but were successful at least once (required further optimization).

Team iBowu-China 2021 Contribution

Group: iBowu-China 2021
Author: Rachel Chen
Introduction:

We used this part as a positive comparison for control group in the measurement of green fluorescence. Carried on a pSB1C3 plasmid, this part can effectively produce green fluorescence, together with a constitutive promoter. Our contribution includes

  1. We transformed both DH5a and BL21(DE3) and produced green fluorescence without any addition of induction reagents such as IPTG.
  2. In both cases, the light intensity is much stronger than the background by about 100 fold.

Protocol: The plasmid was obtained from BNDS-China 2021 team. It was then expressed both in E. coli DH5a and E. coli BL21(DE3) with Cm resistance. The bacteria culture was incubated without addition of any iPTG in both cases overnight at 37 degree Celsius. GFP was measured with a microplate reader and OD600 was taken for normalization of the concentration. A control group was also measured for background light intensity where the E. coli BL21(DE3) without this plasmid was cultured. A picture was taken to show the effect of green fluorescence protein expression.
Results:
Figure 1. Green fluorescence protein expressed by E. coli DH5a and BL21(DE3) under visible light and their measured intensity normalized by their concentration (OD600). The third column was the control group where the E. coli BL21(DE3) without this plasmid was used.


The results show this part can produce green fluorescence protein highly effectively. In BL21(DE3), the expression is stronger than in DH5a. The intensity of the GFP light in BL21(DE3) is about 2x the intensity in DH5a. In both cases, the light intensity is much stronger than the background by about 100 fold.


Summary: This part can be used for effective expression of green fluorescence protein. It is an ideal choice for control group, and it can also be engineered to express other sequences.