Difference between revisions of "Part:BBa J04450"

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	''Figure 4. The calibration curve for absorbance of various concentrations silica particles at 660 nm.  ''		''Figure 4. The calibration curve for absorbance of various concentrations silica particles at 660 nm.  ''

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Revision as of 18:44, 19 October 2019

RFP Coding Device

The colonies are clearly red in color under natural light after about 18 hours. Smaller colonies are visibly red under UV. The RFP part does not contain a degradation tag and the RBS is strong.

• LacI sensitive
• CAP sensitive

This part is commonly used, but can fail if the system contains LacI or CAP protein.
(--Meagan 15:39, 23 July 2009 (UTC))

[http://2012.igem.org/Team:TU_Munich Team TU_Munich 2012] improved this part by making it compatible to RFC10 and RFC25 (see: BBa_K801100)
(--VolkerMorath 15:02, 21 October 2012 (UTC))

[http://2013.igem.org/Team:NRP-UEA-Norwich Team NRP-UEA 2013] improved this part by adding a NdeI restriction site before the RFP gene. (see: BBa_K1041000)
(--holusac 20:46, 14 August 2013 (UTC))

[http://2015.igem.org/Team:Warwick Team Warwick 2015] improved this part by analysing the effect of copy number on gene expression.
(--Lcarroll 20:48, 25 September 2015 (UTC))

[http://2016.igem.org/Team:Leiden Team Leiden 2016] contributed to the characterisation of this part by showing equal functionality in simulated microgravity (0g) as in the normal gravity of the Earth.
(--Valentijn 19:38, 19 October 2016 (UTC))

[http://2017.igem.org/Team:UChicago Team UChicago 2017] contributed to this part by improving/changing the documented sequence through mutagenesis to create blunt-end restriction sites for cloning not within the prefix/suffix region (created BBa_K2428000).
(--pzulueta97 21:14, 25 October 2017 (UTC) )

[http://2017.igem.org/Team:Grenoble-Alpes Team Grenoble-Alpes 2017] contributed to the characterisation of this part by testing the time of apparition of fluorescence, in presence of IPTG or not (because the promoter leaks), as well as they contributed to the improvement of this part by using its fluorescence as a detection signal to be able to detect Vibrio Cholerae.
(--NoreenLouis 20:47, 26 October 2017 (UTC) )

[http://2017.igem.org/Team:Kingsborough_NY Team Kingsborough NY 2017] contributed to the characterization of this part by showing decreased fluorescence when expressed either in a higher salt media - such as LB with 3% sodium chloride - or E. coli that lacks tmRNA, the principal component of the cell's ribosome rescue system. View the data on the experience page or [http://2017.igem.org/Team:Kingsborough_NY/RFP visit our Wiki]
(--djcamenares 17:56, 27 October 2017 (UTC) )

[http://2017.igem.org/Team:iTesla-SoundBio Team iTesla SoundBio 2017] contributed to the characterization of this part by analyzing the rate of false positives when using the coloring of transformed colonies as a red/white screen for determining experimental success.
(--gladish 01:26, 28 October 2017 (UTC) )

[http://2018.igem.org/Team:Grenoble-Alpes Team Grenoble-Alpes 2018] contributed to the characterisation of this part by testing the delay before apparition of fluorescence directly after transformation and the intensity of the leak, in three different E. Coli strains.
(--perrine 15:06, 9 October 2018 (UTC) )

[http://2018.igem.org/Team:H14Z1_Hangzhou Team H14Z1_Hangzhou 2018] contributed to the characterisation of this part by testing the fluorescence idensity in different condition(e.g.temperature,medium volume, IPTG concentration) ,in two different E. Coli strains.
(--ericxu 09:32, 17 October 2018 (UTC) )

[http://2018.igem.org/Team:SKLMT-China Team SKLMT-China 2018] improved this part by replacing the induced promoter lacI with the strong constitutive pf-5 promoter PampC.(see: BBa_K2569029)
(--DDY 16:47, 17 October 2018 (UTC) )

[http://2019.igem.org/Team:Grenoble-Alpes Team Grenoble-Alpes 2019] contributed to the characterisation of this part by testing the effect of cAMP on the promoter’s leakages and protein expression in Escherichia coli.
(--pinerol 19:50, 27 September 2019 (UTC) )

Team Lund 2019 contributed to the charazterization of this part by testing the effect of different media composition in different E. coli strains on RFP expression.
(-- ParselTMatts 18:40, 19 October 2019 (UTC) )

Pictures

• 

  BBa_J04450 visualized under non-UV lightbox

• 

  BBa_J04450 visualized under 254nm wavelength UV lightbox

• 

  BBa_J04450 Colonies

• 

  Pantoea agglomerans colonies in chloramphenicol, containing BBa_J04450 in the pSB1C3 backbone

Team ITB_Indonesia 2017: Red color dynamics of cloned Escherichia coli strains in LB broth

Background

In Team ITB_Indonesia 2017 characterization, we found in normal growth/incubation condition (37 ^oC, LB agar) that BBa_J04450-transformed Escherichia coli BL21 colony appear to need longer incubation time (>18 hours) until it clearly shows red color under natural light.

We then investigate whether this phenomenon is influenced by the strain, and we try if there are lac repressor in the system that can be released by inducing the culture with IPTG, hence increasing the expression of mRFP.

Experimental Design

We used three different strains of transformed E. coli (BL21, DH5alpha, and Top10) for this study. They were incubated in LB broth, 37 ^oC, and sampled every 4 hours for 2 days to determine the red color absorbance at 588 nm. The amount of IPTG added for respective treatment is 500 µM.

Result and Findings

• There are no significant differences of mRFP expression in different strains of E. coli (BL21, DH5alpha, Top10)
• There are no significant effects of mRFP increased expression after IPTG induction.
• The red color absorbance under 588 nm wavelength is recorded around 2.5-3 OD units.
• The broth become red in color under natural light around 16-20 hours of incubation time.

==Pictures==

Team INSA-UPS France 2017 : usage in Vibrio harveyi strain engineered by conjugation

In the context of the iGEM INSA-UPS France project 2017, BBa_J04450 was tested in the Vibrio harveyi background. To the best of our knowledge, RFP has never been used in this strain. BBa_J04450 biobrick was cloned in a broad host range plasmid (pBBR1MCS-4) and conjugated into V. harveyi. The protocol of triparental mating can be found here. Its expression has been studied by fluoresence microscopy in Vibrio harveyi .

The microscopy results demonstrated the fonctional production of RFP in Vibrio harveyi, and hence, the functionality of part BBa_J04450 in this background.

IIT Madras 2016's Characterization

Experimentation

This BioBrick was used along with various GFP producing devices to understand the role of RBS and Promoter parts in giving rise to intrinsic noise in E. coli DH5alpha. Expression data for GFP and RFP proteins were obtained using flow cytometry (BD FACS Aria III) at 3hr, 6hr, 9hr and 12hr stage of growth along with cells expressing only GFP, only RFP and none. Cumulative intrinsic and extrinsic noise were measured using modified [http://2016.igem.org/Team:IIT-Madras/Model#Noise_in_Devices| Elowitz formula]. OD600 values for specific growth rate estimation were obtained using Spectrophotometer over an interval of an hour for 12 hours. Given specific growth rates are in it's logarithmic values. This BioBrick can be used to characterize noise and strength of complex devices by cloning this device with given device, which produces a different reporter protein. In graphs, we have R11-B32, R11-B34, J14-B3, J17-B34, R11-B30 and R11-B31 in pSB1A2 plasmid backbone.

[http://2010.igem.org/Team:Groningen Team Groningen 2010] reports the usage of this part as a cloning tool. When ligating any part, or part assembly, into any standard backbone that contains this part, the non-restricted and single-restricted backbones that self-circularize will produce red colonies on rich media plates (we use TY). These undesired transformants can than be avoided in the screening for the correct construct. With this method, the backbone desired for a new construct does not need to be purified from agarose gel to decrease the amount of undesired tranformants caused by ligation of the original part present in the backbone. The amount of incorrect transformants depends, of course, on the ratio of backbone (mixed with J04450) vs. BioBrick insert, the size of the BioBrick insert, and whether the insert is an assembly of two BioBricks. The images below show two ligations with different efficiencies.

Usage in Chromobacterium Violaceum

[http://2016.igem.org/Team:Tec-Monterrey Team Tec-Monterrey 2016] characterized the output of the part BBa_J04450 in a novel chassis, Chromobacterium Violaceum, as it produces a native purple pigment Violacein, we were curious whether RFP would be useful as a reporter gene. Furthermore, we characterized its expression under lac promoter. We did the transformation of C. Violaceum by a method that has not been reported yet, we made C. Violaceum competent cells with the protocol that is in our wiki, we concluded that the best O.D. for the heat shock transformation is 0.5 since it showed clearly better results than 0.4 or 0.6, we will continue to work in the transformation efficiency.

[Image:]

H14Z1_Hangzhous 2018's Characterization

[http://2018.igem.org/Team:H14Z1_Hangzhou Team H14Z1_Hangzhou 2018]

Purpose

Previous experiments had shown that BBa_J04450-transformed E. coli BL21(DE3) would turn red color under natural light after 18 hours due to leaking expression. This part is useful for characterizing the expression of different proteins using the lacI sensitive promoter. For better application of this promoter in controlling protein expression, the effects of different hosts under various conditions were investigated systematically. The target hosts include E. coli BL21(DE3) and E. coli MC1061. The target culture conditions were temperature, inducer concentration and medium volume in the flask.

Experiment design

• Exploring the effect of temperatures on mRFP expression
  1.Inoculate 5 ml LB with monocolony (BL21(DE3)/MC1061) from the plate. Grow the cells overnight at ^oC，200 rpm.
  2.Dilute pre-culture 1:100 into 5ml LB (BL21(DE3)/MC1061) and cultivate at different temperature (26, 30, 34, 37 ^oC), 200 rpm, respectively.
  3.Measure the fluorescence and OD600 every 4 hours.
• Exploring the effect of medium volume on mRFP expression
  1.Inoculate 5 ml LB with monocolony (BL21/MC1061) from the plate.Grow the cells overnight at 37^oC, 200 rpm.
  2.Dilute pre-culture 1:100 into 30ml, 50ml, 70ml LB (BL21(DE3)/MC1061) and cultivate at 37^oC, 200 rpm.
  3.Measure the fluorescence and OD600 every 2 hours.
• Exploring the effect of concentration of IPTG on mRFP expression
  1.Inoculate 5 ml LB with monocolony(BL21(DE3)/MC1061) from the plate. Grow the cells overnight at 37^oC, 200 rpm.
  2.Dilute pre-culture 1:100 into 5ml LB (BL21(DE3)/MC1061), cultivate at 37^oC, 200 rpm. Protein expression are induced using 0.1, 0.3, 0.5, 0.8, 1.0 mM IPTG, respectively.
  3.Measure the fluorescence and OD600 every 4 hours.

Results

• Exploring the effect of temperatures on mRFP expression

There are some differences of growth rate at different temperatures for both E. coli (BL21(DE3), MC1061) (Fig.1). However, the differences of the fluorescence intensity at different temperature is much higher than those of the growth rate at various temperatures by using two E. coli cells (Fig 2). The fluorescence intensity followed same trend in two E. coli (BL21(DE3)/MC1061).The results indicated that the leaking expression was lower in low temperature (e.g.26 and 30^oC) and higher in high temperature. Highest leak appeared in 34 ^oC. This finding is different from our expectation, where we expected that the highest leaking expression would be at 37 ^oC



Figure.1 Effect of different temperature on cell growth



Figure.2 Efforts of different temperature on mRFP expression







• Exploring the effect of effect of medium volume on mRFP expression

The mRFP expression of two strains showed the same trend in different volume of medium in the flask. After 6 hours, the fluorescence intensity in 30 ml medium-containing flask is significantly higher than others, indicating that mRFP express fastest in this volume scale (Fig 3). This suggest that dissolved oxygen is an important factor which will affect the protein expressin and cell growth, since the lower medium volume in the same type flask will bring about higher level of dissolved oxygen in the medium.



Figure.3 Effects of medium volume in the flask on mRFP expression



• Exploring the effect of concentration of IPTG on mRFP expression

Our results showed that significant fluorescence differences were not observed by using different IPTG induction (Fig 3). However, it is interesting that the fluorescence intensity of E. coli BL21(DE3) after IPTG induction are higher than those of E. coli MC1061, not similar to the trend in our previous experiment (Fig 2). It might be explained that the expression of mRFP can be accelerated by IPTG inductin in BL21(DE3) with regard to other E. coli hosts.



Figure.4 Efforts of different IPTG concentration on mRFP expression




Pictures





Figure.5 Efforts of different temperature on mRFP expression





Figure.6 Effects of medium volume in the flask on mRFP expression





Figure.7 Efforts of different IPTG concentration on mRFP expression

SCAU-China 2018's improvement

Team:SCAU-China 2018

The BBa_K2559005 is a amended eGFP coding part improved from BBa_l714891.

Usage and Biology

The part BBa_K2559005 has a sequence improvement on the basic part submitted by iGEM07_Peking (BBa_l714891) which encodes the SDY_eGFP. However, we found out a 16 bp nucleotides redundancy in the eGFP starting coding region in BBa_I714891, after checking the sequence of BBa_I714891 from NCBI. Therefore, we decided to delete the redundant 16 bp nucleotides in BBa_I714891 to amend the length of eGFP coding sequence. The amended eGFP coding biobrick is the BBa_K2559005. To test the function of BBa_K2559005, we designed a new E.coli expression vector containing our new part termed as BBa_K2559003under a strong E.coli endogenous promoter (PrplJ). Therefore, the amended eGFP in BBa_K2559005 was driven by PrplJ promoter, and expressed in DH10B. In addition, we also applied the BBa_K2559005 in the promoter intensity analysis of our other two new parts, the BBa_K2559004 and BBa_K2559011 which are relatively weaker E.coli endogenous promoters (PdapA and PcaiF) (Figure 1).

We summarized that our improvedpart, the amended eGFP coding biobrick BBa_K2559005 worked well in DH10B. We also hoped that our improvement on the BBa_I714891 can help their future applications by other groups in the future. However, it is difficult for us to perform additional experiments with BBa_K2559005 and BBa_I714891 due to the unavailable BBa_I714891.

To expand the application of BBa_K2559005, we searched theBBa_J04450 stored in registry and do another improvement in the BBa_J04450. The BBa_J04450 is a strong RFP expression vector in E.coli. As the main page of BBa_J04450 mentioned, the E.coli colonies with BBa_J04450 were in red color under normal light after about 18 hour culture on LB plate (Figure 2). We used the BBa_K2559005 to replace the RFP region in BBa_J04450, the modified part is BBa_K2559009. We transferred the BBa_K2559009 to DH5α by heat-shock, and found that the fluorescence signal can be observed under the UV (Figure 2).

So, we confirm that our improved part BBa_K2559005 can work in different E.coli expression system. We are also looking forward to more application of the BBa_K2559009! </html>

Grenoble-Alpes 2019 Characterization

The goal was to characterize the effect of cAMP on the promoter’s leakages and overall proteins expression

MAIN RESULTS

DH5α : standard strain with endogenous adenylate cyclase activity.

BTH101 : strain without endogenous adenylate cyclase activity and so without production of cAMP. BTH101 strain is streptomycin resistant.

BTH101 + adenylate cyclase (BTH101-Zip) to generate cAMP in the strain. To restore the adenylate cyclase activity we used two plasmids: one containing pUT18-LeucineZipper (pUT18-LZ) and the other containing pKT25-LeucineZipper (pKT25-LZ).
The Leucine Zipper’s (LZ) will homodimerize and will bring T18 and T25 closer which will restore the adenylate cyclase activity thus allowing the production of cAMP by BTH101. Both T18 and T25 are under the control of an IPTG inducible promoter (lactose promoter) to create an auto amplifier system.
BBa_J04450 was cloned in pKT25-LeucineZipper, and the bacteria was co- transformed with both plasmid T25-LeucineZipper + BBa_J04450 and T18-LeucineZipper.



It is apparent that without IPTG there is a visible difference between strains that can produce cAMP (DH5α and BTH101-Zip) when compared with the BTH101 strain that do not produce any cAMP.
The DH5α red colonies reveal a substantial RFP expression as the result of the leakage of the promoter.
BTH101 has white colonies indicating that no visible RFP was produced. This data suggest that the promoter does not leak in that condition.
BTH101-Zip presents same red colonies than the DH5α strain.



RFP expression by BTH101 with or without cAMP shows that without cAMP, the lactose promoter has almost no leakage suggesting that the leakage of the lactose promoter is due to cAMP in absence of IPTG. When the cAMP production is restored the promoter is activated and the protein is expressed. It is also interesting to note that with the BT101-Zip there are very little protein expression if no IPTG is added.

Team Lund 2019 charecterization

Aim

The aim of this characterization was to investigate the correlation between the different strains and how the varying amounts of salts and sugars in the media stimulate cell growth and protein expression. RFP was incorporated in the construct to compare the protein expression in various conditions. Three different E.coli strains were tested in three different complex media. The strains that were tested: E. coli TG1 E. coli BL21-DE3 E. coli Nissle 1917 (EcN) While the prepared media were: LB Media TB Media Enriched Complex Media The recipe for the three types of media can be found in our protocols.

Method

The plasmid from the registry was successfully transformed using heat shock to each of the mentioned strains. Three 1 L Erlenmeyer flasks containing 250mL of each media were prepared (9 flasks in total). Cultivation was performed at a temperature of 37 °C and 175 rotations per minute (rpm) , during which the growth was monitored spectrophotometrically. The protein expression could be detected optically from the color formation as well as with the use of Agilent Technologies Cary Eclipse Fluorescence Spectrophotometer at excitation wavelength of 532 nm and emission wavelength of 605 nm. For this experiment, a wavelength of 660 nm was used to measure optical cell density (OD) following the suggestion from iGEM headquarters.

The calibration curves for Rhodamine B (dye equivalent of RFP, obtained from Thermo Fisher Scientific - D1841) and for microbeads (equivalent of cells, provided by iGEM headquarters) were used to create a conversion factor for auxiliary fluorescence unit and Abs 660 nm. Two excitation wavelengths have been used 532 nm (recommended for RFP) and 570 nm (recommended for Rhodamine B). The protocol used to make the standard curves was the iGEM protocol “iGEM 2019 Plate Reader Fluorescence Calibration” for RFP.

Results

Standard curve

The emissions for these two excitation wavelengths can be seen in Figure 1 below.

Figure.1 The emission spectra for various concentrations of Rhodamine B (0 to 50μM): a) excitation at 532nm; b) excitation at 570nm.

One can notice that at excitation wavelength of 532 nm, a clear emission peak can be obtained approximately at 590 nm with the increase in signal intensity for each Rhodamine B concentration. While at excitation wavelength of 570 nm there are no clear peaks but the signal increases with the concentration of the fluorescent dye. Therefore, several standard curves have been plotted. The 532 nm/605 nm (ex./em.) was the one that was used for RFP, the 605 nm was a result of fluorescence shift due to presence of RFP in the cells. The 532 nm/590 nm was the setting to check the theoretical emission Rhodamine B at 590 nm using the same excitation as RFP. The last set of wavelengths 570 nm/590 nm was recommended by the manufacturer of Rhodamine B. The results are presented in Figure 2 below.

Figure 2. Rhodamine B standard curves showing mean fluorescence of four replicates of different concentrations of Rhodamine B (0 µM to 50 µM): a) excitation: 532nm, emission: 605nm; b) excitation: 532nm, emission: 590;c) excitation: 570, emission: 590

It can be clearly seen that in all cases there was an oversaturation of detector at concentrations of 12.5 μM and above. Therefore, it was decided that these concentrations should not be used for creation of standard curve and have been excluded. In case of 532/605, the noise is disturbing the measurement at lower concentrations but both 532/590 and 570/590 do not seem to have the same issues.

Figure 3. Rhodamine B standard curves showing mean fluorescence of four replicates of different concentrations of Rhodamine B (0 µM to 6.25 µM – the higher values were excluded due to oversaturation):a) excitation: 532 nm, emission: 605 nm; b) excitation: 532 nm, emission: 590 nm;c) excitation: 570 nm, emission: 590 nm

It can be seen that all the curves show similar linearity. The data from the curves was used to determine a unit scaling factor (μM Rhodamine B/ fluorescence a.u.) that can be found in Table 1, below.

Table 1. The mean μM of Rhodamine B per fluorescence auxiliary unit (a.u.)

Since all the standard curves have similar linearity but the scaling factors are significantly different, it was decided to choose the manufacturers recommendation and use the date from 570nm excitation and 590nm emission, that is 0.23 μM Rhodamine B/a.u.. It was hence used to convert the fluorescence auxiliary units in the characterisation experiment.

The calibration curve for the particles can be seen in Figure 4. below.

Figure 4. The calibration curve for absorbance of various concentrations silica particles at 660 nm.

The calculated unit conversion factor was 1,23E+09 particles/Abs660 and it was used to convert units in characterization experiments.

Characterization results

Figure 5a below shows the growth of EcN in different media in a fraction of time. Enriched Complex Media appears to be the most suitable for the growth of EcN. The lag phase is shorter compared to the other two media, while the logarithmic phase is longer. In TB Media the lag phase is extended, and the growth is not as high as before but still exhibits a clear logarithmic phase. In sharp contrast to that, the lag phase in LB Media is almost double and the exponential phase is quite brief. These results are expected since the media most enriched with salts and sugars are the media with the highest growth rate. The same applies to all strains (Figure 5b and Figure 5c).

Figure 5. The growth of various E. coli strains expressing RFP in different media: a) EcN in LB (blue), TB (orange), EM (grey). b) E. coli BL21-DE3 in LB (blue), TB (orange), EM (grey) c) E. coli TG1 in LB (blue), TB (orange), EM (grey). The OD was converted to equivalent particle count/100μL.

Figure 6 below shows the expression of RFP in different media. In EcN (Figure 6a), the expression of RFP for TB and Enriched Media are comparable, whereas the RFP production in LB is significantly lower. For E. coli BL21-DE3 (Figure 6b), however, the expression levels in TB and LB Media are similar and in Enriched media the RFP production was considerably higher. Finally, in E. coli TG1 (Figure 6c) contrary to the other two strains the Enriched Media gives the lowest RFP levels. The expression in LB media is only slightly higher than in Enriched media. The protein expression in TB Media, however, is notably higher than the other two media, and in comparison to the other strains in the same media. In all three strains, the production of the protein was initiated in LB Media but it could not reach as high values as in the more fortified media, which was expected.

Figure 6. The expression of RFP in various E. coli strains in various cultivation media: a) EcN in LB (blue), TB (orange), EM (grey). b) E. coli BL21-DE3 EcN in LB (blue), TB (orange), EM (grey) c) E. coli TG1 EcN in LB (blue), TB (orange), EM (grey). The OD was converted to equivalent particle count/100μL.

Figure 7 below shows the expression of RFP (converted to Rhodamine B equivalents) per OD (converted to particle/100uL). When it comes to growth, all strains prefered Enriched media. However, it can be noted that each strain has a different preference regarding the RFP expression. EcN produces the most RFP per cell in TB media, BL21 in LB media while in TG1 the expression is favored in both LB and TB media.

Figure 7. The production of RFP per particle count (OD equivalent) for each strain in various media at the end of cultivation: LB (blue), TB (orange), EM (grey).

Figure 8 displays growth of the three strains of E.coli during the characterization of RFP. Overall, the darkest red color was from cells grown in TB media. This was where the protein expression was higher. The other two media, complex Enriched media and LB media both gave a quite pale pink color and low protein expression, with one exception of the BL21-DE3 cells which gave a dark red color and high protein expression. For BL21-DE3 cells, the complex enriched media showed the darkest color. For TG1 cells the TB media gave the darkest color. For EcN cells the TB media also gave the darkest color. These correspond to the results presented in Figure 7.

Figure 8. Growth of three strains of E.coli cells transformed with RFP plasmid BBa_J04450. The cells were grown in different media. The three flasks on the left were grown in TB media, and the strains were from left to right; BL21-DE3, TG1, EcN. The three flasks in the middle were grown in complex Enriched media, and the strains were from left to right; BL21-DE3, TG1, EcN. The three flasks on the right were grown in LB media, and the strains were from left to right; BL21-DE3, TG1, EcN.

Usage and Biology

Using cAMP depleted strains and T18-leucineZipper/T25-leucineZipper under the control of a lactose promoter is a good alternative to produce inducible proteins without leakage