Part:BBa_M10222
{Pbad.rbs.prepro.StrepTag}{˂GS5-IILK˃}{˂espP(beta)!}{dblTerm}
GS5-IILK passenger part attached to a espP(beta) displayer
Strepavidin-Binding Assay
Goals
1) To measure for the ability of the 16 display constructs to bind Strepavidin on the cell surface
2) To devise a method for quantifying the relative amount of Strepavidin bound by the constructsThe 16 Constructs Tested
M10210 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPG_L6!}{dblTerm} M10211 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<eCPX!}{dblTerm} M10212 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<upaG_short!}{dblTerm} M10213 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<Ag43_short!}{dblTerm} M10214 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<espP(beta)!}{dblTerm} M10215 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<ehaB!]{dblTerm} M10216 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPompX!}{dblTerm} M10217 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<TshA!}{dblTerm} ------------------------------------------------------------------------ M10218 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<CPG_L6!}{dblTerm} M10219 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<eCPX!}{dblTerm} M10220 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<upaG_short!}{dblTerm} M10221 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<Ag43_short!}{dblTerm} M10222 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<espP(beta)!}{dblTerm} M10223 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<ehaB!]{dblTerm} M10224 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<CPompX!}{dblTerm} M10225 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<TshA!}{dblTerm}Controls
1)pBca9145-Bca9494 {AraC-Pbad}{rbs.cpx} (positive control that displays cpx, a streptavidin binding peptide under Pbad) 2)DH10B (no plasmid, negative control) 3)pBca9495CA-Bca1144 {Ptet}{rbs1}{mRFP-3*}{b0015} (negative control: same vector as the other constructs with a part that does not bind Streptavidin)Procedure
Transforming and Plating
1) In a 96-well PCR plate, add to wells a mixture of 220uL competent cells, 30ul KCM salts, and 50 uL ddH2O. 2) Add 1uL of a construct to each well.
2) Incubate for 10' on ice, heat shock at 42C for 1.5', cool for another 2', and then add 90uL of LB media. Cover and shake for 15' at 37C.
3) Plate on chloramphenacol and incubate at 37C for 24h.Inoculating
1) For each construct, pick 1 colony and inoculate in 4 mL of appropriate antibiotic media (CA in most cases), w/ or w/o arabinose (1:1000), in a 24 well block.
2) Shake at 37C for 16-20h.Assaying Strepavidin Binding: First Try
1) Prefill wells in a clean 96-well skirted plate with 300uL PBS, and add 25uL of saturated culture of each construct.
2) Add 15uL Strepavidin-Phycoerythrin to each well and incubate at 37C without shaking for 30min to 1 hour.
3) Spin down the cells at 3,500 RPM for 5 min and note which pellets appear red in normal light and bright white under UV light (have bound streptavidin).
4) Decant and resuspend cells in 150uL, transfer to a microtiter plate, and measure transmittance at 575nm using 488nm excitation (phycoerythrin setting).Assaying Strepavidin Binding: Second Try
1) Spin down 600uL of saturated culture at 5,500 RPM for 5 min in a 96-well skirted plate.
2) Remove media and resuspend in 300uL of PBS.
3) Add 1uL Strepavidin-Phycoerythrin to each well and incubate at 37C without shaking for 30min to 1 hour.
4) Spin down the cells at 5,500 RPM for 5 min and note which pellets appear red in normal light and bright white under UV light (have bound streptavidin).
5) Decant and resuspend cells in 150uL, transfer to a microtiter plate, and measure transmittance at 575nm using 488nm excitation (phycoerythrin setting).Assaying Strepavidin Binding: Third Try
1) Spin down 200uL of saturated culture at 5,500 RPM for 5 min in a 96-well block.
2) Remove media and resuspend in 200uL of PBS.
3) Add 1uL Strepavidin-Phycoerythrin to each well and incubate at 37C without shaking for 30min to 1 hour.
4) Spin down the cells at 5,500 RPM for 5 min and note which pellets appear red in normal light and bright white under UV light (have bound streptavidin).
5) Transfer the supernatant to a microtiter plate to measure how much streptavidin was pulled out of solution by the cells (by measuring transmittance at 575nm using 488nm excitation (phycoerythrin setting)).Assaying Streptavidin Binding: Fourth Try (some quantitative data obtained)
1) In a 96-well PCR plate, add 100uL of PBS and 10uL of saturated culture of each sample (four replicates of each).
2) Add concentrations of 0.5uL, 1uL, 2uL, or 5uL of Streptavidin-phycoerythrin to each replicate.
3) Incubate at 37C without shaking for 30 minutes to 1 hour.
4) Spin down the plate at 5,500rpm for 5 minutes.
5) Use the Tecan to measure fluorescence from the top of the plate (we did depths of 5,100um and 10,100um). 6) Normalize fluorescence to OD measurements of each sample. For accuracy, measure the optical density at 600nm (OD600) of a 10X dilution of the saturated culture and then calculate the actual OD600 of the culture.Results
First Try
Visually, determined that the following four constructs bound to streptavidin (red color in the cell pellet as well as fluorescence under UV). The positive control (pBca9145-Bca9494) and the following 4 constructs bound to Streptavidin:
M10219 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<eCPX!}{dblTerm} M10220 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<upaG_short!}{dblTerm} M10222 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<espP(beta)!}{dblTerm} M10223 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<ehaB!]{dblTerm}After we spun down the cells and resuspended in PBS, we had lost so many of the cells that we were unable to get a meaningful measurement using the Tecan.
Second Try
The same four constructs and positive control showed binding when examined visually. However, when we tried to quantify, the light scattering from the excessive number of cells interfered with the measurement and we got no meaningful data.
Third Try
The same four constructs and positive control showed binding when examined visually. However, we were unable to get a firm enough pellet to remove the supernatant without disturbing the pellet.
Fourth Try: Quantitative Data
We measured fluorescence of the supernatant at two different depths (5,100um and 10,100um). There were higher measurements of fluorescence at lower depths because the unbound streptavidin appears to concentrate lower in the wells after they have been spun down (the average difference in fluorescence intensity at the two depths was 367 for 0.5uL streptavidin and 638 for 1uL streptavidin). We only took measurements for samples that were incubated with 0.5uL or 1uL of streptavidin because higher amounts of streptavidin showed a clear visual gradient of the dye in solution.
The results shown below are an average of the fluorescence measurements obtained at 5,100um and 10,100um. We normalized the values to OD600 measurements of the saturated samples.
M10219 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<eCPX!}{dblTerm} M10220 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<upaG_short!}{dblTerm} M10222 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<espP(beta)!}{dblTerm} M10223 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<ehaB!]{dblTerm} pBca9145-Bca9494 {AraC-Pbad}{rbs.cpx} (positive control) pBca9495CA-Bca1144 {Ptet}{rbs1}{mRFP-3*}{b0015} (negative control)For some undetermined reason the positive control did not work on the fourth try (we could not see red color/fluorescence even when we visually observed it and the quantitative results are comparable with the negative control). In this experimental trial, we visually saw binding for only 3 of the constructs: M10220, M10222, and M10223.
Analysis
The values that we obtained from the quantitative fluorescence measurements were somewhat unexpected. We were unable to explain why the fluorescence intensity measurements at 0.5uL and 1uL for these constructs were not significantly lower than the corresponding measurements for the other constructs. We would have expected such a result because increased binding should have resulted in decreased streptaviding (and fluorescence) in the supernatant that we measured. We feel that the measurements might be erroneous because of the bleed through of fluorescence from the pelleted cells.
We found it difficult to find any significance in the fluorescence intensity measurements unless we looked at the comparison between the values obtained at the two different arabinose concentrations. The three constructs that appeared to be binding streptavidin successfully (M10220, M10222, M10223), all had higher fluorescence intensity in the supernatant with 0.5uL streptavidin than they did with 1uL streptavidin. This seems to be because these constructs are binding to more streptavidin (and thereby, pulling more streptavidin out of the supernatant) as the amount of streptavidin increases. It was visually inconclusive whether or not M10219 had bound streptavidin and the quantitative results for it indicate that it more similar to the constructs that are not binding streptavidin (where the fluorescence intensity of the supernatant increases in the presence of more streptavidin).
The inconsistencies in the data (the unexplained lack of binding of the positive control in the fourth try) as well as the unexpectedly high fluorescence measurements obtained for the supernatant of M10220, M10222, and M10223 warrant further considerations. The best protocol for this experimented still needs to be determined because we were not able to get the expected quantitative data even after four tries. Better data can probably be obtained if we can find a way to separate the pellet and the supernatant without disturbing the pellet or using an excessively high amount of cells.
Growth Curve Assay
The purpose of this assay is to determine the toxicity of various composite constructs. These constructs include:
M10210 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPG_L6!}{dblTerm} M10211 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<eCPX!}{dblTerm} M10212 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<upaG_short!}{dblTerm} M10213 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<Ag43_short!}{dblTerm} M10214 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<espP(beta)!}{dblTerm} M10215 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<ehaB!]{dblTerm} M10216 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPompX!}{dblTerm} M10217 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<TshA!}{dblTerm} M10218 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<CPG_L6!}{dblTerm} M10219 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<eCPX!}{dblTerm} M10220 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<upaG_short!}{dblTerm} M10221 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<Ag43_short!}{dblTerm} M10222 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<espP(beta)!}{dblTerm} M10223 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<ehaB!]{dblTerm} M10224 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<CPompX!}{dblTerm} M10225 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<TshA!}{dblTerm}With all the constructs under an arabinose promoter, toxicity can be inferred from the differences in growth rate (as determined by changes in OD 600 over time) between samples containing LB and LB+arabinose.
Procedures
Plating
The controls are: DH10B and pBca9495CA-Bca1144 (as controls, the DH10B is blank control and pBca9495CA-Bca1144 controls for plasmid effects.)
- Take 2 tubes of 280ul of cell and add 60ul KCM and 100ul water to each.
- Add 20ul of the KCM/water/cell solution into each construct.
- Transform (see Transformation section below)
- Grow plates overnight.
- Pick 5 colonies from each sample.
- Grow to saturation in 96 well blocks with 400 uL LB media in each well.
- Then from each of the 5 unique liquid cultures, make an arabinose sample and a non-arabinose sample.
- Add 50 uL of LB media or LB Media+100ug/mL arabinose per well in 384 well plate.
- Add 1 uL of cell sample to each well.
- Place plate in Tecan and run OD measurements every 10 minutes.
Transformation
- Thaw 2 tubes of 280 uL aliquot of cells on ice
- Add 100 uL of water to each tube
- Add 60 uL of KCM salts to each tube
- Add 1ul of the constructs to 20 uL of the cell cocktail. Pipette up and down gently to mix
- Let sit on ice for 10 min
- Heat shock for 2 min at 42
- Put back on ice for 1 min
- Add 100uL of LB, let shake in the 37 degree incubator for 40 min
- Plate on chloramphenicol/ampicillin selective plates, let incubate overnight
Results
Construct 11 --> M10210 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPG_L6!}{dblTerm}
Construct 12 --> M10211 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<eCPX!}{dblTerm}
Construct 13 --> M10212 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<upaG_short!}{dblTerm}
Construct 14 --> M10213 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<Ag43_short!}{dblTerm}
Construct 15 --> M10214 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<espP(beta)!}{dblTerm
Construct 16 --> M10215 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<ehaB!]{dblTerm}
Construct 17 --> M10216 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPompX!}{dblTerm}
Construct 18 --> M10217 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<TshA!}{dblTerm}
Construct 19 --> M10218 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<CPG_L6!}{dblTerm}
Construct 20 --> M10219 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<eCPX!}{dblTerm}
Construct 21 --> M10220 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<upaG_short!}{dblTerm}
Construct 22 --> M10221 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<Ag43_short!}{dblTerm}
Construct 23 --> M10222 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<espP(beta)!}{dblTerm}
Construct 24 --> M10223 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<ehaB!]{dblTerm}
Construct 25 --> M10224 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<CPompX!}{dblTerm}
Construct 26 --> M10225 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<TshA!}{dblTerm}
Controls are DH10B and pBca9495CA-Bca1144
http://openwetware.org/images/0/09/Bioe140L-GrowthPlotsConstr.pdf
http://openwetware.org/wiki/Image:Bioe140L-GrowthPlotsConstr.pdf
Analysis
Construct 11 --> M10210 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPG_L6!}{dblTerm}
Construct 12 --> M10211 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<eCPX!}{dblTerm}
Construct 13 --> M10212 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<upaG_short!}{dblTerm}
Construct 14 --> M10213 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<Ag43_short!}{dblTerm}
Construct 15 --> M10214 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<espP(beta)!}{dblTerm
Construct 16 --> M10215 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<ehaB!]{dblTerm}
Construct 17 --> M10216 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPompX!}{dblTerm}
Construct 18 --> M10217 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<TshA!}{dblTerm}
Construct 19 --> M10218 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<CPG_L6!}{dblTerm}
Construct 20 --> M10219 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<eCPX!}{dblTerm}
Construct 21 --> M10220 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<upaG_short!}{dblTerm}
Construct 22 --> M10221 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<Ag43_short!}{dblTerm}
Construct 23 --> M10222 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<espP(beta)!}{dblTerm}
Construct 24 --> M10223 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<ehaB!]{dblTerm}
Construct 25 --> M10224 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<CPompX!}{dblTerm}
Construct 26 --> M10225 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<TshA!}{dblTerm}
Controls are DH10B and pBca9495CA-Bca1144
http://openwetware.org/images/5/5f/BioE140L-GrowthRatesConstr.pdf
http://openwetware.org/wiki/Image:BioE140L-GrowthRatesConstr.pdf
http://openwetware.org/images/4/4b/BioE140L-LogFit.pdf
http://openwetware.org/wiki/Image:BioE140L-LogFit.pdf
Autoaggregation: Cell-Cell Adhesion Assay
The purpose of this assay was to determine the ability of recombinant E.coli to aggregate when expressing IILK (leucine zipper) peptides on their outer membranes. The IILK peptide was displayed on the outer membrane via fusion to different displayer peptides. This assay tested the efficacy of the carrier proteins as fusions to functional IILK leucine zippers.
The following composite parts were tested during this assay: M10218 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<CPG_L6!}{dblTerm} M10219 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<eCPX!}{dblTerm} M10220 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<upaG_short!}{dblTerm} M10221 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<Ag43_short!}{dblTerm} M10222 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<espP(beta)!}{dblTerm} M10223 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<ehaB!]{dblTerm} M10224 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<CPompX!}{dblTerm} M10225 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<TshA!}{dblTerm}Procedure
1. Transform DH10B cells with the following plasmids:
-- 1. the IILK test plasmids: pBca9495CA-(M10218-M10225)
-- 2. the AG4 negative controls: pBca9495CA-(M10210-M10217)
-- 3. the Pbad negative control: pBca9495CA-Bca1363
2. Pick 2 colonies per plasmid and grow to saturation in 3mL LB in shaker at 37°C (12 hours)
-- use 24 well block
3a. Dilute the saturated culture 10-fold (100uL saturated cell culture into 1mL LB)
-- Do two replicates of each clone into a 96-well block that will go into the shaker at 37°C
-- Add Arabinose (100ug/mL) to one replicate per clone
Replicates were grown up without arabinose to test for flocculating activity due to basal levels of protein expression. 3b. Seed 2 replicates of each clone (10uL saturated cell culture into 100uL LB) into a V-bottom plate
-- Put plate in incubator at 37°C 6. Grow everything to saturation (12 hours)
7. Look for floculation in the V-bottom plate
7. Take OD measurements (600nm and 660nm) of cells grown in 96-well block
-- Blank the spectrophotometer with LB (or LB with arabinose)
-- Measure OD600 and 660 of shaken cells
-- When taking measurements, pipette 1mL out of 96-well blocks , then pipette each sample up and down 5 times in-order to ensure uniform mixing of each sample for an accurate OD measurements
Cell cultures with more autoaggregation will have less turbidity (lower OD measurements) 8. Flocculating activity = (1− A/B) × 100 (%)
-- where A is the turbidity measured of a sample and B is that of a control.
--Taniguchi et al
Results
V-bottom plates
Saw no clear evidence of flocculation in the V-bottom plates. We expected E.coli to remain suspended in the LB unless they autoaggregated, in which case they would clump at the bottom of the V-bottom plates because clumps of cells are heavier than individual cells. However, all of the E.coli settled at the bottom of the V-bottom plates (even the controls) making it impossible to distinguish a flocculating phenotype.
OD measurements
Flocculating Activity was calculated using the OD600 readings and the equation fromTaniguchi et al.
We defined A as the OD600 measurement of a "sample" (M10218-M10225). -- Samples were constructs with carrier proteins that had the IILK peptide fused to their N-termini. We defined B as the OD600 measurement of a control (M10210-M10217). -- Controls had the same carrier peptides as the samples ({CPG_L6!}, {<eCPX!}, ect.), but they displayed a different peptide ({<AG4>} instead of {<GS5-IILK>}.Here is a chart of Flocculating activity with and without arabinose:
While pipetting the saturated cell culture from the 96-well blocks into the spectrophotometer cuvettes, we made the following observations:
WITH ARABINOSE: M10218 {<CPG_L6!} saw one very large clump M10219 {eCPX!} M10220 {upaG_short!} many small clumps of cells that settled out of solution most in the 96-well block M10221 {Ag43_short!} very small faint clump M10222 {espP(beta)!} M10223 {<ehaB!} many small clumps M10224 {CPompX!} few clumps M10225 {TshA!} one small clump *note: no visible clumps were seen for the "controls"WITHOUT ARABINOSE: *no visible clumps were seen in the "samples" or "controls"
Analysis
The best carrier peptides for display of Leucine Zipper peptides are: {<CPG_L6!}, {<ipaG_short!}, and {<ehaB!}.
Basal levels of protein expression are not enough to see an autoaggregation phenotype.
In the future, the saturated cell cultures should also be mixed a few times before being pipetted into the cuvettes in order to ensure that all the cells (even those that have settled at the bottom of the 96-well block) are re-suspended and part of the OD measurements.
References
1. Kjærgaard, K., Schembri, M.A., Hasman, H., Klemm, P., Antigen 43 from Escherichia coli Induces Inter- and Intraspecies Cell Aggregation and Changes in Colony Morphology of Pseudomonas fluorescens, J Bacteriol.(2000) 182: 4789–4796.
2. Taniguchi, M., Katoa, K., Matsuia, O., Pinga, X., Nakayamaa, H., Usukia, Y., Ichimuraa, A., Fujitaa, K., Tanakaa, T., Taruia, Y., Hirasawaa, E., Flocculating activity of cross-linked poly-γ-glutamic acid against bentonite and Escherichia coli suspension pretreated with FeCl3 and its interaction with Fe3+, J. of Biosci. & Bioeng. (2005) 100:207-211.
3. Veiga, E., Lorenzo, V., Fernández, L.A., Autotransporters as Scaffolds for Novel Bacterial Adhesins: Surface Properties of Escherichia coli Cells Displaying Jun/Fos Dimerization Domains, J Bacteriol. (2003) 185: 5585–5590.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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