Genetic manipulations of nonmodel gut microbes
非模型肠道微生物的基因操作

Materials and devices  材料与设备

Primer star DNA polymerase (Takara, Cat# R045), Blue sapphire DNA polymerase (Takara, Cat# RR350), Plasmid Midiprep Kit (Zymo Research, Cat# D4201), DNA Clean and Concentrator (Zymo Research, Cat# D4003), Tryptic Soy Agar (BD, Cat# 236950), Brain Heart Infusion Agar (BD, Cat# 241830), Columbia Blood Agar (CBA) (BD, Cat# 279240), Horse blood (Hemostat Laboratories, Cat# 637291), Luria–Bertani (LB) broth (BD, Cat# BP1426), glycerol (Fisher Bioreagents, Cat# BP229), the vacuum‐pumping system, phosphate‐buffered saline (PBS) (Gibco, Cat# 10010‐031), centrifuge, polymerase chain reaction (PCR) amplifier, d‐cycloserine (D) (TCI, Cat# C1189), gentamicin (G) (GoldBio, Cat# G‐400‐25), kanamycin (K) (GoldBio, Cat# K‐120‐25), carbenicillin (GoldBio, Cat# C‐103‐25), thiamphenicol (Thiam) (Acros Organics, Cat# 455450250), anaerobic chamber, aerobic incubator, electroporation system, Thermo Scientific Nanodrop 2000, Gibson Assembly Cloning Kit (NEB, Cat# E5510S), Quick DNA fungal/bacterial kit (Zymo Research, Cat# D6005), 50 mL Tube Top Vacuum Filter System (0.22 mm) (Corning Life Sciences, Cat. #430320), and ultralow temperature freezer.
Primer star DNA 聚合酶（Takara，Cat# R045）、Blue sapphire DNA 聚合酶（Takara，Cat# RR350）、质粒中量提取试剂盒（Zymo Research，Cat# D4201）、DNA 净化浓缩液（Zymo Research，Cat# D4003）、胰蛋白酶大豆琼脂（BD，Cat# 236950）、脑心浸液琼脂（BD，Cat# 241830）、哥伦比亚血琼脂 (CBA) (BD，Cat# 279240)、马血（Hemostat Laboratories，Cat# 637291）、Luria–Bertani (LB) 肉汤（BD，Cat# BP1426）、甘油（Fisher Bioreagents，Cat# BP229）、真空抽吸系统、磷酸盐缓冲液 (PBS)（Gibco，Cat# 10010-031）。离心机、聚合酶链式反应 (PCR) 放大器、 d-环丝氨酸 (D) (TCI, Cat# C1189)、庆大霉素 (G) (GoldBio, Cat# G-400-25)、卡那霉素 (K) (GoldBio, Cat# K-120-25)、羧苄西林 (GoldBio, Cat# C-103-25)、甲砜霉素 (Thiam) (Acros Organics, Cat# 455450250)、厌氧室、好氧培养箱、电穿孔系统、Thermo Scientific Nanodrop 2000、Gibson Assembly Cloning Kit (NEB, Cat# E5510S)、Quick DNA 真菌/细菌试剂盒 (Zymo Research, Cat# D6005)、50 mL Tube Top Vacuum Filter System (0.22 mm) (Corning Life Sciences, Cat. #430320) 和超低低温冷冻机。

Screening of culture conditions
培养条件的筛选

The culture of Gram‐negative Bacteroidia strains was incubated in an anaerobic chamber at 37°C under an atmosphere of 5% carbon dioxide (CO₂), 7.5% hydrogen (H₂), and 87.5% nitrogen (N₂). The agar plates were left in the anaerobic chamber for at least one overnight before use. The liquid medium was left in the chamber with a loosened cap for at least 48 h before inoculation.
将革兰氏阴性拟杆菌菌株培养物置于厌氧箱中，在 37°C、5%二氧化碳( _CO2 )、7.5%氢气( _H2 )和 87.5%氮气( _N2 )的环境下培养。琼脂平板在使用前至少在厌氧箱中放置一夜。液体培养基在接种前，将盖子打开的培养箱中放置至少 48 小时。

Strains were restreaked (from original glycerol stock) onto pre‐reduced agar plates (such as Tryptic Soy Agar + 5% blood [TSAB] plates, Brain Heart Infusion Agar + 5% blood [BHIB] plates, or CBA plates). Then, those that can grow on agar plates were subcultured into pre‐reduced liquid medium: Mega, Chopped Meat Medium (CMM), and Reinforced Clostridial Medium (RCM, BD, Cat# 218081). Strains that can grow in any of the four liquid cultures were subjected to the antibiotics test (Figure 1A).
将菌株（从原始甘油原液中）重新划线接种到预还原的琼脂平板上（例如胰蛋白酶大豆琼脂+5%血液[TSAB]平板、脑心浸液琼脂+5%血液[BHIB]平板或 CBA 平板）。然后，将能够在琼脂平板上生长的菌株传代培养到预还原的液体培养基中：Mega 培养基、碎肉培养基(CMM)和强化梭菌培养基(RCM, BD, Cat# 218081)。对能够在四种液体培养基中生长的菌株进行抗生素测试（图 1A ）。

Keynotes: For the screening of the culture conditions of liquid medium, Bacteroidia strains need to be first recovered on agar plates to ensure Bacteroidia strains are activated, instead of inoculating Bacteroidia strains into liquid medium from the frozen glycerol stock directly.
要点 ：液体培养基培养条件的筛选，需要先将拟杆菌菌株在琼脂平板上回收，以确保拟杆菌菌株被激活，而不是直接从冷冻的甘油原液中将拟杆菌菌株接种到液体培养基中。

Potential issues and solutions: If the Bacteroidia strains of interest cannot grow on the common agar plates or in the liquid medium listed above, other specific plates or liquid mediums that favor the growth of the target strains need to be used to screen the culture conditions.
潜在问题和解决方案 ：如果目标拟杆菌菌株不能在普通琼脂平板或上述液体培养基中生长，则需要使用有利于目标菌株生长的其他特定平板或液体培养基来筛选培养条件。

Antibiotic test  抗生素测试

To find the antibiotic that suppresses the growth of conjugation donor E. coli S17, the Bacteroidia (Bacteroides and Prevotella) microbes were restreaked on agar plates supplemented with 200 µg/mL gentamicin (G) or 250 µg/mL d‐cycloserine (D). The tested Bacteroidia microbes are expected to grow on plates with either gentamicin or d‐cycloserine. Most of the Bacteroidia strains we tested so far are sensitive to thiamphenicol (Thiam), so the thiamphenicol‐resistant gene (catP) can be used as a universal marker to select transconjugants whose genome was integrated by the suicide vector. The minimum inhibitory concentrations (MICs) of thiamphenicol of the Bacteroidia microbes were tested on agar plates containing thiamphenicol at different concentrations (Figure 1A).
为了找到抑制结合供体大肠杆菌 S17 生长的抗生素，将拟杆菌属 （ 拟杆菌属和普雷沃氏菌属 ）微生物重新划线接种在添加了 200 µg/mL 庆大霉素 (G) 或 250 µg/mL d- 环丝氨酸 (D) 的琼脂平板上。测试的拟杆菌属微生物预计会在含有庆大霉素或 d- 环丝氨酸的平板上生长。到目前为止，我们测试的大多数拟杆菌属菌株对甲砜霉素 (Thiam) 敏感，因此甲砜霉素抗性基因 ( catP ) 可用作通用标记，以选择基因组由自杀载体整合的转接合子。在含有不同浓度甲砜霉素的琼脂平板上测试了拟杆菌属微生物对甲砜霉素的最低抑菌浓度 (MIC)（图 1A ）。

Keynotes: Antibiotics need to be added into the agar medium before the agar plates are poured and solidified; agar plates supplemented with antibiotics only on the surface of the plates may cause misleading antibiotic test results.
要点 ：琼脂培养基需要在琼脂平板灌注固化前添加抗生素；仅在平板表面添加抗生素的琼脂平板可能会造成抗生素检测结果的误导。

Potential issues and solutions: If the Bacteroidia strains of interest are not resistant against gentamicin or d‐cycloserine, other specific antibiotics that can suppress the growth of E. coli S17 can be used for the test. Likewise, if the Bacteroidia strains of interest are resistant against thiamphenicol, the antibiotic marker on the suicide vector can be replaced by other markers, whose corresponding antibiotics can suppress the growth of the Bacteroidia strains.
潜在问题及解决方案 ：如果目标拟杆菌菌株对庆大霉素或 d- 环丝氨酸不耐药，则可以使用其他能够抑制大肠杆菌 S17 生长的特异性抗生素进行测试。同样，如果目标拟杆菌菌株对甲砜霉素耐药，则自杀载体上的抗生素标记可以用其他标记替换，这些标记对应的抗生素可以抑制拟杆菌菌株的生长。

Vector assembly  向量组装

By assembling the synthesized conserved ~1 kb chi‐16s rRNA sequence (chi‐16s) for Bacteroidia with the suicide vector pExchange [21], plasmids pGM‐NACB (targeting 16s rRNA gene of Bacteroides, GM: genetic manipulation, N: no gram‐positive replication origin, A: R6K gram‐negative replication origin, C: catP antibiotic marker, B: Bacteroides) and pGM‐NACP (targeting 16s rRNA gene of Prevotella, P: Prevotella) were generated [2] (Figure 1A). Likewise, to target other specific genes in Bacteroidia strains, an ~1 kb fragment of the target gene could be amplified by PCR and assembled with the backbone amplified from the suicide vector pGM‐NACB/P to get the plasmid for mutating the target gene via insertion–deletion (Figure 1A).
通过将合成的保守的约 1 kb chi‐16s rRNA 序列 (chi‐16s) 与自杀载体 pExchange [ 21 ] 进行组装， 生成质粒 pGM‐NACB（靶向拟杆菌 16s rRNA 基因，GM：基因操作，N：无革兰氏阳性菌复制起点，A：R6K 革兰氏阴性菌复制起点，C： catP 抗生素标记，B： 拟杆菌 ）和 pGM‐NACP（靶向普雷沃氏菌 16s rRNA 基因，P： 普雷沃氏菌 ）[ 2 ]（图 1A ）。同样，为了针对拟杆菌菌株中的其他特定基因，可以通过 PCR 扩增目标基因的约 1 kb 片段，并将其与从自杀载体 pGM‐NACB/P 扩增的骨架组装在一起，以获得通过插入-缺失来突变目标基因的质粒（图 1A ）。

Keynotes: In the case the size of the target gene is smaller than 1 kb, a ~0.5 kb fragment of the target gene is also functional for the single crossover, the key is that the fragment has to be an incomplete fraction (both upstream and downstream) of the target gene.
重点 ：在目标基因小于 1 kb 的情况下，目标基因的~0.5 kb 片段对于单交换也具有功能，关键是该片段必须是目标基因的不完整部分（上游和下游）。

Introduction of suicide vectors into the Bacteroidia microbes
将自杀载体引入拟杆菌微生物

The suicide vectors pGM‐NACB/P were introduced into the target Bacteroides/Prevotella using E. coli conjugation following the previously published protocol [21]. A single colony of the target Bacteroidia microbe was inoculated in 3 mL liquid broth and cultured in an anaerobic chamber at 37°C. The E. coli S17 harboring the pGM‐NACB/P vector was inoculated in the LB broth supplemented with carbenicillin (100 µg/mL) and grown at 37°C with aerobic shaking at 220 rpm. After ~12–16 h, when the OD₆₀₀ of E. coli S17 reached 0.8–1.0, 6 mL of E. coli S17 culture was centrifuged at 1500g for 2 min. The supernatant was discarded, and the cell pellet was washed twice with 3 mL PBS buffer (pH = 7.4). The washed E. coli S17 cell pellet was resuspended in a 3 mL overnight culture of the target Bacteroidia strain and gently mixed by pipetting. The mixture was filtered through a 0.2 µm filter. The filtered liquid was discarded. The filter with the mixture of donor and recipient cells was placed onto the surface of a pre‐reduced agar plate (cell surface facing down). The plate was incubated aerobically in a 37°C incubator.
按照先前发表的方案 [ 21 ]，将自杀载体 pGM-NACB/P 通过大肠杆菌接合法引入目标拟杆菌 / 普雷沃菌 。将目标拟杆菌单菌落接种于 3 mL 液体肉汤中，并在 37°C 的厌氧培养箱中培养。将携带 pGM-NACB/P 载体的大肠杆菌 S17 接种于添加了羧苄青霉素 (100 µg/mL) 的 LB 肉汤中，并在 37°C 的有氧摇床上以 220 rpm 的转速培养。约 12-16 小时后，当大肠杆菌 S17 的 OD ₆₀₀ 达到 0.8-1.0 时，取 6 mL 大肠杆菌 S17 培养物，以 1500 g 的转速离心 2 分钟。弃去上清液，用 3 mL PBS 缓冲液（pH = 7.4）洗涤细胞沉淀两次。将洗涤后的大肠杆菌 S17 细胞沉淀重悬于 3 mL 目标拟杆菌菌株的过夜培养液中，并用移液器轻轻吹打混匀。混合物经 0.2 µm 滤膜过滤。弃去滤液。将装有供体细胞和受体细胞混合物的滤膜置于预还原琼脂平板表面（细胞表面朝下）。将平板置于 37°C 培养箱中需氧培养。

After incubation aerobically at 37°C for 24 h, the filter was soaked in 2 mL pre‐reduced liquid broth. The cell on the filter was resuspended into the broth by gentle vortexing. The mixture was then transferred into the anaerobic chamber, and 100 µL (or serial diluted suspension) was plated onto a pre‐reduced agar plate with 200 µg/mL gentamicin + 15 µg/mL thiamphenicol (or MICs). Colonies of the target strain typically appeared after 36–48 h. Four colonies were picked and restreaked on a pre‐reduced agar plate with 200 µg/mL gentamicin + 15 µg/mL thiamphenicol (or MICs) to isolate single colonies.
在 37°C 下需氧培养 24 小时后，将滤膜浸泡在 2 毫升预还原液体肉汤中。轻轻涡旋使滤膜上的细胞重悬于肉汤中。然后将混合物转移至厌氧培养箱，取 100 µL（或连续稀释的悬浮液）接种于预还原琼脂平板上，平板上含有 200 µg/mL 庆大霉素+15 µg/mL 甲砜霉素（或 MIC）。目标菌株菌落通常在 36-48 小时后出现。挑取四个菌落，在预还原琼脂平板上划线接种，平板上含有 200 µg/mL 庆大霉素+15 µg/mL 甲砜霉素（或 MIC），以分离单个菌落。

Keynotes: The target Bacteroidia microbes are cultured in the anaerobic chamber overnight (~12 h). Do not culture the Bacteroidia strains for too long before conjugation, it will lead to the lysis of the strains.
要点 ：将目标拟杆菌菌株在厌氧培养箱中培养过夜（约 12 小时）。在接合前，请勿将拟杆菌菌株培养过久，否则会导致菌株裂解。

Potential issues and solutions: For some Bacteroidia strains that are pretty sensitive to oxygen, the aerobic conjugation will lead to the death of the majority of bacteria, in this case, there are two solutions: (1) expend the Bacteroidia strains by recovering the bacteria on the filter in liquid medium (supplemented with antibiotics to suppress the growth of E. coli) in the anaerobic chamber, then plate the growth in the liquid medium onto plates with antibiotics to isolate transconjugants; and (2) perform the conjugation on the filter in the anaerobic chamber.
可能出现的问题及解决办法 ：对于一些对氧气比较敏感的拟杆菌菌株，有氧结合将导致大部分细菌死亡，对于这种情况，解决办法有两种：（1）在厌氧室的液体培养基（添加抗生素以抑制大肠杆菌的生长）中，在滤膜上回收细菌，然后将液体培养基中的生长物接种到添加抗生素的平板上，分离转化子；（2）在厌氧室的滤膜上进行结合。

Diagnostic PCR and sequencing to verify the single crossover integration
诊断 PCR 和测序以验证单交叉整合

The isolated single colony was inoculated in a 3 mL liquid broth supplemented with 200 µg/mL gentamicin + 15 µg/mL thiamphenicol (or MICs). After 12 h, genomic DNA was extracted using a Quick DNA fungal/bacterial kit (Zymo Research). Diagnostic PCR was performed using primers 16s_27F and R6K_R to verify the single crossover integration of pGM‐NACB/P at their 16s rRNA loci. An ~2.5 kb PCR band would be seen in the transconjugants, whose chromosomal 16s rRNA loci were integrated by pGM‐NACB/P. The 2.5 kb PCR product was purified using a DNA Clean & Concentrator kit (Zymo Research) and sent for sequencing using primer R6K_F_RC. The sequencing results would show that a partial sequence of the 2.5 kb fragment came from the synthetic chi‐16s in pGM‐NACB/P and a partial sequence of the original 16s rRNA gene of the target strain, suggesting a single crossover of pGM‐NACB/P into one of its 16s rRNA loci (Figure 1A).
将分离出的单菌落接种于 3 mL 液体培养基中，培养基中添加 200 µg/mL 庆大霉素+15 µg/mL 甲砜霉素（或 MIC）。12 小时后，使用 Quick DNA 真菌/细菌试剂盒（Zymo Research）提取基因组 DNA。使用引物 16s_27F 和 R6K_R 进行诊断性 PCR，以验证 pGM‐NACB/P 在其 16s rRNA 基因位点的单交换整合。在已通过 pGM‐NACB/P 整合 16s rRNA 基因位点的转化接合子中可见约 2.5 kb 的 PCR 条带。使用 DNA 清洁浓缩试剂盒（Zymo Research）纯化 2.5 kb 的 PCR 产物，并使用引物 R6K_F_RC 送去测序。测序结果显示，2.5 kb 片段的部分序列来自 pGM‐NACB/P 中的合成 chi‐16s，以及目标菌株原始 16s rRNA 基因的部分序列，这表明 pGM‐NACB/P 与其 16s rRNA 基因位点之一发生了单次交叉（图 1A ）。

This single crossover integration strategy readily applies to other genes of interest in Bacteroidia microbes. To inactive a target gene in Bacteroidia strains, ~1 kb fragment of the target gene is amplified and the purified PCR product is then Gibson‐assembled, with the backbone amplified from the suicide vector pGM‐NACB, to get the plasmid for the target gene (Figure 1A). The plasmid is transferred into the conjugation donor E. coli S17 and introduced into the recipient microbe via conjugation. The transconjugants that undergo the expected single crossover integration are identified by diagnostic PCR and sequencing.
这种单交换整合策略可轻松应用于拟杆菌属微生物中的其他目标基因。为了使拟杆菌属菌株中的目标基因失活，需要扩增约 1 kb 的目标基因片段，然后将纯化的 PCR 产物与从自杀载体 pGM-NACB 扩增的骨架进行 Gibson 组装，以获得目标基因的质粒（图 1A ）。将该质粒转移到接合供体大肠杆菌 S17 中，并通过接合引入受体微生物。通过诊断性 PCR 和测序鉴定出发生预期单交换整合的转接合子。

Leveraging this protocol, after using the 16s rRNA‐targeting strategy to identify the gene transfer method for a nonmodel gut microbe Prevotella bivia DSM 20514, we were able to inactivate the asparaginase gene (ansB, which catalyzes the conversion of asparagine to aspartic acid) via single crossover insertion, as shown in Figure 1B,C. In diagnostic PCR, the mutant strain (ΔansB) had the PCR product of ~2 kb, while the wide type (WT) strain had no band (Figure 1B), and we also demonstrated that the mutant strain lost the ability to convert substrate asparagine to aspartic acid by liquid chromatography–mass spectrometry (LC‐MS) (Figure 1C).
利用该方案，在使用 16s rRNA 靶向策略确定非模型肠道微生物 Prevotella bivia DSM 20514 的基因转移方法后，我们能够通过单交换插入使天冬酰胺酶基因（ ansB ，该基因催化天冬酰胺转化为天冬氨酸）失活，如图 1B,C 所示。在诊断 PCR 中，突变菌株 ( ΔansB ) 的 PCR 产物约为 2 kb，而野生型 (WT) 菌株没有条带（图 1B ），我们还通过液相色谱-质谱法 (LC-MS) 证明突变菌株失去了将底物天冬酰胺转化为天冬氨酸的能力（图 1C ）。

On the basis of the essential first step that assesses the tractability of nonmodel Bacteroidia microbes, including Prevotella, our approach also paves the way for developing more advanced genetic tools, such as the double crossover recyclable marker system. As a proof of concept, after the establishment of the 16s rRNA‐targeting strategy in gut bacteria Bacteroides sp. 1_1_30, we further developed the double crossover recyclable marker system to knock out the thymidine kinase gene (tdk, which phosphorylates both thymidine and deoxyuridine). As shown in Figure 1D,E, following the integration of the suicide plasmid guided by the left arm and right arm of the targeted gene via HR, the antibiotic marker catP will be removed in the second step (double crossover) to get the markerless mutant strain (Figure 1D), in diagnostic PCR, the mutant strain (Δtdk) had the expected shorter PCR product compared with the WT strain (Figure 1E).
在评估非模型拟杆菌属 （包括普雷沃氏菌） 微生物的可处理性这一关键的第一步的基础上，我们的方法也为开发更先进的遗传工具（例如双交换可回收标记系统）铺平了道路。作为概念验证，在肠道细菌拟杆菌属 1_1_30 中建立 16s rRNA 靶向策略后，我们进一步开发了双交换可回收标记系统，以敲除胸苷激酶基因（ tdk ，该基因可同时磷酸化胸苷和脱氧尿苷）。如图 1D,E 所示，自杀质粒在目的基因左臂和右臂的引导下，通过 HR 整合后，抗生素标记 catP 在第二步（双交换）中被去除，得到无标记的突变菌株（图 1D ），在诊断 PCR 中，突变菌株（ Δtdk ）与野生菌株相比，具有预期的更短的 PCR 产物（图 1E ）。

Potential issues and solutions: If there is no correct transconjugant out of the four restreaked colonies upon diagnostic PCR, suggesting that the integration efficiency is low, in this case, solution 1 is to pick more colonies (like 24 colonies) to restreak and then do diagnostic PCR, solution 2 is to amplify another version of a fragment from the target gene.
可能出现的问题及解决方案 ：如果在诊断性 PCR 中，4 个重新划线的菌落没有出现正确的转化子，说明整合效率低，这种情况下，解决方案 1 是挑取更多的菌落（比如 24 个菌落）重新划线，然后进行诊断性 PCR，解决方案 2 是从目标基因中扩增另一个版本的片段。

Experimental results interpretation: For the result of diagnostic PCR of the transconjugants, as shown in Figure 1A,B, because the forward diagnostic primer binds the sequence on the genome and the reverse diagnostic primer binds the sequence on the plasmid, only transconjugants that undergo the expected integration would have the 2.5 kb PCR product; WT strain or transconjugants that undergo the unexpected insertion would not have the 2.5 kb PCR product.
实验结果解释 ：对于图 1A,B 所示的接合子诊断 PCR 结果，由于正向诊断引物与基因组上的序列结合，反向诊断引物与质粒上的序列结合，因此只有发生预期插入的接合子才会有 2.5 kb 的 PCR 产物；WT 菌株或发生意外插入的接合子不会有 2.5 kb 的 PCR 产物。

Materials and devices  材料与设备

Primer star DNA polymerase (Takara, Cat# R045), Blue sapphire DNA polymerase (Takara, Cat# RR350), Plasmid Midiprep Kit (Zymo Research, Cat# D4201), DNA Clean and Concentrator (Zymo Research, Cat# D4003), Tryptic Soy Agar (BD, Cat# 236950), Brain Heart Infusion Agar (BD, Cat# 241830), CBA (BD, Cat# 279240), Horse blood (Hemostat Laboratories, Cat# 637291), LB broth (BD, Cat# BP1426), glycerol (Fisher Bioreagents, Cat# BP229), PBS (Gibco, Cat# 10010‐031), centrifuge, PCR amplifier, tetracycline (GoldBio, Cat# T‐101‐25), chloramphenicol (VWR, Cat# 0230), d‐cycloserine (D) (TCI, Cat# C1189), gentamicin (G) (GoldBio, Cat# G‐400‐25), kanamycin (K) (GoldBio, Cat# K‐120‐25), carbenicillin (GoldBio, Cat# C‐103‐25), thiamphenicol (Thiam) (Acros Organics, Cat# 455450250), anaerobic chamber, aerobic incubator, electroporation system, Thermo Scientific Nanodrop 2000, Gibson Assembly Cloning Kit (NEB, Cat# E5510S), Quick DNA fungal/bacterial kit (Zymo Research, Cat# D6005), and ultralow temperature freezer.
Primer star DNA 聚合酶（Takara，Cat# R045）、Blue sapphire DNA 聚合酶（Takara，Cat# RR350）、质粒中量提取试剂盒（Zymo Research，Cat# D4201）、DNA 净化浓缩液（Zymo Research，Cat# D4003）、胰蛋白酶大豆琼脂（BD，Cat# 236950）、脑心浸液琼脂（BD，Cat# 241830）、CBA（BD，Cat# 279240）、马血（Hemostat Laboratories，Cat# 637291）、LB 肉汤（BD，Cat# BP1426）、甘油（Fisher Bioreagents，Cat# BP229）、PBS（Gibco，Cat# 10010-031）、离心机、PCR 扩增子、四环素（GoldBio，Cat# T-101-25）。氯霉素 (VWR，Cat# 0230)、 d- 环丝氨酸 (D) (TCI，Cat# C1189)、庆大霉素 (G) (GoldBio，Cat# G-400-25)、卡那霉素 (K) (GoldBio，Cat# K-120-25)、羧苄西林 (GoldBio，Cat# C-103-25)、甲砜霉素 (Thiam) (Acros Organics，Cat# 455450250)、厌氧室、好氧培养箱、电穿孔系统、Thermo Scientific Nanodrop 2000、Gibson Assembly Cloning Kit (NEB，Cat# E5510S)、Quick DNA 真菌/细菌试剂盒 (Zymo Research，Cat# D6005) 和超低温冰箱。

Screening of culture conditions
培养条件的筛选

The culture conditions for the Gram‐positive Clostridia microbes were screened. Strains were restreaked (from the original glycerol stock) onto pre‐reduced agar plates (such as TSAB, BHIB, or CBA plates). Then, microbes that can grow on these agar plates were subcultured into 1 mL pre‐reduced liquid medium: Mega, CMM, and RCM. Strains that can grow in any one of the four liquid cultures were subject to the antibiotics test (Figure 2).
筛选了革兰氏阳性梭菌的培养条件。将菌株（从原始甘油原液中）重新划线接种到预还原琼脂平板（例如 TSAB、BHIB 或 CBA 平板）上。然后将能够在这些琼脂平板上生长的微生物传代培养到 1 mL 预还原液体培养基：Mega、CMM 和 RCM。在四种液体培养基中均能生长的菌株需进行抗生素测试（图 2 ）。

Keynotes: For the screening of the culture conditions of liquid medium, Clostridia strains need to be first recovered on agar plates to ensure that Clostridia strains are activated, instead of inoculating Clostridia strains into the liquid medium from the frozen glycerol stock directly.
要点 ：液体培养基培养条件的筛选，需要先在琼脂平板上回收梭菌菌株，以确保梭菌菌株被激活，而不是直接从冷冻的甘油原液中将梭菌菌株接种到液体培养基中。

Potential issues and solutions: If the Clostridia strains of interest cannot grow on the common agar plates or in the liquid medium listed above, other specific plates or liquid mediums that favor the growth of the target strains need to be used to screen the culture conditions.
潜在问题和解决方案 ：如果目标梭菌菌株不能在普通琼脂平板或上述液体培养基中生长，则需要使用有利于目标菌株生长的其他特定平板或液体培养基来筛选培养条件。

Antibiotic test  抗生素测试

The Clostridia strains were restreaked on agar plates supplemented with 250 µg/mL d‐cycloserine (D), 200 µg/mL gentamicin (G), or 200 µg/mL kanamycin (K). d‐cycloserine or gentamicin will be used to inhibit the growth of conjugation donor E. coli CA434 after conjugation, and kanamycin will be used to inhibit the growth of conjugation donor E. coli HB101/pRK24. Both E. coli CA434 and HB101/pRK24 have been shown to successfully conjugate exogenous genomic DNA into Clostridium bacteria like Clostridium sporogenes or Clostridium acetobutylicum in previous studies [10, 11, 15].
将梭菌菌株重新划线接种于添加了 250 µg/mL d- 环丝氨酸(D)、200 µg/mL 庆大霉素(G)或 200 µg/mL 卡那霉素(K)的琼脂平板上。d- 环丝氨酸或庆大霉素用于抑制结合供体大肠杆菌 CA434 在结合后的生长，卡那霉素用于抑制结合供体大肠杆菌 HB101/pRK24 的生长。先前的研究表明， 大肠杆菌 CA434 和 HB101/pRK24 均能成功地将外源基因组 DNA 结合到梭菌属细菌（如产孢梭菌或丙酮丁醇梭菌） 中[ 10 , 11 , 15 ]。

The majority of the Clostridia strains we tested so far are sensitive to thiamphenicol, so the thiamphenicol‐resistant gene (catP) can be exploited as a universal marker to select transconjugants that can uptake and maintain exogenous genomic DNA. The MICs of thiamphenicol of the Clostridia microbes were tested on agar plates containing thiamphenicol at different concentrations (Figure 2).
到目前为止，我们测试的大多数梭菌菌株对甲砜霉素敏感，因此可以利用甲砜霉素抗性基因 ( catP ) 作为通用标记，筛选能够摄取并维持外源基因组 DNA 的转化接合子。在含有不同浓度甲砜霉素的琼脂平板上，我们测试了梭菌微生物对甲砜霉素的最低抑菌浓度 (MIC)（图 2 ）。

Keynotes: Antibiotics need to be added into the agar medium before the agar plates are poured and solidified; agar plates supplemented with antibiotics only on the surface of the plates may cause misleading antibiotic test results.
要点 ：琼脂培养基需要在琼脂平板灌注固化前添加抗生素；仅在平板表面添加抗生素的琼脂平板可能会造成抗生素检测结果的误导。

Potential issues and solutions: If the Clostridia strains of interest are not resistant against d‐cycloserine, gentamicin, or kanamycin, other specific antibiotics that can suppress the growth of E. coli CA434 or HB101/pRK24 can be used for the test. Likewise, if the Clostridia strains of interest are resistant against thiamphenicol, the antibiotic marker on the conjugation vector can be replaced by other markers, whose corresponding antibiotics can suppress the growth of the Clostridia strains.
潜在问题及解决方案 ：如果目标梭菌菌株对 d- 环丝氨酸、庆大霉素或卡那霉素不耐药，则可以使用其他能够抑制大肠杆菌 CA434 或 HB101/pRK24 生长的特异性抗生素进行检测。同样，如果目标梭菌菌株对甲砜霉素耐药，则可将结合载体上的抗生素标记替换为其他标记，其对应的抗生素可以抑制梭菌菌株的生长。

Vector assembly  向量组装

A series of vectors pGM‐xBCM (pGM‐ABCM, BBCM, CBCM, DBCM, EBCM, FBCM, GBCM, HBCM, and IBCM) harboring different rep oris for Clostridia microbes were generated to screen the compatible rep oris for the recipient Clostridia strains [2]. To include more rep oris for screening, the rep ori sequences could be amplified by PCR and assembled with the backbone amplified from pGM‐xBCM.
构建了一系列携带不同梭菌属细菌 rep oris 的载体 pGM‐xBCM（pGM‐ABCM、BBCM、CBCM、DBCM、EBCM、FBCM、GBCM、HBCM 和 IBCM），用于筛选与受体梭菌菌株相容的 rep oris [ 2 ]。为了纳入更多 rep oris 进行筛选，可以通过 PCR 扩增 rep oris 序列，并将其与从 pGM‐xBCM 扩增出的骨架序列进行组装。

Mixed‐conjugation strategy to identify Clostridia microbes that uptake and maintain exogenous plasmid DNA
混合结合策略鉴定吸收和维持外源质粒 DNA 的梭菌微生物

The series of vectors pGM‐ABCM, BBCM, CBCM, DBCM, EBCM, FBCM, GBCM, HBCM, and IBCM harboring different rep oris, but the same antibiotic marker catP (against thiamphenicol) were transformed into chemical competent E. coli CA434 or E. coli HB101/pRK24. Mixed‐conjugation strategies separating these E. coli donors into three groups (Group I: pGM‐ABCM, BBCM, and CBCM; Group II: pGM‐DBCM, EBCM, and FBCM; and Group III: pGM‐GBCM, HBCM, IBCM) were established to identify the compatible rep ori for each Clostridia microbe of interest. For the Clostridia microbes resistant to d‐cycloserine (250 μg/mL) or gentamicin (200 µg/mL), E. coli CA434 would be used as the conjugation donor. For the microbes that are not resistant to d‐cycloserine (250 μg/mL) or gentamicin (200 µg/mL) but resistant to kanamycin (200 μg/mL), E. coli HB101/pRK24 would be used as their conjugation donors.
将一系列载体 pGM-ABCM、BBCM、CBCM、DBCM、EBCM、FBCM、GBCM、HBCM 和 IBCM（它们携带不同的 rep ori ，但具有相同的抗生素标记 catP （针对甲砜霉素））转化到化学感受态大肠杆菌 CA434 或大肠杆菌 HB101/pRK24 中。建立了混合结合策略，将这些大肠杆菌供体分为三组（第 I 组：pGM-ABCM、BBCM 和 CBCM；第 II 组：pGM-DBCM、EBCM 和 FBCM；第 III 组：pGM-GBCM、HBCM、IBCM），以确定与每种目标梭菌微生物相容的 rep ori 。对于对 d- 环丝氨酸（250 μg/mL）或庆大霉素（200 μg/mL）具有抗性的梭菌 ，可使用大肠杆菌 CA434 作为结合供体。对于对 d- 环丝氨酸（250 μg/mL）或庆大霉素（200 μg/mL）不具有抗性但对卡那霉素（200 μg/mL）具有抗性的梭菌，可使用大肠杆菌 HB101/pRK24 作为结合供体。

The Clostridia microbe was restreaked on a pre‐reduced agar plate. After 24–48 h, a single colony was inoculated in 1 mL of liquid broth that supported its growth in an anaerobic chamber. On the same day, E. coli containing plasmids with different rep oris were inoculated into 6 mL of LB supplemented with tetracycline (15 µg/mL) and chloramphenicol (25 µg/mL) and shaken aerobically at 37°C for 12–18 h (overnight). The next day, these E. coli donors were separated into three groups, as mentioned above. For conjugating one Clostridia microbe, a 1.0 mL culture of each E. coli within the same group was mixed and centrifuged at 1500g for 2 min. The culture supernatant was discarded, and the cell pellet was gently washed with 500 µL PBS buffer (pH = 7.4). The PBS supernatant was then removed after centrifugation at 1500g for 2 min, and the cell pellet was transferred on ice into the anaerobic chamber. Next, the cell pellet (a total of three cell pellets) was mixed gently with 300 μL overnight culture of the targeting Clostridia microbe, and a 35 μL cell mixture was dotted on pre‐reduced agar plates. After 48 h, the cell dots were scraped using a sterile inoculation loop and resuspended in 300 μL pre‐reduced PBS (pH = 7.4) buffer. The cell suspension (100 µL) was plated on agar plates supplemented with 15 µg/mL thiamphenicol (or MICs) and 250 µg/mL d‐cycloserine or 200 µg/mL gentamicin (if E. coli CA434 is the conjugation donor), or 200 µg/mL kanamycin (if E. coli HB101/pRK24 is the conjugation donor). Colonies typically appeared after 36–48 h. Four colonies were picked and restreaked onto agar plates with the same antibiotics to isolate single colonies (Figure 2).
将梭菌微生物重新划线接种于预还原琼脂平板上。24-48 小时后，将单菌落接种于 1 mL 液体肉汤中，该液体肉汤可在厌氧培养箱中生长。同一天，将含有不同复制位点质粒的大肠杆菌接种于 6 mL 添加四环素（15 µg/mL）和氯霉素（25 µg/mL）的 LB 培养基中，并在 37°C 下有氧振荡培养 12-18 小时（过夜）。第二天，将这些大肠杆菌供体按上述方法分成三组。为了接合一株梭菌微生物，将同一组内每种大肠杆菌的 1.0 mL 培养物混合，并以 1500 g 离心 2 分钟。弃去培养上清液，用 500 μL PBS 缓冲液（pH = 7.4）轻轻洗涤细胞沉淀。然后以 1500 g 离心 2 分钟后去除 PBS 上清液，并将细胞沉淀在冰上转移至厌氧培养箱。接下来，将细胞沉淀（共三个细胞沉淀）与 300 μL 目标梭菌过夜培养物轻轻混合，并将 35 μL 细胞混合物点在预还原的琼脂平板上。48 小时后，用无菌接种环刮下细胞点，并将其重新悬浮于 300 μL 预还原的 PBS（pH = 7.4）缓冲液中。将 100 µL 细胞悬液接种于添加 15 µg/mL 甲砜霉素（或 MIC）和 250 µg/mL d- 环丝氨酸或 200 µg/mL 庆大霉素（若大肠杆菌 CA434 为结合供体）或 200 µg/mL 卡那霉素（若大肠杆菌 HB101/pRK24 为结合供体）的琼脂平板上。菌落通常在 36-48 小时后出现。 挑取四个菌落，重新划线接种到含有相同抗生素的琼脂平板上，以分离单个菌落（图 2 ）。

Keynotes: (1) The target Clostridia microbes are cultured in the anaerobic chamber overnight (~12 h), do not culture the Clostridia strains for too long before conjugation, which will lead to the lysis of the strains; (2) air dry the agar plates for conjugation a little bit is good for conjugation, if the plates are wet, the cell mixture dot will spread all over the plate, in this case, the cell mixture is diluted on the plate, which will reduce the conjugation efficiency.
要点 ：（1）将目标梭菌微生物在厌氧箱中培养过夜（~12 h），在进行结合前不要将梭菌菌株培养太长时间，这会导致菌株裂解；（2）将用于结合的琼脂平板稍微风干以利于结合，如果平板潮湿，细胞混合物点会遍布整个平板，在这种情况下，细胞混合物在平板上被稀释，这会降低结合效率。

Potential issues and solutions: In our experience, conjugation donor E. coli CA434 works better than E. coli HB101/pRK24. For specific strains of interest, if the two E. coli donors cannot transfer plasmids into the recipient strains, other E. coli donors could be utilized.
潜在问题及解决方案 ：根据我们的经验，接合供体大肠杆菌 CA434 的效果优于大肠杆菌 HB101/pRK24。对于特定目标菌株，如果两种大肠杆菌供体无法将质粒转移至受体菌株，则可以使用其他大肠杆菌供体。

Diagnostic PCR and sequencing to verify the plasmid uptake
诊断性 PCR 和测序以验证质粒的摄取

The isolated single colony was cultivated in 3 mL liquid broth supplemented with the corresponding antibiotics 250 µg/mL d‐cycloserine (or 200 µg/mL gentamicin/kanamycin) + 15 µg/mL thiamphenicol (or MICs). The genomic DNA was isolated from the resulting cell material using the Quick DNA fungal/bacterial kit (Zymo Research). Then multiplex diagnostic PCRs were performed to assess which plasmid was uptaken by the conjugation recipient Clostridia microbe. For the mixed‐conjugation with Group I (Groups II and III are performed likewise), primers pMTL_laz_diag_F (universal forward primer) + pGM‐ABCM_rep_R_1500bp + pGM‐BBCM_rep_R_1000bp + pGM‐CBCM_rep_R_2000bp (for 15 µL PCR reaction, the amount of the four primers is 0.75, 0.3, 0.3, and 0.3 µL [10 µM]) were used for diagnostic PCR. A PCR band of 1.5 kb (or 1.0 or 2.0 kb) would be seen if pGM‐ABCM (or BBCM or CBCM) is uptaken by the Clostridia microbe. In the meantime, to confirm that the picked and restreaked colonies are the target Clostridia strain but not the E. coli conjugation donor, the 16s rRNA region of the colony was amplified using primers 16s_27F + 16s_1391R. The PCR product was purified and sent for Sanger sequencing using primer 16s_1391R, and the colonies were further restreaked aerobically to confirm not to be E. coli (if the colonies cannot grow aerobically, they will be considered not to be E. coli) (Figure 2).
将分离出的单菌落置于 3 mL 液体肉汤中培养，并添加相应的抗生素：250 µg/mL d- 环丝氨酸（或 200 µg/mL 庆大霉素/卡那霉素）+ 15 µg/mL 甲砜霉素（或 MIC）。使用 Quick DNA 真菌/细菌试剂盒（Zymo Research）从所得细胞材料中分离基因组 DNA。然后进行多重诊断 PCR，以评估接合受体梭菌微生物摄取了哪种质粒。对于与 I 组（II 组和 III 组类似）的混合结合，使用引物 pMTL_ laz _diag_F（通用正向引物）+ pGM‐ABCM_rep_R_1500bp + pGM‐BBCM_rep_R_1000bp + pGM‐CBCM_rep_R_2000bp（对于 15 µL PCR 反应，四个引物的用量分别为 0.75、0.3、0.3 和 0.3 µL [10 µM]）进行诊断 PCR。如果梭菌微生物摄取了 pGM‐ABCM（或 BBCM 或 CBCM），则会看到 1.5 kb（或 1.0 或 2.0 kb）的 PCR 带。同时，为了确认挑取并重新划线的菌落为目标梭菌菌株而非大肠杆菌接合供体，使用引物 16s_27F + 16s_1391R 扩增菌落的 16s rRNA 区。PCR 产物纯化后，使用引物 16s_1391R 进行桑格测序，并进一步在有氧条件下重新划线，以确认菌落并非大肠杆菌 （如果菌落无法在有氧条件下生长，则视为非大肠杆菌 ）（图 2 ）。

Keynotes: When performing the diagnostic PCR to figure out which plasmid in a group was transferred into the recipient strain, to avoid a false‐positive conclusion, it is necessary to include the genome of the recipient strain to serve as the negative control.
重点 ：在进行诊断性 PCR 以确定某一组中的哪个质粒转移到受体菌株中时，为避免假阳性结论，有必要加入受体菌株的基因组作为阴性对照。

Experimental results interpretation: For the diagnostic PCR of each group (I, II, and III), when different plasmids are transferred into recipient strains, there will be PCR products of different sizes (1, 1.5, and 2 kb), the corresponding size of each plasmid in each group is annotated at the end of the “Name of primers” in Table S1.
实验结果解释 ：对于各组（I、II、III）的诊断性 PCR，当不同的质粒转移到受体菌株中时，会有不同大小（1、1.5、2kb）的 PCR 产物，各组各质粒对应的大小在表 S1 中“引物名称”末尾标注。

Single E. coli donor‐conjugation validation
单个大肠杆菌供体结合验证

Next, single E. coli donor‐conjugation (one E. coli donor to one Clostridia recipient) was performed to validate that the PCR‐identified plasmid(s) can be conjugated into the targeted Clostridia microbe. A single colony of the targeted Clostridia strain was inoculated in a 1 mL liquid broth in an anaerobic chamber. The conjugation donor E. coli (CA434 or HB101/pRK24) harboring the PCR‐identified plasmid was inoculated into 6 mL of LB supplemented with tetracycline (15 µg/mL) and chloramphenicol (25 µg/mL) and shaken aerobically at 37°C for 12–18 h (overnight). After 12–18 h, 1.5 mL of the E. coli culture was centrifuged at 1500g for 2 min. The supernatant was discarded and the cell pellet was washed with 500 µL PBS buffer (pH = 7.4). The PBS supernatant was then removed after centrifugation at 1500g for 2 min, and the cell pellet was transferred on ice into the anaerobic chamber. Next, the cell pellet was mixed gently with a 300 μL overnight culture of the targeting Clostridia microbe, and a 35 μL cell mixture was dotted on pre‐reduced agar plates. After 48 h, the cell dots were scraped using a sterile inoculation loop and resuspended in 300 μL pre‐reduced PBS (pH = 7.4) buffer. The cell suspension (100 µL) was plated on agar plates supplemented with 15 µg/mL thiamphenicol (or MICs) and 250 µg/mL d‐cycloserine or 200 µg/mL gentamicin (if E. coli CA434 is the conjugation donor), or 200 µg/mL kanamycin (if E. coli HB101/pRK24 is the conjugation donor). Colonies typically appeared after 36–48 h. Four colonies were picked and restreaked onto agar plates with the same antibiotics to isolate single colonies. The isolated single colonies will be cultured in 1 mL of pre‐reduced liquid broth with the same antibiotics, and the glycerol stock will be prepared using the culture (Figure 2).
接下来，进行单大肠杆菌供体-受体接合实验（一个大肠杆菌供体与一个梭菌受体），以验证 PCR 鉴定的质粒能否与目标梭菌微生物接合。将目标梭菌菌株的单菌落接种于厌氧培养箱中的 1 mL 液体肉汤中。将携带 PCR 鉴定质粒的接合供体大肠杆菌 （CA434 或 HB101/pRK24）接种于 6 mL 添加了四环素（15 µg/mL）和氯霉素（25 µg/mL）的 LB 培养基中，在 37°C 下有氧振荡培养 12-18 小时（过夜）。12-18 小时后，取 1.5 mL 大肠杆菌培养物，以 1500 g 离心 2 分钟。弃去上清液，用 500 μL PBS 缓冲液（pH = 7.4）洗涤细胞沉淀。然后以 1500 g 离心 2 分钟后，除去 PBS 上清液，并将细胞沉淀在冰上转移至厌氧培养箱。接下来，将细胞沉淀与 300 μL 目标梭菌过夜培养物轻轻混合，并将 35 μL 细胞混合物点在预还原的琼脂平板上。48 小时后，用无菌接种环刮下细胞点，并将其重新悬浮于 300 μL 预还原的 PBS（pH = 7.4）缓冲液中。将 100 µL 细胞悬液接种于添加 15 µg/mL 甲砜霉素（或 MIC）和 250 µg/mL d- 环丝氨酸或 200 µg/mL 庆大霉素（如果大肠杆菌 CA434 为结合供体）或 200 µg/mL 卡那霉素（如果大肠杆菌 HB101/pRK24 为结合供体）的琼脂平板上。菌落通常在 36-48 小时后出现。挑取四个菌落，重新划线接种于添加相同抗生素的琼脂平板上，以分离单个菌落。 将分离的单菌落培养在 1 mL 预先还原的含有相同抗生素的液体肉汤中，并使用该培养物制备甘油原液（图 2 ）。

Keynotes: For the step of scraping cell dots and suspending cells in PBS to be plated onto agar plates with selective antibiotics, in the case that a lot of conjugations plates need to be scraped, it is better to scrap at most three plates and suspend in PBS at one time, keeping cells in PBS for too long will reduce the conjugation efficiency.
要点 ：对于刮取细胞点并将细胞悬浮在 PBS 中以接种到含有选择性抗生素的琼脂平板上的步骤，如果需要刮取大量结合板，则最好一次刮取最多三块板并悬浮在 PBS 中，细胞在 PBS 中停留时间过长会降低结合效率。

Potential issues and solutions: In the step of plating conjugation cells onto agar plates with d‐cycloserine (or gentamicin, kanamycin) + thiamphenicol (MICs), some recipient strains may overgrow because the MICs of thiamphenicol are not enough to suppress the growth of the plated cells that contain a high concentration of recipient strains; in this case, plates with a higher concentration of thiamphenicol need to be used (e.g., if the MICs of thiamphenicol are 7.5 µg/mL, 10 µg/mL thiamphenicol can be added into the agar plate).
潜在问题和解决方案 ：在将接合细胞接种到含有 d- 环丝氨酸（或庆大霉素、卡那霉素）+甲砜霉素（MIC）的琼脂平板上时，一些受体菌株可能会过度生长，因为甲砜霉素的 MIC 不足以抑制含有高浓度受体菌株的接种细胞的生长；在这种情况下，需要使用具有更高浓度甲砜霉素的平板（例如，如果甲砜霉素的 MIC 为 7.5 µg/mL，则可以将 10 µg/mL 甲砜霉素添加到琼脂平板中）。

Materials and devices  材料与设备

Primer star DNA polymerase (Takara, Cat# R045), Blue sapphire DNA polymerase (Takara, Cat# RR350), Plasmid Midiprep Kit (Zymo Research, Cat# D4201), DNA Clean and Concentrator (Zymo Research, Cat# D4003), Tryptic Soy Agar (BD, Cat# 236950), Brain Heart Infusion Agar (BD, Cat# 241830), CBA (BD, Cat# 279240), Horse blood (Hemostat Laboratories, Cat# 637291), LB broth (BD, Cat# BP1426), glycerol (Fisher Bioreagents, Cat# BP229), PBS (Gibco, Cat# 10010‐031), centrifuge, PCR amplifier, tetracycline (GoldBio, Cat# T‐101‐25), chloramphenicol (VWR, Cat# 0230), d‐cycloserine (D) (TCI, Cat# C1189), gentamicin (G) (GoldBio, Cat# G‐400‐25), kanamycin (K) (GoldBio, Cat# K‐120‐25), thiamphenicol (Thiam) (Acros Organics, Cat# 455450250), anaerobic chamber, aerobic incubator, electroporation system, Thermo Scientific Nanodrop 2000, Gibson Assembly Cloning Kit (NEB, Cat# E5510S), Quick DNA fungal/bacterial kit (Zymo Research, Cat# D6005), ultralow temperature freezer, Direct‐zol RNA Microprep kit (Zymo Research, Cat# R2062), PrimeScript RT Reagent Kit (Takara, Cat# RR047A), real‐time quantitative PCR system (Applied Biosystems, ABI 7500).
Primer star DNA 聚合酶（Takara，Cat# R045）、Blue sapphire DNA 聚合酶（Takara，Cat# RR350）、质粒中量提取试剂盒（Zymo Research，Cat# D4201）、DNA 净化浓缩液（Zymo Research，Cat# D4003）、胰蛋白酶大豆琼脂（BD，Cat# 236950）、脑心浸液琼脂（BD，Cat# 241830）、CBA（BD，Cat# 279240）、马血（Hemostat Laboratories，Cat# 637291）、LB 肉汤（BD，Cat# BP1426）、甘油（Fisher Bioreagents，Cat# BP229）、PBS（Gibco，Cat# 10010-031）、离心机、PCR 扩增子、四环素（GoldBio，Cat# T-101-25）。氯霉素 (VWR, Cat# 0230)、 d- 环丝氨酸 (D) (TCI, Cat# C1189)、庆大霉素 (G) (GoldBio, Cat# G-400-25)、卡那霉素 (K) (GoldBio, Cat# K-120-25)、甲砜霉素 (Thiam) (Acros Organics, Cat# 455450250)、厌氧箱、好氧培养箱、电穿孔系统、Thermo Scientific Nanodrop 2000、Gibson Assembly Cloning Kit (NEB, Cat# E5510S)、Quick DNA 真菌/细菌试剂盒 (Zymo Research, Cat# D6005)、超低温冰箱、Direct-zol RNA Microprep 试剂盒 (Zymo Research, Cat# R2062)、PrimeScript RT 试剂盒 (Takara, Cat# RR047A)、实时定量 PCR 系统（Applied Biosystems，ABI 7500）。

Vector assembly  向量组装

Three sets of plasmids pGM‐xBCD (pGM‐ABCD, BBCD, pGM‐CBCD, DBCD, EBCD, FBCD, GBCD, HBCD, and IBCD) carrying the CRISPRi‐dCpf1 machinery, plasmids pGM‐xBCL (pGM‐ABCL, BBCL, pGM‐CBCL, DBCL, EBCL, FBCL, GBCL, HBCL, and IBCL) carrying CRISPRi‐dCpf1 and the lacZα reporter gene, and plasmids pGM‐xBCF (pGM‐ABCF, BBCF, pGM‐CBCF, DBCF, EBCF, FBCF, GBCF, HBCF, and IBCF) carrying CRISPRi‐dCpf1, lacZα, and the lacZα targeting guide RNA (gRNA) were generated to test the CRISPRi‐dCpf1 lacZα system for Clostridia microbes [2] (Figure 3A–D). To target other specific genes in Clostridia strains, the gRNA locus targeting the promoter region and coding sequence (CDS) of the target gene (as in Figure 3D) could be amplified by PCR and assembled with the backbone amplified from pGM‐xBCD to get the plasmid carrying CRISPRi‐dCpf1 and the gRNA for the target gene.
生成了三组携带 CRISPRi‐dCpf1 机制的质粒 pGM‐xBCD（pGM‐ABCD、BBCD、pGM‐CBCD、DBCD、EBCD、FBCD、GBCD、HBCD 和 IBCD）、携带 CRISPRi‐dCpf1 和 lacZα 报告基因的质粒 pGM‐xBCL（pGM‐ABCL、BBCL、pGM‐CBCL、DBCL、EBCL、FBCL、GBCL、HBCL 和 IBCL）以及携带 CRISPRi‐dCpf1、 lacZα 和 lacZα 靶向引导 RNA（gRNA）的质粒 pGM‐xBCF（pGM‐ABCF、BBCF、pGM-CBCF、DBCF、EBCF、FBCF、GBCF、HBCF 和 IBCF）来测试 CRISPRi‐dCpf1 lacZα 系统对梭菌的作用微生物 [ 2 ]（图 3A–D ）。为了靶向梭菌菌株中的其他特定基因，可以通过 PCR 扩增针对目标基因启动子区和编码序列（CDS）的 gRNA 基因座（如图 3D 所示），并将其与从 pGM‐xBCD 扩增的骨架组装在一起，以获得携带 CRISPRi‐dCpf1 和目标基因 gRNA 的质粒。

Utilization of dCpf1 to suppress the lacZα transcription in Clostridia strains
利用 dCpf1 抑制梭菌菌株中的 lacZα 转录

Using a Gram‐positive strain Clostridium bolteae DSM 29485 as an example, pGM‐ABCL and pGM‐ABCF were transformed into chemically competent E. coli CA434, respectively. E. coli CA434 harboring pGM‐ABCL and pGM‐ABCF were conjugated to Clostridium bolteae DSM 29485 (Figure 3E). The transconjugants were picked and restreaked onto a TSAB agar plate supplemented with d‐cycloserine (250 µg/mL) + thiamphenicol (15 µg/mL). Then, three isolated single colonies were cultivated in 5 mL Mega liquid broth supplemented with 15 µg/mL thiamphenicol for 36 h. The bacterial RNA was extracted using a Direct‐zol RNA Microprep kit (Zymo Research) and quantitative polymerase chain reaction (qPCR) was performed to quantify the relative expression of lacZα after normalizing to 16s rRNA gene, using primers dCpf1‐lacZα_qPCR_F and dCpf1‐lacZα_qPCR_R for lacZα gene and S74_16 s_qPCR_F and S74_16s_qPCR_R for the control 16s rRNA (Figure 3F).
以革兰氏阳性菌株 Clostridium bolteae DSM 29485 为例，将 pGM-ABCL 和 pGM-ABCF 分别转化至化学感受态大肠杆菌 CA434 中。将携带 pGM-ABCL 和 pGM-ABCF 的大肠杆菌 CA434 与 Clostridium bolteae DSM 29485 偶联（图 3E ）。挑取转接合子，重新划线接种于添加了 d- 环丝氨酸（250 µg/mL）+甲砜霉素（15 µg/mL）的 TSAB 琼脂平板上。然后，将分离出的三个单菌落置于添加了 15 µg/mL 甲砜霉素的 5 mL Mega 液体肉汤中培养 36 小时。使用 Direct‐zol RNA Microprep 试剂盒 (Zymo Research) 提取细菌 RNA，并进行定量聚合酶链式反应 (qPCR) 以量化 lacZα 的相对表达，在标准化为 16s rRNA 基因后，使用引物 dCpf1‐ lacZα _qPCR_F 和 dCpf1‐ lacZα _qPCR_R 作为 lacZα 基因，使用 S74_16 s_qPCR_F 和 S74_16s_qPCR_R 作为对照 16s rRNA（图 3F ）。

This CRISPRi‐dCpf1 gene repression tool is readily applicable to other genes of interest in Clostridia microbes. To knock down a target gene in Clostridia strains, the gRNA locus targeting the promoter region and CDS of the target gene is introduced into the plasmid harboring dCpf‐1 and the corresponding compatible rep ori (the set of vectors pGM‐xBCD). As in targeting lacZα, the synthetic fragment containing the terminator region (Figure 3D, in red) was amplified to get a PCR product that has one direct repeat sequence (Figure 3D, in green) and gRNA (Figure 3D, in blue) fused with the terminator. Then, this PCR product was purified and used as the template for the second PCR to get the gRNA locus with two gRNAs for the target gene. This gRNA locus was then assembled with the backbone amplified from pGM‐xBCD to get the plasmid for the target gene. The plasmid was transformed into donor E. coli CA434 (or HB101/pRK24) and introduced into the recipient microbe via conjugation. The transconjugants harboring the CRISPRi‐dCpf1 plasmid are identified by antibiotic selection, and the gene knockdown is validated by qPCR of the target gene or other readout like LC‐MS measurement of metabolites.
CRISPRi‐dCpf1 基因抑制工具可轻松应用于梭菌属微生物中的其他目标基因。为了敲低梭菌属菌株中的目标基因，将靶向目标基因启动子区域和 CDS 的 gRNA 基因座引入含有 dCpf‐1 和相应兼容 rep ori 的质粒（载体组 pGM‐xBCD）。与靶向 lacZα 一样，扩增含有终止子区域的合成片段（图 3D ，红色）以获得具有一个直接重复序列（图 3D ，绿色）和与终止子融合的 gRNA（图 3D ，蓝色）的 PCR 产物。然后，纯化该 PCR 产物并用作第二次 PCR 的模板，以获得具有两个针对目标基因的 gRNA 的 gRNA 基因座。然后将该 gRNA 基因座与从 pGM‐xBCD 扩增的骨架组装以获得目标基因的质粒。将质粒转化至供体大肠杆菌 CA434（或 HB101/pRK24）中，并通过接合导入受体菌。通过抗生素筛选鉴定出携带 CRISPRi‐dCpf1 质粒的接合子，并通过靶基因的 qPCR 或其他方法（例如 LC-MS 代谢物测定）验证基因敲减效果。

Keynotes: To transfer plasmids containing the CRISPRi‐dCpf1 machinery into the target strains, in the step of plating conjugation cells onto agar plates with selective antibiotics, it is recommended to plate all 300 µL scraped‐cell suspension in PBS onto three plates, because the conjugation efficiency will decrease when the CRISPRi‐dCpf1 machinery is introduced into plasmids to make these plasmids bigger.
要点 ：为了将含有 CRISPRi-dCpf1 机制的质粒转移到目标菌株中，在将接合细胞接种到含有选择性抗生素的琼脂平板上的步骤中，建议将 PBS 中 300µL 刮下的细胞悬浮液全部接种到三个平板上，因为当 CRISPRi-dCpf1 机制引入质粒并使这些质粒变大时，接合效率会降低。

Potential issues and solutions: In some cases, the knockdown efficiency is low because the CRISPRi‐dCpf1 system has off‐target effects. To address this problem, one option is to test several different gRNA designs (construct different plasmids containing different gRNAs); the other option is to construct multiple gRNAs containing plasmids, an extended version of our current duplex gRNA design, introducing multiple gRNAs, like four gRNAs, would reduce the off‐target effects and enhance the suppressive efficiency of the CRISPRi‐dCpf1 system.
潜在问题及解决方案 ：在某些情况下，由于 CRISPRi‐dCpf1 系统存在脱靶效应，导致敲减效率较低。为了解决这个问题，一种方案是测试几种不同的 gRNA 设计（构建包含不同 gRNA 的不同质粒）；另一种方案是构建包含多个 gRNA 的质粒，这是我们目前双链 gRNA 设计的扩展版本，引入多个 gRNA（例如四个 gRNA），可以降低脱靶效应并增强 CRISPRi‐dCpf1 系统的抑制效率。

Experimental results interpretation: To determine the suppressive efficiency of target genes by the CRISPRi‐dCpf1 system, the expression of target genes is quantified by qPCR and normalized to reference 16s rRNA gene of the strain. In the case the introduced lacZα gene is the target gene, the strain harboring plasmid pGM‐xBCL (carrying CRISPRi‐dCpf1 + lacZα) serves as the control, and expression of the lacZα gene in the strain harboring plasmid pGM‐xBCF (carrying CRISPRi‐dCpf1 + lacZα + lacZα targeting gRNAs) is normalized to the expression of lacZα gene in control (as shown in Figure 3F). In the case, where a specific gene on the genome of the strain of interest is the target gene, the strain harboring plasmid pGM‐xBCD (carrying CRISPRi‐dCpf1) serves as the control, and expression of the target gene in the strain harboring plasmids that carry CRISPRi‐dCpf1 + gene targeting gRNAs is normalized to the expression of the target gene in control.
实验结果解读 ：为确定 CRISPRi‐dCpf1 系统对靶基因的抑制效率，通过 qPCR 定量检测靶基因的表达量，并以菌株的参考 16s rRNA 基因为标准进行标准化。以导入的 lacZα 基因为靶基因的情况为例，以质粒 pGM‐xBCL（携带 CRISPRi‐dCpf1 + lacZα ）的菌株为对照，以质粒 pGM‐xBCF（携带 CRISPRi‐dCpf1 + lacZα + lacZα 靶向 gRNA）的菌株中 lacZα 基因的表达量为标准进行标准化（如图 3F 所示）。当目标菌株基因组上的特定基因为目标基因时，以含有质粒 pGM‐xBCD（携带 CRISPRi‐dCpf1）的菌株为对照，将含有携带 CRISPRi‐dCpf1 + 基因靶向 gRNA 的质粒的菌株中目标基因的表达标准化为对照中目标基因的表达。

Materials and devices  材料与设备

Primer star DNA polymerase (Takara, Cat# R045), Blue sapphire DNA polymerase (Takara, Cat# RR350), Plasmid Midiprep Kit (Zymo Research, Cat# D4201), DNA Clean and Concentrator (Zymo Research, Cat# D4003), Tryptic Soy Agar (BD, Cat# 236950), Brain Heart Infusion Agar (BD, Cat# 241830), CBA (BD, Cat# 279240), Horse blood (Hemostat Laboratories, Cat# 637291), LB broth (BD, Cat# BP1426), glycerol (Fisher Bioreagents, Cat# BP229), PBS (Gibco, Cat# 10010‐031), centrifuge, PCR amplifier, tetracycline (GoldBio, Cat# T‐101‐25), chloramphenicol (VWR, Cat# 0230), d‐cycloserine (D) (TCI, Cat# C1189), gentamicin (G) (GoldBio, Cat# G‐400‐25), kanamycin (K) (GoldBio, Cat# K‐120‐25), thiamphenicol (Thiam) (Acros Organics, Cat# 455450250), erythromycin (VWR, Cat# 0219), spectinomycin (Cat# BML‐A281‐0010), anaerobic chamber, aerobic incubator, electroporation system, Thermo Scientific Nanodrop 2000, Gibson Assembly Cloning Kit (NEB, Cat# E5510S), Quick DNA fungal/bacterial kit (Zymo Research, Cat# D6005), and ultralow temperature freezer.
Primer star DNA 聚合酶（Takara，Cat# R045）、Blue sapphire DNA 聚合酶（Takara，Cat# RR350）、质粒中量提取试剂盒（Zymo Research，Cat# D4201）、DNA 净化浓缩液（Zymo Research，Cat# D4003）、胰蛋白酶大豆琼脂（BD，Cat# 236950）、脑心浸液琼脂（BD，Cat# 241830）、CBA（BD，Cat# 279240）、马血（Hemostat Laboratories，Cat# 637291）、LB 肉汤（BD，Cat# BP1426）、甘油（Fisher Bioreagents，Cat# BP229）、PBS（Gibco，Cat# 10010-031）、离心机、PCR 扩增子、四环素（GoldBio，Cat# T-101-25）。氯霉素 (VWR，Cat# 0230)、 d- 环丝氨酸 (D) (TCI，Cat# C1189)、庆大霉素 (G) (GoldBio，Cat# G-400-25)、卡那霉素 (K) (GoldBio，Cat# K-120-25)、甲砜霉素 (Thiam) (Acros Organics，Cat# 455450250)、红霉素 (VWR，Cat# 0219)、壮观霉素 (Cat# BML-A281-0010)、厌氧室、好氧培养箱、电穿孔系统、Thermo Scientific Nanodrop 2000、Gibson Assembly Cloning Kit (NEB，Cat# E5510S)、Quick DNA 真菌/细菌试剂盒 (Zymo Research，Cat# D6005) 和超低温冰箱。

Vector assembly  向量组装

Two sets of plasmids (1) pGM‐xCAQ (pGM‐ACAQ, BCAQ, CCAQ, DCAQ, ECAQ, FCAQ, GCAQ, HCAQ, and ICAQ) whose conjugation‐selection marker is catP, and RAM is ermB, and (2) plasmids pGM‐xCBQ (pGM‐ACBQ, DCBQ, ECBQ, FCBQ, HCBQ, and ICBQ) whose conjugation‐selection marker is catP, and RAM is aad9, were generated to test the 16s‐tron strategy for Clostridia microbes [2] (Figure 4A–C). To target other specific genes in Clostridia strains, the introns for the target gene (~300 bp, Figure 4D) could be designed using the ClosTron website and synthesized and assembled with the backbone amplified from pGM‐xCAQ to get the plasmid for the target gene.
生成了两组质粒 (1) pGM‐xCAQ (pGM‐ACAQ、BCAQ、CCAQ、DCAQ、ECAQ、FCAQ、GCAQ、HCAQ 和 ICAQ)，其结合选择标记为 catP ，RAM 为 ermB ，以及 (2) 质粒 pGM‐xCBQ (pGM‐ACBQ、DCBQ、ECBQ、FCBQ、HCBQ 和 ICBQ)，其结合选择标记为 catP ，RAM 为 aad9 ，以测试梭菌微生物的 16s-tron 策略 [ 2 ]（图 4A–C ）。为了针对梭菌菌株中的其他特定基因，可以使用 ClosTron 网站设计目标基因的内含子（~300 bp，图 4D ），并与从 pGM‐xCAQ 扩增的骨架进行合成和组装，以获得目标基因的质粒。

Keynotes: Different combinations of conjugation‐selection markers and RAM could be selected and used to replace the antibiotic markers in those established plasmids.
主题 ：可以选择不同组合的结合选择标记和 RAM 来替换已建立的质粒中的抗生素标记。

Introduction of the assembled 16s‐tron vectors and selection of the RAM‐integrated mutants
组装的 16s-tron 载体的引入和 RAM 整合突变体的选择

Using the strain Blautia luti DSM 14534 (S54) as an example, the plasmid pGM‐FCAQ was transformed into chemically competent E. coli CA434. Then E. coli CA434 harboring plasmid pGM‐FCAQ was conjugated to S54. The transconjugants were picked and restreaked onto a TSAB agar plate supplemented with d‐cycloserine (250 µg/mL) + thiamphenicol (15 µg/mL). Then, three single colonies were cultivated into 1 mL Mega liquid broth supplied with 15 µg/mL thiamphenicol and 250 µg/mL d‐cycloserine. After 24–36 h, 50 µL of cultures were spread onto TSAB agar plates supplemented with 250 µg/mL d‐cycloserine and 10 µg/mL erythromycin. The transconjugants typically appeared after 36–48 h. Eight colonies were picked to inoculate 3 mL Mega liquid broth supplemented with 250 µg/mL d‐cycloserine and 10 µg/mL erythromycin. After 24–36 h, genomic DNA was extracted using Quick DNA fungal/bacterial kit (Zymo Research) and diagnostic PCR was performed using primers 16s_tron_diagR_v4 + 16s_1391R + 16s_1391R_3to5 (with 16s_tron_diagR_v4 binding the integrated intron part and 16s_1391R + 16s_1391R_3to5 binding the target 16s site, only colonies that undergo RAM integration will have the band of ~2.5 kb) (Figure 4D).
以菌株 Blautia luti DSM 14534 (S54) 为例，将质粒 pGM-FCAQ 转化至化学感受态大肠杆菌 CA434 中。然后将携带质粒 pGM-FCAQ 的大肠杆菌 CA434 与 S54 连接。挑取连接子，重新划线接种于添加了 d- 环丝氨酸 (250 µg/mL) + 甲砜霉素 (15 µg/mL) 的 TSAB 琼脂平板上。然后，将三个单菌落培养至 1 mL 含有 15 µg/mL 甲砜霉素和 250 µg/mL d- 环丝氨酸的 Mega 液体肉汤中。24-36 小时后，取 50 µL 培养物涂布于添加了 250 µg/mL d- 环丝氨酸和 10 µg/mL 红霉素的 TSAB 琼脂平板上。转接合子通常在 36-48 小时后出现。挑取 8 个菌落接种于 3 mL Mega 液体肉汤中，并添加 250 µg/mL d- 环丝氨酸和 10 µg/mL 红霉素。24-36 小时后，使用 Quick DNA 真菌/细菌试剂盒（Zymo Research）提取基因组 DNA，并使用引物 16s_tron_diagR_v4 + 16s_1391R + 16s_1391R_3to5 进行诊断性 PCR（其中 16s_tron_diagR_v4 与整合的内含子部分结合，16s_1391R + 16s_1391R_3to5 与目标 16s 位点结合，只有进行 RAM 整合的菌落才会出现约 2.5 kb 的条带）（图 4D ）。

This Group II intron strategy also readily applies to other genes of interest in Clostridia microbes. To mutate a target gene in Clostridia strains, the introns for the target gene (~300 bp, Figure 4D) are designed using the design tool on the ClosTron website and synthesized. The synthesized fragment was assembled with the backbone amplified from the set of plasmids pGM‐xCAQ with a compatible rep ori and antibiotic marker to get the plasmid for mutating the target gene. The plasmid was transferred into donor E. coli CA434 (or HB101/pRK24) and introduced into the recipient microbe via conjugation. The transconjugants harboring the plasmid are identified by antibiotic selection with d‐cycloserine and thiamphenicol. Then, colonies are cultivated into liquid broth with d‐cycloserine and thiamphenicol, and 50 µL of cultures are spread onto agar plates supplemented with d‐cycloserine and erythromycin (or other antibiotic markers to select for RAM insertion). The insertion mutants are validated by diagnostic PCR and sequencing as with 16s rRNA.
这种 II 组内含子策略也适用于梭菌属微生物中其他感兴趣的基因。为了突变梭菌属菌株中的目标基因，需要使用 ClosTron 网站上的设计工具设计并合成目标基因的内含子（约 300 bp，图 4D ）。将合成的片段与从质粒 pGM‐xCAQ 中扩增出的骨架（该骨架带有兼容的复制位点和抗生素标记）组装在一起，得到用于突变目标基因的质粒。将该质粒转移到供体大肠杆菌 CA434（或 HB101/pRK24）中，并通过接合引入受体微生物。通过 d‐ 环丝氨酸和甲砜霉素进行抗生素筛选，鉴定出携带该质粒的接合子。然后，将菌落培养至含有 d- 环丝氨酸和甲砜霉素的液体肉汤中，取 50 µL 培养物涂布于添加了 d- 环丝氨酸和红霉素（或其他用于筛选 RAM 插入的抗生素标记物）的琼脂平板上。插入突变体通过诊断性 PCR 和测序进行验证，验证方法与 16s rRNA 类似。

Keynotes: The Group II intron‐based plasmids are about 10 kb, which is much bigger than the plasmids used for the screening of plasmid uptake and will reduce the conjugation efficiency, in the step of plating conjugation cells onto agar plates with selective antibiotics, it is better to plate all 300 µL scraped‐cell suspension in PBS onto three different plates (100 µL onto each plate).
重点 ：II 组内含子的质粒约为 10 kb，比用于筛选质粒摄取的质粒大得多，并且会降低结合效率，在将结合细胞接种到含有选择性抗生素的琼脂平板上的步骤中，最好将 PBS 中刮下的 300 µL 细胞悬浮液全部接种到三个不同的平板上（每个平板上 100 µL）。

Potential issues and solutions: For some strains, the efficiency of the RAM selection step is low, and the above‐mentioned plating volume (50 µL) is not enough, in this case, more liquid could be plated onto the RAM selection plates, and is recommended to plate several plates.
潜在问题和解决方案 ：对于某些菌株，RAM 选择步骤的效率较低，并且上述接种体积（50 µL）不够，在这种情况下，可以在 RAM 选择平板上接种更多的液体，并建议接种多个平板。

Experimental results interpretation: For the result of diagnostic PCR of the transconjugants up on RAM selection, as shown in Figure 4D, because the forward diagnostic primer binds the sequence on the plasmid and the reverse diagnostic primer binds the sequence on the genome, only transconjugants that undergo the expected integration would have the 2.5 kb PCR product; WT strain or transconjugants that undergo the unexpected insertion would not have the 2.5 kb PCR product.
实验结果解释 ：对于图 4D 所示的基于 RAM 选择的转接合子诊断 PCR 结果，由于正向诊断引物与质粒上的序列结合，反向诊断引物与基因组上的序列结合，因此只有发生预期整合的转接合子才会有 2.5kb 的 PCR 产物；而 WT 菌株或发生意外插入的转接合子则不会有 2.5kb 的 PCR 产物。