DOI: 10.6084/m9.figshare.24940631
title: Additional file 1 of A novel in-situ-process technique constructs whole circular cpDNA library

description: Additional file 1: Figure S1. (a) Sample of crop leaves. Using Hydroponics with the repeated-short time scale of lighting, reduced the carbohydrate content of the leaves. Respectively, the labels (1 to 4) are Barley (3–4 leaf stage), Wheat (3–4 leaf stage), Maize (V2–V3 stage), and Soybean (V3–V4 stage). The yellow and white arrowheads represent younger (V2 stage) and older (V6 stage) age groups. The soybean older leaves (white line), before and after the V6 stage, the leaves have turned yellow and entered the leaf senescence development. Whereas, under natural conditions, the V6 stage of soybean is at the beginning of vegetative growth. (b) Chloroplast images. Soybean cell and chloroplast are distinguished by labels 1 and 2, respectively. The black arrowheads denote the cell, and the red arrowheads denote the chloroplast. Left bar = 20 μm, right bar = 50 μm. Figure S2. (a) Circular cpDNA extraction. The remaining gel (Lane 1, 2 and 3, red arrowhead) was stained with ethidium bromide before reconstructing the integrated gel to cut the elutable gel slices that contained the unstained circular cpDNA (Lane 4, 5 and 6). Subjecting to PFGE using the following procedure: Reverse Voltage Gradient: 4.5 V/cm, Int. Sw. Tm: 60 s, Fin. Sw. Tm:90 s, Included angle: 120°, Run Time: 18 h, Ramping factor: a=: ENTER. The HMW lambda ladder loaded into the far left. Attention circular DNA size cannot be marked up with a linearized DNA ladder because of the circular topology properties, whereas it can serve as a relative reference standard. (b) Recovering the circular cpDNA from gel slices using the D-tube Dialyzer. A supporting tray containing the unstained gel slice is built by placing the unstained gel slice into the D-Tube dialyzer. Filling the D-tube dialyzer with 5 mM Tris-HCl running buffer to the top of membranes. Avoid introducing air bubbles in the tube, as they interfere with electroelution. The optimum electroelution time must be determined according to the sample and gel concentration. (c) The mono-restriction endonuclease. SrfI, BssHII, NaeI and SacII are screened to digest the circular cpDNA of Wheat (Label 1), Maize (Label 2), Soybean (Label 3) and Barley (Label 4), respectively. End-blunting the cpDNA fragments by the Klenow Fragment when the end of the fragments is a cohesive end. The redly label numbers are marked with the enzyme-digested products. (d) Electroporation of vector/insert recombinant DNA. Transform the ligate into E. coil (DH10B) by electrotransformation. A respectable amount of recombinant clones per culture dish is achieved with the circular cpDNA from Wheat (Label 1), Maize (Label 2), Soybean (Label 3) and Barley (Label 4), respectively. Figure S3. Illustration of the mono-restriction endonuclease selection from Wheat chloroplast Ref-genome. Figure S4. Illustration of the mono-restriction endonuclease selection from Maize chloroplast Ref-genome. Figure S5. Illustration of the mono-restriction endonuclease selection from Soybean chloroplast Ref-genome. Figure S6. Illustration of the mono-restriction endonuclease selection from Barley chloroplast Ref-genome. Figure S7. Illustration of the wheat circular cpDNA library. To create a vector/insert recombinant DNA, a cloneable wheat cpDNA fragment of the desired size is introduced into the modifying vector. Figure S8. Illustration of the maize circular cpDNA library. To create a vector/insert recombinant DNA, a cloneable maize cpDNA fragment of the desired size is introduced into the modifying vector. Figure S9. Illustrations of the barley circular cpDNA library. To create a vector/insert recombinant DNA, a cloneable barley cpDNA fragment of the desired size is introduced into the modifying vector. Figure S10. Lysate efficacy of the old lysate in the In-gel chloroplast. The reagents and the steps, which are unaltered and quoted from the in-situ bacterial lysis method, are used to lyse the In-gel chloroplast (soybean). Labels 1, 2, and 3 are three repetitions. Subjecting to PFGE using the following procedure: Reverse Voltage Gradient: 6 V/cm, Int. Sw. Tm: 60 s, Fin. Sw. Tm: 90 s, included angle: 120°, Run Time: 18 h, Ramping factor: a=: ENTER. The HMW lambda ladder loaded into the far left. Figure S11. Comparison of whole circular cpDNA extraction methods conventional technique and in-situ-process technique. (a) Discontinuous sucrose gradient is prepared for chloroplast purification. The upper layer of Label 1 is soybean crude chloroplast, and the lower layer of Label 1 is discontinuous sucrose density gradients (8%, 25%, 45%, 60%, and 80%). (b) Purification of crude chloroplast. The intact chloroplasts approach the equilibrium position in the lower band (Label 3), whereas non-target pollutants in the label 1 and damaged chloroplat in the label 2. Label 1 is the larger non-target pollutants like surviving leaves tissue, intact cells, and other debris. Centrifugal condition: 10,000×g/60 min/4 °C. (c) Detecting the extracting effect of circular cpDNA by PFGE-gel. The soybean circular cpDNA from the in-situ chloroplast lysis technique cannot be separated as discrete bands under PGFE conditions, showing the target band (line 2) in PFGE-gel. Whereas the conventional technique has separated as discrete bands (line 1) under PGFE. PFGE conditions: Reverse Voltage Gradient: 6 V/cm, Int. Sw. Tm: 1 s, Fin. Sw. Tm: 20 s, included angle: 120°, Run Time: 14 h, Ramping factor: a=: ENTER). line NA, not available. The HMW lambda ladders are loaded into the first and second on the left corner.